Agonists and antagonists for excitatory amino acid receptors

Behind the recent explosion of interest in the field, there lies a long period of relatively slow progress in the characterization of excitatory amino acid receptors. In this article, Jeff Watkins and Harry Olverman summarize the emergence of current ideas relating to receptor differentiation and describe some of the molecular features of agonists and antagonists that activate and block them.     

Illness-induced hyperalgesia is mediated by spinal neuropeptides and excitatory amino acids

The spinal cord dorsal horn contains neural mechanisms which can greatly facilitate pain. We have recently shown that ‘illness’-inducing agents, such as intraperitoneally administered lipopolysaccharide (LPS; bacterial endotoxin), can produce prolonged hyperalgesia. This hyperalgesic state is mediated at the level of the spinal cord via activation of the NMDA-nitric oxide cascade. However, prolonged neuronal depolarization is required before such a cascade can occur. The present series of experiments were aimed at identifying spinal neurotransmitters which might be responsible for creating such a depolarized state. These studies show that LPS hyperalgesia is mediated at the level of the spinal cord by substance P, cholecystokinin and excitatory amino acids acting at non-NMDA sites. No apparent role for serotonin or kappa opiate receptors was found.     

Regulation of excitatory amino acid release by N-methyl-D-aspartate receptors in rat striatum: in vivo microdialysis studies.

The microdialysis technique was utilized to study the effects of N-methyl-D-aspartate (NMDA) receptor ligands on the in vivo release of endogenous glutamate (Glu) and aspartate (Asp) from the rat striatum. Addition of NMDA (250 and 500 microM) to the dialysis perfusion solution resulted in a striking dose-dependent increase in extracellular concentrations of Glu and Asp in the striatum. The NMDA-induced effects were reduced in a dose-related way by prior perfusion with 75 microM dizocilpine (MK-801), a non-competitive NMDA receptor antagonist. MK-801, at 75 microM, produced no changes on basal levels of Glu and Asp. However, 100 microM MK-801 did increase Glu and Asp extracellular concentrations. Local infusion with 500 microM D-serine, an agonist at the glycine site associated to the NMDA receptor, significantly increased basal level of Glu, but not Asp. Such D-serine-induced effects were reduced by 7-Cl-kynurenic acid (200 microM), a selective blocker of the glycine site present in the NMDA receptor. It is proposed that activation of NMDA receptors by endogenous Glu and Asp enhances the subsequent release of these excitatory amino acids in the striatum. Part of these NMDA receptors might be located presynaptically on cortico-striatal nerve endings. In addition, postsynaptic NMDA receptors present in the striatum may also indirectly modulate the release of Glu and Asp, through trans-synaptic mechanism.     

In vivo modulation of excitatory amino acid receptors: microdialysis studies on N-methyl-D-aspartate-evoked striatal dopamine release and effects of antagonists.

Striatal dopamine (DA) release was measured following intrastriatal (i.s.) administration of N-methyl-D-aspartate (NMDA) to unanesthetized, freely-moving rats. One hour after insertion of a removable microdialysis probe and perfusion with normal Ringer's solution, a modified Ringer's solution containing 100 mM potassium (high-K+ Ringer's) was used to standardize the preparation. DA release following i.s. administration of NMDA (12.5 mM in normal Ringer's) was dose-dependent. When NMDA (12.5 mM) was administered in high-K+ Ringer's, DA release was greatly potentiated. Administration of the competitive NMDA receptor antagonist aminophosphonovalerate (APV) in normal Ringer's prior to treatment with NMDA in high-K+ Ringer's resulted in a significant reduction of DA release compared to control animals. In contrast, administration of APV priot to treatment with NMDA in normal Ringer's resulted in a significantly increased release of DA compared to controls. Administration of the non-competitive NMDA antagonist, dextromethorphan (DXT) prior to treatment with NMDA in normal Ringer's or NMDA in high-K+ Ringer's caused significant reductions of DA release compared to controls. Intrastriatal DXT also caused dose-dependent inhibition of high-K+ Ringer's-induced DA release. Similarly, administration of the non-specific calcium channel blocker, cadmium, prior to treatment with NMDA resulted in a significant decrease when compared to control values. Results of this study indicate that dose-dependent NMDA-induced striatal DA release is greatly potentiated by potassium suggesting that under physiological conditions in vivo, striatal NMDA receptors are mostly inactivated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebral synthesis and release of kynurenic acid: an endogenous antagonist of excitatory amino acid receptors

Excitatory amino acid (EAA)-mediated synaptic transmission is the most prevalent excitatory system within the mammalian brain. Activation of EAA receptors has been postulated to contribute to neuronal cell death in stroke, epilepsy, hypoglycemia, and Huntington's disease. Kynurenic acid is an endogenous substance that inhibits EAA receptors and may therefore influence important physiologic and pathologic processes. The release of intracerebrally synthesized kynurenic acid into the extracellular fluid (ECF), where it may act at EAA receptors, has not been established in vivo. We studied the synthesis and release of kynurenic acid in the rat striatum using intracerebral microdialysis coupled with high performance liquid chromatography and fluorescence detection. The basal ECF concentration of kynurenic acid in the rat corpus striatum was 17.1 +/- 1.1 nM. Peripheral administration of the immediate biosynthetic precursor of kynurenic acid, L-kynurenine, resulted in marked dose-dependent increases in striatal ECF concentrations of kynurenic acid, peaking at 2-2.5 hr. The highest dose of L-kynurenine (100 mg/kg), administered peripherally, resulted in a 108-fold increase in plasma kynurenic acid levels and a 37-fold increase in cerebral ECF levels. Peripheral administration of kynurenic acid, at a dose that caused plasma levels to increase 430-fold, resulted in only 4-fold increases in striatal ECF concentrations. The precursor responsiveness of striatal ECF kynurenic acid to peripherally infused L-kynurenine was blocked by the central application (via the dialysis probe) of aminooxyacetic acid, an inhibitor of the immediate synthetic enzyme for kynurenic acid, kynurenine aminotransferase. Administration of L-tryptophan was less effective than L-kynurenine in increasing ECF kynurenic acid concentrations and did so at a considerably later time interval (6 hr).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of neonatal cholinergic basal forebrainlesions on excitatory amino acid receptors in neocortex

The role of cholinergic basal forebrain projections in the modulation of corticalplasticity and associated functional changes is currently the subject of renewed attention.Excitatory amino acid receptors have been identified as mediators of cortical topographic efferentand afferent information. In addition some of these receptors, notably the NMDA andmetabotropic [mGluR] type, participate in cortical plasticity. Growing evidence suggests thatinteractions between cholinergic and glutamatergic systems contribute to cognitive corticalfunctions and their anatomical and physiological substrates. Though cholinergic and glutamatergicmechanisms have both been shown to be involved in cortical morphogenesis, few studies haveattempted to study their interactions in development. The present study investigates the effect ofneonatal lesions to the cholinergic basal forebrain on NMDA, AMPA and mGluR receptors inBALB/CByJ mice, at two different developmental ages. We demonstrated previously that nBMlesions at birth result in transient cholinergic depletion for the first two postnatal weeks,substantial morphogenetic alterations in neocortex and cognitive deficits by adulthood. We showhere that unilateral neonatal lesions result in decreases in NMDA and AMPA receptors butincreases in mGluRs during the second postnatal week (PND 14). At 30 days postnatal, lesionmediated changes were attenuated, compared with PND 14, but significant sex differences incontrol and nBM lesioned mice were apparent. These data support the notion thatcholinergic/glutamatergic interactions are important during early cortical morphogenesis.Moreover, our results highlight the fact that cholinergic as well glutamatergic developmentalmechanisms are sexually dimorphic.     

Further evidence for excitatory amino acid transmission in lamprey reticulospinal neurons: selective retrograde labeling with (3H)D-aspartate

The distribution of radiolabeled neurons in the brain stem of Lampetra fluviatilis was studied following unilateral injections of (3H)D-aspartate in the rostral spinal cord. After survival periods of 1-3 days, labeled perikarya were present within and nearby the posterior, middle, and anterior rhombencephalic reticular nuclei and in the mesencephalic reticular nucleus. The highest number of (3H)D-aspartate labeled cell bodies were present in the posterior rhombencephalic reticular nucleus. The labeled reticulospinal neurons were distributed mainly ipsilateral to the injection site and included the giant Müller cells as well as medium-sized and small neurons. Contralateral labeling occurred in cell bodies scattered along the lateral margin of the rhombencephalic reticular formation, the most rostral of these contralaterally projecting neurons being the Mauthner cell. The (3H)D-aspartate labeling correlates with previous electrophysiological studies showing that lamprey reticulospinal neurons utilize excitatory amino acid transmission.     

Antibodies to glutamate and aspartate recognize non-endogenous ligands for excitatory amino acid receptors

Antisera raised against glutaraldehyde conjugates of glutamate (Glu) and aspartate (Asp) with hemocyanin proved highly specific for their respective unconjugated amino acid haptens when tested in immunocytochemical blocking experiments on sections of the rat spinal cord. In addition, immunocytochemical staining by the Glu antiserum was effectively blocked by quisqualate but not by kainate or N-methyl-D-aspartate (NMDA); staining with the Asp antiserum was effectively blocked by kainate, to a lesser extent by quisqualate, and was not affected by NMDA. These results may be explained by assuming that the specific binding regions of the antibodies tested share certain recognition characteristics with endogenous binding sites or receptors for excitatory amino acids and their agonists.     

Behavioral and biochemical evidence for a functional role of excitatory amino acids in the median raphe nucleus

Recent experiments have suggested the existence of excitatory amino acid (EAA)-containing afferents to the median raphe nucleus. In the present study we investigated the functional significance of EAAs in the median raphe (MR) by examining the behavioral and biochemical effects of intra-raphe injections of EAA antagonists. Injections of kynurenic acid, gamma-glutamylglycine, 2-amino-5-phosphonovaleric acid (2-APV) and 2-amino-7-phosphonoheptanoic acid (2-APH) into the median raphe resulted in marked hyperactivity. In contrast to the effect of 2-APV and 2-APH, intra-raphe injections of the homologues of these compounds with 4, 6 or 8 carbon atoms, which have a lower affinity for excitatory amino acid receptors, were without significant effects on activity. Additionally, the effects of 2-APV were shown to be stereospecific to the active D-isomer further suggesting receptor mediation of the effect. Injections of EAA antagonists into the dorsal raphe nucleus or the ventral tegmental area were much less effective in increasing activity than were injections into the MR, suggesting anatomical specificity of the effect. Injections of 2-APV into the median raphe were also shown to result in a reduction of serotonin metabolism within the hippocampus and an increase in dopamine metabolism within the nucleus accumbens and the magnitude of both of these effects was positively correlated with the behavioral responses to the injections. These findings suggest that cells within the median raphe may be subject to a tonic excitation exerted through EAA receptors.     

Rods and cones activate different excitatory amino acid receptors on the mudpuppy retinal horizontal cell

We have studied rod and cone neurotransmission onto horizontal cells (HCs) of the mudpuppy retina by: (1) dual whole-cell recordings from synaptically connected pairs in a superfused retinal slice preparation, and (2) intracellular recordings from HCs in a superfused retina-eyecup preparation. In the presence of relatively weak concentrations of broad spectrum excitatory amino acid antagonists, HC recordings show that cone-mediated responses were significantly more attenuated than those mediated by rods. These observations suggest that the HC postsynaptic receptors for rods and cones are pharmacologically different. One advantage of having distinct rod- and cone-activated receptors on second-order neurons may be to preserve different rod and cone functions under varying levels of light stimulation and provide separate means of regulating rod and cone neurotransmission onto the second-order cells.     

Interaction between beta-N-methylamino-L-alanine and excitatory amino acid receptors in brain slices and neuronal cultures.

beta-N-Methylamino-L-alanine (BMAA) stimulated the hydrolysis of polyphosphoinositides (PPI) in hippocampal slices prepared from 8-day old rats. The action of BMAA was antagonized by D,L-2-amino-3-phosphonopropionate (an antagonist of metabotropic receptors) and was largely reduced after lowering the concentration of bicarbonate ions from 25 to 1 mM. In cultured cerebellar neurons, stimulation of PPI hydrolysis by BMAA was mediated by the activation of both metabotropic and N-methyl-D-aspartate (NMDA) receptors. However, BMAA exhibited low activity as an NMDA receptor agonist, as reflected by its low efficacy in increasing cGMP formation in cultures incubated in the absence of extracellular Mg2+. A preferential interaction of BMAA with non-NMDA receptors was confirmed by binding studies on crude synaptic membranes from rat brain. Accordingly, BMAA was more potent in displacing specifically bound [3H]glutamate than 3-(2-carboxypiperazin-4-yl)[1,23H]propyl-1-phosphonic acid (CPP) (a selective NMDA receptor ligand). As expected, the affinity of BMAA for [3H]glutamate or [3H]CPP binding sites was greater in the presence of 25 mM bicarbonate. BMAA weakly displaced specifically bound [3H]glycine in the absence of bicarbonate and, in cultured neurons incubated with buffer containing 1 mM bicarbonate, mimicked glycine in reversing the inhibitory action of kynurenic acid on glutamate-stimulated 45Ca2+ influx. Taken collectively, these results suggest that BMAA acts as a mixed agonist of 'metabotropic' and NMDA receptors.     

The role of NMDA and non-NMDA excitatory amino acid receptors in the excitation of primate spinothalamic tract neurons by mechanical, chemical, thermal, and electrical stimuli.

The role of excitatory amino acids (EAAs) in the excitation of monkey spinothalamic tract (STT) neurons following activation of cutaneous primary afferent fibers by noxious and non-noxious stimuli was investigated. The responses of STT neurons to either NMDA or non-NMDA EAA ligands were blocked by infusion of specific antagonists through a microdialysis fiber into the region surrounding the cells. Our results show that blockade of non-NMDA receptors results in a nearly complete elimination of the responses of STT neurons to all stimuli. Blockade of NMDA receptors results in an attenuation of the responses to noxious stimuli but, in addition, prevents the development of the sensitization of STT neurons that is often observed after intradermal injection of capsaicin. These observations further support a role of EAAs in the transmission of sensory information from primary afferent fibers to dorsal horn neurons and a role for NMDA receptors in the generation of hyperalgesia.     

Electrophysiological evidence for the presence of ionotropic and metabotropic excitatory amino acid receptors on dopaminergic neurons of the rat mesencephalon: an in vitro study.

The actions of the ionotropic and metabotropic excitatory amino acid agonists AMPA, quisqualate, kainate, NMDA and trans-ACDP were studied by means of intracellular electrophysiological recordings from dopaminergic neurons of rat mesencephalon in brain slices. It was observed that all these agents evoked an inward current in cells which were voltage-clamped near the resting potential (-50, -60 mV). The membrane responses produced by AMPA, kainate and quisqualate were associated with an increase of the apparent input conductance while the responses induced by NMDA and trans-ACDP were associated with a decrease in the apparent input conductance. Therefore, stimulation of ionotropic and metabotropic amino acid receptors activates inward currents in the dopaminergic cells by different mechanisms.     

3-Aminopropanephosphinic acid is a potent agonist at peripheral and central presynaptic GABAB receptors

The actions of the GABA analog 3-aminopropanephosphinic acid (3-APA) were studied in the guinea-pig isolated ileal preparation and at synapses between cultured rat hippocampal neurons. Like the GABAB receptor agonist, baclofen, 3-APA inhibited the electrically evoked ileal twitch. The EC50 for 3-APA was 0.8 microM; the EC50 for baclofen was 9 microM. In addition, the depressant responses to 3-APA and baclofen were blocked by the GABAB receptor antagonists phaclofen, saclofen, 2-hydroxy-saclofen and delta-aminovaleric acid. 3-APA also mimicked the presynaptic action of baclofen at GABAergic synapses between embryonic rat hippocampal neurons in culture. 3-APA reduced the amplitude of inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) by greater than 50% at a concentration of 1 microM, while baclofen reduced synaptic transmission to a similar degree at 10 microM. 3-APA did not alter membrane conductance, nor did the drug alter postsynaptic responses to GABA. These data show that 3-APA is a potent agonist at presynaptic GABAB receptors in the periphery and on GABAergic neurons from the central nervous system. The activity of 3-APA at central postsynaptic GABAB receptors remains to be studied.     

Effects of birth insult and stress at adulthood on excitatory amino acid receptors in adult rat brain

Birth complications involving fetal hypoxia and stress at adulthood, which are risk factors for schizophrenia, can produce alterations in subcortical dopamine (DA) function in rat models. As adults, rats born either by cesarean section (C-section) or by C-section with added global anoxia show increased stress-induced DA release from nucleus accumbens and increased amphetamine-induced locomotion, compared to vaginally born controls. Moreover, stress at adulthood interacts with these birth insults to modulate DA receptor and transporter levels. Glutamatergic transmission at the level of the nucleus accumbens, prefrontal cortex, and hippocampus are known to modulate subcortical DA activity. Thus, altered excitatory amino acid (EAA) function might contribute to the dopaminergic changes observed in rats after birth insult and/or stress at adulthood. To test this possibility, rats born vaginally, by C-section, or by C-section with 15 min of anoxia, were either repeatedly stressed (15 min of tail pinch daily for 5 days) at adulthood or received no stress, and levels of EAA receptor binding were measured by ligand autoradiography in limbic brain regions. As adults, rats born by C-section showed increases in AMPA receptor binding in nucleus accumbens shell, NMDA receptor binding in cingulate cortex, and kainate receptor binding in the hippocampal CA1 region. Anoxic rats showed increases in CA1 kainate receptor and anterior olfactory NMDA receptor binding. Stress at adulthood increased AMPA receptor binding in several regions of prefrontal cortex and reduced NMDA receptor binding in infralimbic cortex and dentate gyrus, across all birth groups. Two instances of interactions between birth insult and stress at adulthood were observed. Stress reduced cingulate cortex NMDA receptor binding and increased olfactory tubercle kainate receptor binding only in C-sectioned animals, but not in controls. The possibility that the observed EAA receptor changes contribute to dopaminergic dysfunction in these animal models is discussed, in light of known glutamate-DA interactions.     

Treatment with excitatory amino acids or high K+ and NMDA receptors in cerebellar granule cells.

N-methyl-D-aspartate (NMDA) receptor activity was examined, in terms of 45Ca2+ influx, during the development of cerebellar granule cells grown under 'non-trophic' [10 mM potassium (K10)] or 'trophic' conditions [25 mM potassium (K25), NMDA and kainate (KA)]. NMDA receptor activity increased sharply between 2 and 4 days in vitro (DIV) irrespective of growth conditions which upon further cultivation exerted a powerful influence, the NMDA response increasing progressively in K25 and NMDA grown cells, while remaining at a constant level in KA treated cells. In contrast, in K10 grown cells the NMDA response declined by 7 DIV to about 20% of the estimates in K25 at 9 DIV. Trophic conditions are, therefore, essential for the proper functional expression of NMDA receptors.