Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury.

We describe here a new strategy for the treatment of stroke, through the inhibition of NAALADase (N-acetylated-alpha-linked-acidic dipeptidase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. We demonstrate that the newly described NAALADase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) robustly protects against ischemic injury in a neuronal culture model of stroke and in rats after transient middle cerebral artery occlusion. Consistent with inhibition of NAALADase, we show that 2-PMPA increases NAAG and attenuates the ischemia-induced rise in glutamate. Both effects could contribute to neuroprotection. These data indicate that NAALADase inhibition may have use in neurological disorders in which excessive excitatory amino acid transmission is pathogenic.

External validation, extension and recalibration of Braunwald's simple risk index in a community-based cohort of patients with both STEMI and NSTEMI.

BACKGROUND: Using the simple risk index (SRI) that is based on age, heart rate and systolic blood pressure, we sought to evaluate the ability to predict outcome in AMI patients in a community-based population. METHODS AND RESULTS: We identified and evaluated 3684 consecutive patients with an admission diagnosis of possible AMI, who attended between 1st September and 30th November 1995. Two thousand one hundred fifty three patients had confirmed evidence of WHO definition AMI, of whom 1656 survived to hospital discharge. We evaluated the ability of the SRI to predict mortality over 30 days using the score generated by the equation (heart ratex[age/10]2)/systolic blood pressure. The SRI was a strong (c-statistic = 0.77 CI 0.74-0.79) predictor of 30-day mortality in both STEMI and all consecutive cases of WHO definition AMI. However, the model showed poor calibration when used on a community-based population with 30-day mortality being underestimated across all risk quintiles. Consequently we sought to recalibrate the quantitative aspects of the model for the total AMI population in the following way (Risk Index; 30-day mortality) (< or = 29.2; 9.2%), (29.3-37.8; 23.9%), (37.9-47.3; 34.6%), (47.4-61.5; 40.3%), (> or = 61.6; 65.5%). CONCLUSION: We have externally validated the SRI in an unselected cohort of consecutive WHO definition AMI patients. However, the original model consistently underestimated the likelihood of death at 30 days regardless of the calculated risk score. We have therefore suggested corrections to the risk estimates, to allow its application in an unselected community cohort.

Acute regulation of sodium-dependent glutamate transporters: a focus on constitutive and regulated trafficking.

The acidic amino acid glutamate activates a family of ligand-gated ion channels to mediate depolarization that can be as short-lived as a few milliseconds and activates a family of G protein-coupled receptors that couple to both ion channels and other second messenger pathways. Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and is required for essentially all motor, sensory, and cognitive functions. In addition, glutamate-mediated signaling is required for development and the synaptic plasticity thought to underlie memory formation and retrieval. The levels of glutamate in brain approach 10 mmol/kg and most cells in the CNS express at least one of the receptor subtypes. Unlike acetylcholine that mediates "rapid" excitatory neurotransmission at the neuromuscular junction, there is no evidence for extracellular inactivation of glutamate. Instead, glutamate is cleared by a family of Na(+)-dependent transport systems that are found on glial processes that sheath the synapse and found on the pre- and postsynaptic elements of neurons. These transporters ensure crisp excitatory transmission by maintaining synaptic concentrations below those required for tonic activation of glutamate receptors under baseline conditions (approximately 1 microM) and serve to limit activation of glutamate receptors after release. During the past few years, it has become clear that like many of the other neurotransmitter transporters discussed in this volume of Handbook of Experimental Pharmacology, the activity of these transporters can be rapidly regulated by a variety of effectors. In this chapter, a broad overview of excitatory signaling will be followed by a brief introduction to the family of Na(+)-dependent glutamate transporters and a detailed discussion of our current understanding of the mechanisms that control transporter activity. The focus will be on our current understanding of the mechanisms that could regulate transporter activity within minutes, implying that this regulation is independent of transcriptional or translational control mechanisms. The glutamate transporters found in forebrain are regulated by redistributing the proteins to or from the plasma membrane; the signals involved and the net effects on transporter activity are being defined. In addition, there is evidence to suggest that the intrinsic activity of these transporters is also regulated by mechanisms that are independent of transporter redistribution; less is known about these events. As this field progresses, it should be possible to determine how this regulation affects physiologic and pathologic events in the CNS.

Transcriptional Regulation of Glutamate Transporters: From Extracellular Signals to Transcription Factors.

Glutamate is the predominant excitatory neurotransmitter in the mammalian CNS. It mediates essentially all rapid excitatory signaling. Dysfunction of glutamatergic signaling contributes to developmental, neurologic, and psychiatric diseases. Extracellular glutamate is cleared by a family of five Na(+)-dependent glutamate transporters. Two of these transporters (GLAST and GLT-1) are relatively selectively expressed in astrocytes. Other of these transporters (EAAC1) is expressed by neurons throughout the nervous system. Expression of the last two members of this family (EAAT4 and EAAT5) is almost exclusively restricted to specific populations of neurons in cerebellum and retina, respectively. In this review, we will discuss our current understanding of the mechanisms that control transcriptional regulation of the different members of this family. Over the last two decades, our understanding of the mechanisms that regulate expression of GLT-1 and GLAST has advanced considerably; several specific transcription factors, cis-elements, and epigenetic mechanisms have been identified. For the other members of the family, little or nothing is known about the mechanisms that control their transcription. It is assumed that by defining the mechanisms involved, we will advance our understanding of the events that result in cell-specific expression of these transporters and perhaps begin to define the mechanisms by which neurologic diseases are changing the biology of the cells that express these transporters. This approach might provide a pathway for developing new therapies for a wide range of essentially untreatable and devastating diseases that kill neurons by an excitotoxic mechanism.

Differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors.

Mouse models of ornithine transcarbamylase (OTC) deficiency are being used to test the efficacy of viral vectors as possible vehicles for gene therapy. However, it has been demonstrated that virus containing the human OTC cDNA failed to express functional OTC enzyme in the recipient animals. Because functional OTC is assembled as a homotrimer in the mitochondria, there are at least two possible explanations for these results. Either endogenous mutant protein coassembles with the human OTC and has a "dominant-negative effect," or the human version of the protein is not appropriately imported or processed in the mouse mitochondria. To test the importance of processing, which in rodents is thought to depend on the leader peptide, adenoviral vectors containing chimeric OTC cDNAs were prepared. These vectors were evaluated in the OTC-deficient sparse fur mouse models. Although comparable levels of transgene expression were observed in all groups of mice, the only mice that had high levels of OTC activity and mitochondrial OTC immunoreactivity were those mice injected with the vectors containing the mouse leader peptide (mouse OTC and a mouse-human chimera of OTC). To address possible dominant-negative effects, adenoviruses containing mutant human or mouse OTC cDNAs were prepared and evaluated in cell lines or normal C3H mice, respectively. No inhibition of normal OTC activity was observed in either model system. Together, these studies provide no evidence of a dominant-negative effect and suggest that the human and rodent enzymes responsible for transporting of OTC and possibly other mitochondrial proteins have different specificity.

Effects of quisqualic acid analogs on metabotropic glutamate receptors coupled to phosphoinositide hydrolysis in rat hippocampus.

L-Glutamic acid (L-Glu) and L-aspartic acid (L-Asp) activate several receptor subtypes, including metabotropic Glu receptors coupled to phosphoinositide (PI) hydrolysis. Quisqualic acid (Quis) is the most potent agonist of these receptors. There is evidence that activation of these receptors may cause a long lasting sensitization of neurons to depolarization, a phenomenon called the Quis effect. The purpose of the current studies was to use Quis analogs and the recently identified metabotropic receptor antagonist, (+)-alpha-methyl-4-carboxy-phenylglycine((+)-MCPG), to define the structural properties required for interaction with the metabotropic receptors coupled to PI hydrolysis and to determine if the Quis effect is mediated by these receptors. The effects of Quis analogs on PI hydrolysis were studied in the absence or presence of the metabotropic receptor-specific agonist 1SR,3RS-1-amino-1,3-cyclopentanedicarboxylic acid (1SR,3RS-ACPD) in neonatal rat hippocampus. Some of the compounds that induce the Quis effect also stimulate PI hydrolysis, including Quis itself and 9 (homoquisqualic acid). Not all of the Quis analogs that stimulate PI hydrolysis, however, induce the Quis effect, including 7A (EC50 = 750 +/- 150 microM) and (RS)-4-bromohomoibotenic acid (BrHI) (EC50 = 130 +/- 40 microM). Although (+)-MCPG blocked PI hydrolysis stimulated by Quis (IC50 = 370 +/- 70 microM), it had no effect on the induction of the Quis effect. Other Quis analogs did not stimulate PI hydrolysis but rather blocked the effects of 1SR,3RS-ACPD. The IC50 values were 240 +/- 70 microM for 2, 250 +/- 90 microM for 3, and 640 +/- 200 microM for 4. Data for inhibition by 2 and 3 were consistent with non-competitive mechanisms of action. These studies provide new information about the structural features of Quis required for interaction with metabotropic receptors coupled to PI hydrolysis and provide evidence that the Quis effect is not mediated by (+)-MCPG sensitive subtypes of these receptors.

Cyclic analogues of 2-amino-4-phosphonobutanoic acid (APB) and their inhibition of hippocampal excitatory transmission and displacement of [3H]APB binding.

Conformationally restricted analogues of 2-amino-4-phosphonobutanoic acid (APB,2) were prepared where the structure of APB was incorporated into cyclopentane (3) or cyclohexane (4) rings. Hydrophosphinylation of the appropriate cycloalkenones followed by Strecker amino acid syntheses provided the desired analogues. Assignments of the relative configurations for 3a (trans), 3b (cis), 4a (cis), and 4b (trans) were determined through 13C NMR studies. Compounds 3b, 4a, and 4b possessed low activity as inhibitors of excitatory synaptic field potentials in the rat hippocampal perforant path. Analogues 4a and 4b also showed little activity in displacing [3H]APB from synaptic plasma membranes. The cyclopentyl APB analogue 36, on the other hand, was extremely potent in inhibiting the binding of [3H]APB, possessing an IC50 = 4.7 microM, thus giving further credence to the idea that the APB binding site in the rat brain synaptosomal membrane preparation is not the same as the receptor mediating APB-induced inhibition of the lateral perforant path. Of the four cyclic APB analogues, 3a most resembled APB in its spectrum of biological activity. It showed significant potency (IC50 = 130 microM) in inhibiting lateral entorhinal projections to hippocampal granule cells. Analogous to APB, 3a also showed selectivity for the lateral perforant path over the medial perforant path. Its activity in the radioligand binding assay paralleled its activity in inhibiting the lateral perforant path. It thus appears that 3a comes closest to mimicking the active conformation of APB and suggests that a folded conformation wherein the amino and phosphonate moieties are in a cis relationship to one another may approximate the active conformation of APB.

Cyclobutane quisqualic acid analogues as selective mGluR5a metabotropic glutamic acid receptor ligands.

The conformationally constrained cyclobutane analogues of quisqualic acid (Z)- and (E)-1-amino-3-[2'-(3',5'-dioxo-1',2', 4'-oxadiazolidinyl)]cyclobutane-1-carboxylic acid, compounds 2 and 3, respectively, were synthesized. Both 2 and 3 stimulated phosphoinositide (PI) hydrolysis in the hippocampus with EC50 values of 18 +/- 6 and 53 +/- 19 microM, respectively. Neither analogue stimulated PI hydrolysis in the cerebellum. The effects of 2 and 3 were also examined in BHK cells which expressed either mGluR1a or mGluR5a receptors. Compounds 2 and 3 stimulated PI hydrolysis in cells expressing mGluR5a but not in those cells expressing mGluR1a. The EC50 value for 2 was 11 +/- 4 microM, while that for 3 was 49 +/- 25 microM. Both 2 and 3 did not show any significant effect on cells expressing the mGluR2 and mGluR4a receptors. In addition, neither compound blocked [3H]glutamic acid uptake into synaptosomal membranes, and neither compound was able to produce the QUIS effect as does quisqualic acid. This pharmacological profile indicates that 2 and 3 are selective ligands for the mGluR5a metabotropic glutamic acid receptor.

Depression of retinal glutamate transporter function leads to elevated intravitreal glutamate levels and ganglion cell death.

PURPOSE: Elevated levels of extracellular glutamate have been implicated in the pathophysiology of neuronal loss in both central nervous system and ophthalmic disorders, including glaucoma. This increase in glutamate may result from a failure of glutamate transporters (molecules that ordinarily regulate extracellular glutamate; E:xcitatory A:mino A:cid T:ransporter; EAAT). Elevated glutamate levels can also lead to alterations in glutamate receptor expression. It was hypothesized that selective blockade of glutamate transporters would be toxic to retinal ganglion cells. METHODS: Glutamate transporters were blocked either pharmacologically or with subtype-specific antisense oligonucleotides against EAAT1. Glutamate levels, transporter levels and ganglion cell survival were assayed. RESULTS: Pharmacological inhibition of glutamate transporters with either an EAAT2 specific inhibitor or a nonspecific inhibitor of all the subtypes of transporters was toxic to ganglion cells. Treatment with oligonucleotides against the glutamate transporter EAAT1 decreased the levels of expression of the transporter, increased vitreal glutamate, and was toxic to ganglion cells. CONCLUSIONS: These results demonstrate that normal function of EAAT1 and EAAT2 is necessary for retinal ganglion cell survival and plays an important role in retinal excitotoxicity. Manipulation of retinal glutamate transporter expression may become a useful tool in understanding retinal neuronal loss.

Expression patterns and regulation of glutamate transporters in the developing and adult nervous system.

Glutamate and aspartate are the primary excitatory neurotransmitters in the mammalian central nervous system and have also been implicated as mediators of excitotoxic neuronal injury and death. The precise control of extracellular glutamate and aspartate is crucial to the maintenance of normal synaptic transmission and the prevention of excitotoxicity following acute insults to the brain, such as stroke or head trauma, or during the progression of neurodegenerative diseases such as amyotrophic lateral sclerosis. The removal of excitatory amino acids (EAAs) from the extracellular space is primarily mediated by a family of sodium-dependent glutamate transporters. These transporters use the sodium electrochemical gradients of the cell to actively concentrate EAAs in both neurons and glia. Five members of this transporter family have been cloned recently and include both 'glial'-specific and 'neuron'-specific subtypes. Although these subtypes share many common functional properties, there are considerable differences in developmental expression, chronic and acute regulation by cellular signaling pathways, and contribution to disease processes among the subtypes. In this review recent studies of glutamate transporter expression, regulation, function, and pathological relevance are summarized, and some of the discrepancies and unexpected results common to any rapidly progressing field are discussed.

The family of sodium-dependent glutamate transporters: a focus on the GLT-1/EAAT2 subtype.

The acidic amino acids, glutamate and aspartate, are the predominant excitatory neurotransmitters in the mammalian CNS. Under many pathologic conditions, these excitatory amino acids (EAAs) accumulate in the extracellular fluid in CNS and the resultant excessive activation of EAA receptors contributes to brain injury through a process known as 'excitotoxicity'. Unlike many other neurotransmitters, there is no evidence for extracellular metabolism of EAAs, rather, they are cleared by Na+-dependent transport mechanisms. Therefore, this transport process is important for ensuring crisp synaptic signaling as well as limiting the excitotoxic potential of EAAs. With the cloning of five distinct EAA transporters, a variety of tools were developed to characterize individual transporter subtypes, including specific antibodies, expression systems, and probes to delete/knock-down expression of each subtype. These tools are beginning to provide fundamental information that has the potential to impact our understanding of EAA physiology and pathophysiology. For example, biophysical studies of the cloned transporters have led to the observation that some subtypes function as ligand-gated ion channels as well as transporters. With these reagents, it has also been possible to explore the relative contributions of each transporter to the clearance of extracellular EAAs and to begin to examine the regulation of specific transporter subtypes. In this review, an overview of the properties of the transporter subtypes will be presented. The evidence which suggests that the transporter, GLT1/EAAT2, may be sufficient to explain a large percentage of forebrain transport will be critically reviewed. Finally, the studies of regulation of GLT-1 in vitro and in vivo will be described.

Substrate-induced up-regulation of Na(+)-dependent glutamate transport activity.

Sodium-dependent transporters regulate extracellular glutamate in the CNS. Recent studies suggest that the activity of several different neurotransmitter transporters can be rapidly regulated by a variety of mechanisms. In the present study, we report that pre-incubation of primary 'astrocyte-poor' neuronal cultures with glutamate (100 microM) for 30 min nearly doubled the V(max) for Na(+)-dependent accumulation of L-[(3)H]-glutamate, but had no effect on Na(+)-dependent [(3)H]-glycine transport. Pre-incubation with glutamate also increased the net uptake of non-radioactive glutamate, providing evidence that the increase in accumulation of L-[(3)H]-glutamate was not related to an increase in intracellular glutamate and a subsequent increase in exchange of intracellular non-radioactive glutamate for extracellular radioactive glutamate. The glutamate receptor agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, quisqualate, and (1 S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid did not mimic the effect of pre-incubation with glutamate and the glutamate-induced increase was not blocked by receptor antagonists. However, compounds known to interact with the transporters, including L-aspartate, D-aspartate, L-(-)-threo-3-hydroxyaspartate (L-THA) and L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC), caused variable increases in transport activity and attenuated the increase induced by glutamate, suggesting that the increase is related to the interaction of glutamate with the transporters. Several studies were attempted to define the mechanism of this regulation. We found no evidence for increases in transporter synthesis or cell surface expression. Inhibitors of signaling molecules known to regulate other neurotransmitter transporters had no effect on this stimulation. Using a variety of cultures, evidence is provided to suggest that this substrate-induced up-regulation of glutamate transport is specific for the GLT-1 and GLAST subtypes and does not influence transport mediated by EAAC1. These studies suggest that the interaction of glutamate with some of the subtypes of glutamate transporters causes an increase in transport activity. Conceivably, this phenomenon provides an endogenous mechanism to increase the clearance of glutamate during periods of prolonged elevations in extracellular glutamate.

The glutamate transporter, GLT-1, is expressed in cultured hippocampal neurons.

There are multiple subtypes of Na+-dependent glutamate transporters. Several studies suggest that EAAC1 and EAAT4 are expressed in neurons, while GLT-1 and GLAST expression is thought to be restricted to glia. In the present study, expression of GLT-1 and EAAC1 was examined in cultured rat hippocampal neurons using single cell mRNA amplification and immunocytochemistry with subtype specific antibodies. GLT-1 and EAAC1 mRNAs were observed in all neurons examined. Neuronal phenotype was confirmed in these cells by expression of neurofilament (NF-L) mRNA and absence of glial fibrillary acidic protein (GFAP) mRNA. EAAC1 immunoreactivity was observed in essentially all cells which expressed neuron specific enolase (NSE) and GLT-1 immunoreactivity was detected in the majority (approximately 90%) of NSE-positive cells. Consistent with the glial expression of GLT-1, GLT-1 immunoreactivity was also observed in NSE-negative cells. These studies provide evidence that GLT-1 expression is not intrinsically restricted to glial cells, but can occur in neurons under certain circumstances.

Identifying acute myocardial infarction: effects on treatment and mortality, and implications for National Service Framework audit.

BACKGROUND: The National Service Framework (NSF) for Coronary Heart Disease requires annual clinical audit of the care of patients with myocardial infarction, with little guidance on how to achieve these standards and monitor practice. AIM: To assess which method of identification of acute myocardial infarction (AMI) cases is most suitable for NSF audit, and to determine the effect of the definition of AMI on the assessment of quality of care. DESIGN: Observational study. METHODS: Over a 3-month period, 2153 consecutive patients from 20 hospitals across the Yorkshire region, with confirmed AMI, were identified from coronary care registers, biochemistry records and hospital coding systems. The sensitivity and positive predictive value of AMI patient identification using clinical coding, biochemistry and coronary care registers were compared to a 'gold standard' (the combination of all three methods). RESULTS: Of 3685 possible cases of AMI singled out by one or more methods, 2153 patients were identified as having a final diagnosis of AMI. Hospital coding revealed 1668 (77.5%) cases, with a demographic profile similar to that of the total cohort. Secondary preventative measures required for inclusion in NSF were also of broadly similar distribution. The sensitivities and positive predictive values for patient identification were substantially less in the cohorts identified through biochemistry and coronary care unit register. Patients fulfilling WHO criteria (n=1391) had a 30-day mortality of 15.9%, vs. 24.2% for the total cohort. DISCUSSION: Hospital coding misses a substantial proportion (22.5%) of AMI cases, but without any apparent systematic bias, and thus provides a suitably representative and robust basis for NSF-related audit. Better still would be the routine use of multiple methods of case identification.

Repeated exposure to hyperbaric oxygen sensitizes rats to oxygen-induced seizures.

Repeated exposure to increased partial pressure of oxygen (PO2) is the standard of care for several medical conditions. The side-effects of repeated exposure to hyperbaric oxygen (HBO), however, are not well defined. Previous studies have demonstrated that acute exposure of rats to HBO causes hypothermia that precedes convulsions. In the present studies, rats that were repeatedly exposed to 100% oxygen at 4 atmospheres absolute (ATA) pressure developed convulsions earlier than naive controls. There was also a trend toward less hypothermia in the rats repeatedly exposed to oxygen. The purpose of this study was to test the hypothesis that repeated exposure to HBO increases sensitivity to convulsions induced by HBO and to determine if the time to onset of convulsions is affected by the hypothermia caused by exposure to HBO. Rats were repeatedly exposed to 2 ATA oxygen for a total of 10 days. After 72 h, these rats were challenged by exposure to 100% oxygen at 4 ATA pressure. Rats repeatedly exposed to HBO had convulsions significantly earlier than the naive controls (84 +/- 8 min compared to 147 +/- 11 min), and they developed significantly less hypothermia. Control studies suggested that the decrease in the degree of hypothermia was caused by both repeated exposure to oxygen and adaptation to the mild restraint used during oxygen re-exposures. Adaptation to restraint eliminated the hypothermia induced by oxygen but did not change the time to onset of convulsions. Increased sensitivity to convulsions was present after five exposures to 2 ATA oxygen and persisted for 10 days after the last 2 ATA oxygen re-exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacologically distinct sodium-dependent L-[3H]glutamate transport processes in rat brain.

The transport of L-[3H]glutamate into crude synaptosomal membrane fractions prepared from cerebellum, brainstem, hippocampus, cortex, striatum, and midbrain was characterized. In all brain regions, greater than 95% of the accumulation of radiolabel was sodium-dependent and the concentration-dependence was consistent with a single high affinity site. Dihydrokainate and L-alpha-aminoadipate were region specific inhibitors of uptake; this inhibition was consistent with a competitive mechanism. In the forebrain regions examined, dihydrokainate inhibited transport with IC50s of approx. 100 microM (range from 80 to 170 microM). Transport in cerebellum was essentially dihydrokainate-insensitive L-alpha-Aminoadipate inhibited transport in forebrain regions with IC50s of approx. 700 microM (range from 590 to 800 microM) and inhibited transport in cerebellum with an IC50 of 40 microM. The inhibition data obtained with forebrain and cerebellar tissues were consistent with nearly homogeneous (greater than 80%) populations of non-interacting sites. Inhibition data obtained with tissue prepared from brainstem were best fit to a mixture of the two sites (35-50% of the type observed in cerebellum). Other previously identified uptake inhibitors, including DL-threo-hydroxyaspartate, L-aspartate-beta-hydroxamate, beta-glutamate, and L-cysteine sulfinate were not selective for the two types of transport. These data demonstrate that there are two pharmacologically distinct sodium-dependent high affinity transport systems with heterogeneous regional distributions.

Kynurenic acid as an antagonist of hippocampal excitatory transmission.

Kynurenate, an endogenous tryptophan metabolite, was bath-applied to hippocampal slices while recording extracellular synaptic field potentials. Kynurenate antagonized the medial and lateral entorhinal projections to dentate granule cells, the Schaffer collateral projections to CA1 pyramidal cells, and inputs to the CA3 stratum radiatum of regio inferior with similar potencies. Concentration-response curves for these pathways paralleled theoretical antagonist curves with a Hill coefficient of 1, and the KdS were in the range of 130-400 microM. Projections to the stratum lucidum of regio inferior were much less sensitive to kynurenate. Inputs to CA3 pyramidal cells showed varying sensitivities to kynurenate, L-2-amino-4-phosphonobutanoic acid (L-APB), and (-)-baclofen depending on the placements of the stimulating and recording electrodes. When both electrodes were located in area CA3, outside the hilus of area dentata, all responses were insensitive to inhibition by L-APB. Under these conditions, responses recorded within the stratum radiatum were sensitive to inhibition by kynurenate and baclofen, while responses recorded within the stratum lucidum were insensitive to these drugs. When the stimulating electrode was placed within the hilus of area dentata, variable patterns of sensitivity to APB, baclofen, and kynurenate were observed from recording electrodes in area CA3. These results suggest that stimulation in the hilus, while recording in the stratum lucidum, produces responses that show composite effects resulting both from direct stimulation of mossy fibers and from stimulation of neuronal elements in the hilus which produce outputs to mossy fibers.

Exposure of hippocampal slices to quisqualate sensitizes synaptic responses to phosphonate-containing analogues of glutamate.

Exposure of transverse slices of rat hippocampus to quisqualate (Quis) resulted in a marked increase in the potency of D- and L-2-amino-4-phosphonobutanoate (APB) and D- and L-2-amino-5-phosphonopentanoate (APV) for depression of extracellular synaptic field potentials recorded from CA1 pyramidal cells. L-APB depressed the amplitude of CA1 field potentials with an IC50 = 1800 microM before exposure to Quis. After a brief (4 min) exposure to sufficient Quis (16 microM) to depress the response by 50%, L-APB depressed these responses with an IC50 = 54 microM. These phosphonate-containing glutamate analogues transiently induced population-spiking after the tissue was pretreated with Quis. This suggests that APB and APV can act as agonists at micromolar concentrations.

Intrastriatal injections of the succinate dehydrogenase inhibitor, malonate, cause a rise in extracellular amino acids that is blocked by MK-801.

The effects of intrastriatal injections of a reversible inhibitor of succinate dehydrogenase, malonate, on the extracellular concentrations of amino acid neurotransmitters were examined using a microdialysis probe that was positioned a fixed distance from an injection cannula. Malonate (2 mumol) caused a 23 +/- 5-fold increase in extracellular glutamate (Glu), a 18 +/- 6-fold increase extracellular gamma-aminobutyric acid (GABA) and a modest increase in extracellular aspartate (Asp, 2.9 +/- 0.8-fold increase). Administration of the NMDA receptor antagonist MK-801 (5 mg/kg) prior to injection of malonate almost completely blocked these increases. This study provides direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular amino acid neurotransmitters and further evidence that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through an excitotoxic mechanism.

Seizures decrease regional enzymatic hydrolysis of N-acetyl-aspartylglutamate in rat brain.

Previous results have shown that kindled seizures increase N-acetyl-aspartylglutamate (NAAG) levels in the entorhinal cortex, while non-kindled convulsions have no effect. To further explore possible relationships between epilepsy and the physiology of NAAG, the effect of amygdaloid kindling on the activity of a NAAG-hydrolyzing enzyme was examined in specific brain regions associated with limbic seizures. NAAG is hydrolyzed into glutamate (Glu) and N-acetyl-aspartate (NAA) by N-acetylated-alpha-linked acidic dipeptidase (NAALADase), a membrane-bound peptidase. We found that convulsions decreased NAALADase activity and these effects were generalized to several brain regions. While small decreases in the hippocampus were specific to kindling, the decreases in other limbic regions were larger, non-specific, and appear to be aftereffects of convulsions; i.e. not specific to kindling. Although there is evidence that NAAG may be an excitatory neurotransmitter, it could also function as a storage form of Glu. Thus, a reduction in NAALADase activity could reduce the availability of Glu at certain synapses, which might be a homeostatic mechanism for lessening susceptibility to further seizures.