James F. Koerner

Micromolar L-2-amino-4-phosphonobutyric acid selectively inhibits perforant path synapses from lateral entorhinal cortex

Transverse slices of the rat hippocampus were used to examine the ability of phosphonate analogues of acidic amino acids to inhibit perforant path synaptic transmission. Micromolar concentrations of L-2-amino-4-phosphonobutyric acid (APB), an analogue of L-glutamic acid, inhibited transmission from the lateral entorhinal cortex. Two other less-sensitive components were detected in projections from the medial entorhinal cortex. The component from the lateral entorhinal cortex showed high stereospecificity for the L-isomer of APB and was relatively insensitive to phosphonate homologues of shorter and longer chain length.

Response of Schaffer collateral-CA1 pyramidal cell synapses of the hippocampus to analogues of acidic amino acids

Analogues of the putative excitatory transmitters aspartic acid and glutamic acid were tested for antagonism against stimulus-evoked activation of Schaffer collateral-CA 1 pyramidal cell synapses in slices of rat hippocampus. Responses to the analogues, applied via the superfusing medium, were extracellularly recorded. The compounds examined included D- and L-alpha-aminodicarboxylic acids, diaminodicarboxylic acids, phosphonate analogues of acidic amino acids, D- and L-gamma-glutamyl glycine, and the cis- and trans-isomers of piperidine 2,3-, and 2,4-dicarboxylic acid. Many of these compounds are known to be potent and selective antagonists for excitatory amino acids and a few excitatory pathways. In this hippocampal pathway most of these analogues showed relatively low and similar potency. The most potent antagonist uncontaminated with agonist activity was D-alpha-aminosuberate, with an apparent antagonist dissociation constant (Kd) of 3 mM. Only 5 of the analogues, 3 of the piperidine dicarboxylates, kainic acid, and L-alpha-aminopimelic acid, reduced the amplitude of the extracellularly recorded field potentials more than 30% at 0.5 mM. However, all of the others reduced the potential by more than 30% at 5 mM. Most analogues also evoked extracellular responses which can be attributed to depolarization of the pyramidal neurons. Agonist activity was particularly strong among the most potent analogues. These results contrast with the responses documented by others for the N-methyl-D-aspartate receptor of the dorsal-ventral root excitatory pathway of the spinal cord in which the higher homologues tested here were the most potent antagonists, and the D-isomers were more potent than the L-isomers. It also contrasts with the response of the perforant path synapses to granule cells of the dentate gyrus in which the portion derived from the lateral entorhinal cortex is sensitive to L-2-amino-4-phosphonobutyric acid. Thus the Schaffer-CA 1 pyramidal cell synaptic field utilizes a novel excitatory transmitter receptor which interacts detectably but only weakly with a variety of acidic amino acids with potent specific inhibitory action for receptors elsewhere in the central nervous system.

NMDA-, kainate- and quisqualate-stimulated release of taurine from electrophysiologically monitored rat hippocampal slices

While excitatory amino acids (EAAs) are known to evoke the release of taurine in the hippocampus, we have found that taurine is localized primarily in dendrites and only to a lesser extent in terminals in this region. To determine whether taurine is released as a neurotransmitter by non-toxic concentrations of EAAs, or exclusively as a neuroprotectant in response to excitotoxicity, we monitored the release of amino acids from hippocampal slices during simultaneous electrophysiological recording in the CA1 region to assess tissue viability. N-methyl-D-aspartate (NMDA) was the most potent of the EAA agonists tested for stimulating release of taurine. Exposure of slices to 120 microM NMDA increased the concentration of taurine in the perfusate to 1325% of its basal value. Kainate (KA) at a concentration of 128 microM increased taurine to 543% of baseline while quisqualate (Quis) at a concentration of 120 microM increase taurine to only 202% of its baseline value. Release of taurine in response to NMDA and KA peaked during the period when the concentration of the agonist was declining in the bath and did not return to its baseline value until 20 min after removal of the agonist. Increases in release of taurine were associated with concentrations of NMDA, KA, and Quis that caused an incomplete recovery of the CA1 field potential. These results suggest that taurine is primarily released by concentrations of glutamate receptor agonists that exhibit evidence of excitotoxicity in the CA1 region.

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.

A microperfusion chamber for brain slice pharmacology.

The construction of a chamber for extracellular recording from submerged CNS tissue is described. Its operation is illustrated using hippocampal slices prepared from rat brains. The total volume of perfusing medium is less than 0.4 ml, and a drug solution can be uniformly distributed throughout this volume in less than 1 min. Drug-laden medium can also be rapidly replaced with fresh medium. The perfusing medium is continuously stirred with a jet of 95% O2-5% CO2, maintaining submerged slices viable for many hours. The system has exceptional advantages for investigating synaptic pharmacology of scarce and expensive drugs.

Structure - function relationships for gamma-substituted glutamate analogues on dentate granule cells.

We previously demonstrated in the Schaffer collateral-CA1 region of the hippocampus that bath-applied agonists could be distinguished from antagonists among a group of acidic amino acid analogues by extracellular recording techniques. Here we report the use of the extracellular signs of agonist activity for discerning agonists and antagonists among several gamma-substituted glutamate analogues tested in the perforant path. The two-pathway composition of the perforant path offers the advantage over CA1 in that pathway-specificity, a postulated characteristic of antagonists, may be tested. By extracellular recording, D- and L-homocysteic acid, L-serine-O-sulfate, and L-2-amino-4-(5-tetrazolyl)-butanoic acid [L-glutamate tetrazole] were identified as agonists, and all 4 analogues were more potent than L-glutamate for inhibiting synaptic field potentials. Two previously identified antagonists, L-2-amino-4-phosphonobutyric acid and L-O-phosphoserine, exhibited the pathway-specificity and inhibitory kinetics consistent with properties expected for antagonists; both compounds detected 3 perforant path components with the same rank in sensitivity, suggesting that they are acting on the same set of receptors.

L-Quisqualic acid transport into hippocampal neurons by a cystine-sensitive carrier is required for the induction of quisqualate sensitization.

A brief exposure of hippocampal slices to L-quisqualic acid sensitizes CA1 pyramidal neurons 30-250-fold to depolarization by two classes of excitatory amino acid analogues: (1) those whose depolarizing effects are rapidly terminated following washout, e.g. L-2-amino-4-phosphonobutanoic acid (L-AP4) and L-2-amino-6-phosphonohexanoic acid (L-AP6) and (2) those whose depolarizing effects persist following washout, e.g. L-aspartate-beta-hydroxamate (L-AbetaH). This process has been termed quisqualate sensitization. In this study we directly examine the role of amino acid transport systems in the induction of quisqualate sensitization. We report that L-quisqualate is a low-affinity substrate (K(M)=0.54 mM) for a high capacity (V(max)=0.9 nmol (mg protein)(-1) min(-1)) Na(+)-dependent transport system(s) and a high-affinity substrate (K(M)=0.033 mM) for a low-capacity (V(max)=0.051 nmol (mg protein)(-1) min(-1)) transporter with properties similar to the cystine/glutamate exchange carrier, System x(c-). We present evidence that suggests that System x(c-) participates in quisqualate sensitization. First, simultaneous application of L-quisqualate and inhibitors of System x(c-), but not inhibitors of Na(+)-dependent glutamate transporters, prevents the subsequent sensitization of hippocampal neurons to phosphonates or L-AbetaH. Second, L-quisqualic acid only sensitizes hippocampal neurons to other substrates of System x(c-), including cystine. Third, immunocytochemical analysis of L-quisqualate uptake demonstrates that only inhibitors of System x(c-) inhibit the highly concentrative uptake of L-quisqualate into a widely dispersed group of GABAergic hippocampal interneurons. We conclude that quisqualate sensitization is a direct consequence of the unique interaction of various excitatory amino acids, namely L-quisqualate, cystine, and phosphonates, with the exchange carrier, System x(c-). Therefore, the results of this study have important implications for the mechanism by which L-quisqualate, and other substrates of this transporter which are also excitatory amino acid agonists (such as glutamate and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, beta-L-ODAP) may trigger neurotoxicity.

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.

Utilization of the resolved L-isomer of 2-amino-6-phosphonohexanoic acid (L-AP6) as a selective agonist for a quisqualate-sensitized site in hippocampal CA1 pyramidal neurons.

Brief exposure of rat hippocampal slices to quisqualic acid (QUIS) sensitizes neurons to depolarization by the α-amino-ω-phosphonate excitatory amino acid (EAA) analogues AP4, AP5 and AP6. These phosphonates interact with a novel QUIS-sensitized site. Whereasl-AP4 andd-AP5 cross-react with other EAA receptors,dl-AP6 has been shown to be relatively selective for the QUIS-sensitized site. This specificity ofdl-AP6, in conjuction with the apparent preference of this site forl-isomers, suggested that the hitherto unavailablel-isomer of AP6 would be a potent and specific agonist. We report the resolution of thed- andl-enantiomers of AP6 by fractional crystallization of thel-lysine salt ofdl-AP6. We also report the pharmacological responses of kainate / AMPA, NMDA, lateral perforant pathl-AP4 receptors and the CA1 QUIS-sensitized site tod- andl-AP6, and compare these responses to thed- andl-isomers of AP3, AP4, AP5 and AP7. Thed-isomers of AP4, AP5 and AP6 were 5-, 3- and 14-fold less potent for the QUIS-sensitized site than their respectivel-isomers. Whilel-AP4 andl-AP5 cross-reacted with NMDA andl-AP4 receptors,l-AP6 was found to be highly potent and specific for the QUIS-sensitized site (IC50 = 40 μM). Its IC50 values for kainate / AMPA, NMDA andl-AP4 receptors were > 10, 3 and 0.8 mM, respectively. As with AP4 and AP5, sensitization tol-AP6 was reversed byl-α-aminoadipate.