N-methyl D-aspartate acts as an antagonist of the photoreceptor transmitter in the carp retina

Glutamate, aspartate, and their agonists, kainate, quisqualate, cysteine sulfinate and N-methyl-D-aspartate (NMDA), were applied to the isolated carp retina while recording from horizontal cells. All these agents, except NMDA, depolarized horizontal cells membrane and reduced responses to light, thus mimicking the effect of the endogenous photoreceptor transmitter. Application of NMDA, on the other hand, caused a membrane hyperpolarization of horizontal cells in the dark, an effect different from its depolarizing effect as observed elsewhere in the central nervous system. NMDA also reduced or blocked the light responses of these cells as well as the depolarizing responses to applications of glutamate, aspartate or kainate. Effects of NMDA on the spectral properties of the horizontal cell responses were identical to the effects of the acidic amino acid receptor antagonists alpha-methyl glutamate, and alpha-amino adipate. Thus, NMDA appears to act as a weak antagonist to the photoreceptor transmitter, whose receptors on the horizontal cell membrane interact with a glutamate-like substance but appear atypical of glutamate receptors described elsewhere in the brain.     

Action of 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic aci (CPP): a new and highly potent antagonist of N-methyl-d-aspartate receptors in the hippocampus

A new compound, 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), has been evaluated as an excitatory amino acid receptor antagonist using electrophysiological assays and radioligand binding. In autoradiographic preparations, CPP reduces l-[³H]glutama binding in regions of the hippocampus rich in N-methyl-d-aspartate (NMDA) receptors, but not in regions richin kainate sites. In isolated membrane fraction preparations, CPP displaces l-[³H]glutamate binding to NMDA sites, but does not compete with the binding of selective kainate or quisqualate site ligands. CPP potently reduces depolarizations produced by application of NMDA but not depolarizations produced by quisqualate or kainate. Its order of potency against excitatory amino acid-induced responses in the hippocampus is NMDA > homocysteate > aspartate > glutamate > quisqualate. CPP has no efect on lateral perforant path responses or on inhibition of these responses by 2-amino-4-phosphonobutyrate. Finally, at doses that do not affect Schaffer collateral synpatic transmission, CPP reversibly blocks the induction of long-term potentiation of Schaffer synaptic responses. This new compounds is, therefore, a higly selective brain NMDA receptor blocker, and the most potent such by nearly an order of magnitude.     

Postsynaptic responses of horizontal cells in the tiger salamander retina are mediated by AMPA-preferring receptors

The postsynaptic responses of sign-preserving second-order retinal neurons (horizontal cells (HCs) and off-bipolar cells) are mediated by CNQX-sensitive AMPA/KA glutamate receptors. In this study we used receptor-specific allosteric regulators of desensitization and selected antagonists to determine the glutamate receptor subtypes in tiger salamander horizontal cells. Two approaches were employed in this study. The first was to measure postsynaptic currents induced by exogenously applied glutamate under voltage clamp conditions in living retinal slices; and the second was to record voltage responses controlled by endogenous glutamate released from photoreceptors in whole retinas. Application of 100 μM cyclothiazide (a specific AMPA receptor desensitization blocker) enhanced the glutamate-induced current by about 5 fold. In contrast, 300 μg ml⁻¹ ConA (a specific kainate receptor desensitization blocker), had no effect. GYKI 52466 (a specific AMPA receptor antagonist) at 30 μM almost completely suppressed the glutamate-induced inward current in HCs. Cyclothiazide at 100 μM depolarized the HC dark membrane potential by about 5 mV and reduced the amplitudes of the voltage responses to dim lights, but enhanced the voltage responses to bright lights. Cyclothiazide had no effect on either the dark potential or the light responses of rods and cones. Con A at 300 μg ml⁻¹ had no effect on either the dark potential or the light responses of the HC. GYKI 52466 (30 μM) hyperpolarized the HC dark membrane potential by about 55 mV and almost completely suppressed the light responses. We conclude from these results that the postsynaptic glutamate- and light-induced responses in the tiger salamander retinal horizontal cells are mediated by AMPA-preferring, and not kainate-preferring glutamate receptors. The functional roles of AMPA receptors and their desensitization kinetics in visual information processing are discussed.     

‘Chemical ischemia' in cultured retina cells: the role of excitatory amino acid receptors and of energy levels on cell death

In this study, we determined whether the retina cell death observed in response to an ischemic-like insult is related to an overactivation of the ionotropic glutamate receptors and/or to a collapse of the energy levels. Cultured chick retina cells were submitted to ‘chemical ischemia' by metabolic inhibition with sodium cyanide and iodoacetic acid, which block oxidative phosphorylation and glycolysis, respectively. The assessment of neuronal injury was made spectrophotometrically by quantification of cellularly reduced MTT, which gives information about mitochondrial function, or by staining with fluorescein diacetate (FDA), which correlates with changes in the plasma membrane permeability. ‘Chemical ischemia' induced both an acute and a delayed time-dependent degeneration of chick retina cells. We observed that 2 min after the ischemic insult, the levels of ATP were reduced to a minimum. On the other hand, the metabolic inhibition induced the release of aspartate, glutamate and γ-aminobutyric acid, and the activation of AMPA/kainate receptors during the period of metabolic arrest was partially responsible for the loss of mitochondrial function. However, the NMDA and non-NMDA receptor antagonists (MK-801 and CNQX) did not prevent the plasma membrane damage caused by sodium cyanide and iodoacetic acid. The results show that the collapse of the energy levels, rather than the increase in excitatory amino acids, appears to underlie the observed cell injury, suggesting an important relationship between ischemia-induced depletion of high-energy metabolites and retina cell degeneration.     

Presynaptic kainate and N-methyl-d-aspartate receptors regulate excitatory amino acid release in the olfactory cortex

The potassium-evoked release of endogenous aspartate, glutamate and GABA from olfactory cortex slices has been monitored. Release of aspartate alone is significantly reduced by N-methyl-d-aspartate (NMDA) whilst kainate significantly increases release of both aspartate and glutamate. These effects, which are blocked by appropriate receptor antagonists, suggest that presynaptic NMDA and kainate receptors regulate excitatory amino acid release in the olfactory cortex.     

Origin of Aspartate-induced Responses in Rat Cerebellar Purkinje Cells

The effects of glutamate, aspartate and N-methyl-d -aspartate (NMDA) on Purkinje cells and interneurons were investigated in cerebellar slice cultures using the whole-cell configuration of the patch-clamp technique. l -Glutamate and l -aspartate induced inward currents in Purkinje cells voltage-clamped at -60 mV. In standard external solution, the amplitude of the responses induced by these two amino-acids was a linear function of the membrane potential. l -Aspartate-induced currents were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective antagonist of non-NMDA receptors. NMDA, a selective agonist of NMDA receptors, had no effect of its own on the excitability of Purkinje cells, but was effective in blocking the responses induced by aspartate in Purkinje cells in a voltage-independent manner. In contrast, d -(–)-2-amino-5-phosphonovaleric acid (d -APV), a selective antagonist of NMDA receptors, had no effect on aspartate-induced responses. d -Aspartate also induced responses in Purkinje cells, and the amplitude of these responses was a linear function of the membrane potential. Currents induced by l - and d -aspartate were inhibited by dihydrokainate, a glutamate uptake blocker. In sodium-free external solution, glutamate still induced outward currents in Purkinje cells, whereas l - and d -aspartate no longer evoked any current. When sodium was replaced by lithium in the external medium, no change in the holding current could be detected in Purkinje cells maintained at -60 mV; moreover, in this bathing medium l -aspartate no longer evoked any current whereas glutamate-induced responses were still present. In contrast, interneurons were sensitive to both NMDA and aspartate applications, and these responses were antagonized by d -APV. In addition, aspartate still induced an outward current in sodium-free external solution. This study presents rather direct evidence in favour of l -aspartate as being a very selective NMDA receptor agonist in the cerebellum. l -Aspartate-induced currents in Purkinje cells are not due to activation of mixed NMDA/non-NMDA receptors, but are probably due to the release of l -glutamate induced by aspartate through glutamate uptake.     

Effect of selective kainate lesions on the release of glutamate and aspartate from chick retina

In order to contribute evidence leading to establishing the excitatory pathways in the vertebrate retina, we selectively lesioned chick retinas by intraocular injection of 6, 60, 120, and 200 nmol of kainate, which selectively damages OFF-bipolars, amacrines, horizontals, and ON-bipolars, and measured the K+-stimulated, Ca++-dependent release of L-(3H)-glutamate and L-(3H)-aspartate. We also measured (3H)-GABA release as a marker for horizontal cells and a population of amacrines, as well as (14C)-glycine release as a tracer of a different subpopulation of amacrines. All four amino acids were released from control retinas by a depolarizing K+ concentration in a Ca++-dependent fashion. GABA and glycine, however, showed an additional Ca++-independent component of release. Lesion induced by 6 nmol of kainate decreased by 50% the release of glutamate and by 20% that of aspartate; glycine release was reduced 40% while GABA release was unaffected. Injection of 60 nmol of kainate reduced glutamate release a further 20% and significantly decreased GABA (50%) and glycine (75%) release; aspartate release remained unmodified; 120 nmol of kainate caused a further 30% reduction in aspartate and GABA release. Neither compound was significantly released after treatment with 200 nmol of kainate. These results seem to suggest that while OFF-bipolars could release glutamate as transmitter, aspartate is released from a different cell population which is less sensitive to kainate, probably ON-bipolars.     

Functional properties of spontaneous excitatory currents and encoding of light/dark transitions in horizontal cells of the mouse retina

As all visual information is represented in the spatio‐temporal dynamics of transmitter release from photoreceptors and the combined postsynaptic responses of second‐order neurons, appropriate synaptic transfer functions are fundamental for a meaningful perception of the visual world. The functional contribution of horizontal cells to gain control and organization of bipolar and ganglion cell receptive fields can only be evaluated with an in‐depth understanding of signal processing in horizontal cells. Therefore, a horizontal slice preparation of the mouse retina was established to record from horizontal cell bodies with their dendritic fields intact and receiving functional synaptic input from cone photoreceptors. Horizontal cell bodies showed spontaneous excitatory currents (spEPSCs) of monophasic and more complex multi‐peak waveforms. spEPSCs were induced by quantal release of glutamate from presynaptic cones with a unitary amplitude of 3 pA. Non‐stationary noise analysis revealed that spEPSCs with a monoexponential decay were mediated by 7–8 glutamate receptors with a single‐channel amplitude of 1.55 pA. Responses to photopic full‐field illumination were characterized by reduction of a tonic inward current or hyperpolarization, inhibition of spEPSCs, followed by a fast and transient inward current at light offset. The response to periodic dark/light transitions of different frequencies was dependent on the adaptational status of the cell with a limiting frequency of 10 Hz. Both on and off components of the light response were mediated by AMPA and kainate receptors. Detailed analysis of horizontal cell synaptic physiology is a prerequisite for understanding signal coding and processing at the photoreceptor ribbon synapse.     

Retinal neurones containing kainate receptors are influenced by exogenous kainate and ischaemia while neurones lacking these receptors are not - melatonin counteracts the effect of ischaemia and kainate

The present experiments were carried out on three types of neurone in primary rabbit retinal cultures. One cell-type, bipolar neurones, have glutamate APB-type metabotropic receptors and can be identified by the presence of θPKC-immunoreactivity. The other two cell-types are primarily amacrine cells and can be ‘stained' for the localisation of GABA immunoreactivity or for serotonin taken up from the medium. Most of the serotonin-accumulating and GABA-containing neurones contain glutamate kainate-type receptors. Exposure of the cultures to treatment of kainate (50 μM) or experimental ischaemia (8 h followed by 16 h reoxygenation) produced essentially similar findings. The serotonin-accumulating and GABA cells were affected as they were drastically reduced in numbers while the numbers of θPKC-containing cells were unaffected. Inclusion of the kainate/AMPA antagonist CNQX (100 μM) or melatonin (100 μM) to the medium during kainate or ischaemia treatments largely prevented the detrimental influences on the serotonin-accumulating and GABA cells. It is concluded that during experimental ischaemia excessive glutamate is released to influence cells which contain kainate and APB-type receptors. However, only the neurones containing the kainate receptors are negatively affected with the generation of free radicals. Melatonin or CNQX protects against this effect by scavenging free radicals or acting at the receptor level, respectively.© 1997 Elsevier Science B.V. All rights reserved.     

Characterization of excitatory amino acid receptors expressed by embryonic chick motoneurons in vitro

We have examined the effect of L-glutamate and other excitatory amino acids on embryonic chick motoneurons maintained in cell culture along with other types of spinal cord cells. When the motoneuron membrane is clamped at -50 mV, glutamate induces a dose-dependent inward current. Although the dose-response curve is hyperbolic with an ED50 of 78 microM, glutamate apparently activates 2 types of receptors on motoneurons. The first, G1, is activated by N-methyl-D-aspartate (NMDA) and aspartate and inhibited by 2-amino-5-phosphonovaleric acid (2-APV). The second, G2, is activated by kainate and quisqualate and is not inhibited by 2-APV. At -50 mV, 38% of the glutamate current is due to activation of G1 receptors and the remaining 62% to G2 activation. In contrast to motoneurons grown with other spinal cord cells, sorted motoneurons grown in isolation apparently exhibit only G2 receptor-mediated currents. Both G1 and G2 currents reverse polarity between -10 and -5 mV. However, they could be distinguished when the membrane was hyperpolarized. G2 currents increased but G1 currents decreased when the membrane potential was increased beyond -50 mV. Consistent with the mixed agonist action of glutamate, glutamate currents remained nearly constant on hyperpolarization. No evidence was obtained that the G2 class of receptors on motoneurons could be subdivided: Quisqualate and kainate apparently compete for the same sites; gamma-glutamylglycine blocked quisqualate as effectively as it blocked kainate currents when the different potencies of the 2 agonists were taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitatory amino acid-evoked membrane currents and excitatory synaptic transmission in lamprey reticulospinal neurons

The characteristics of excitatory amino acid-evoked currents and of excitatory synaptic events have been examined in lamprey Müller neurons using voltage clamp and current clamp recording techniques. Application of glutamate evoked depolarizations associated with a decrease in input resistance. The reversal potential of the responses was -35 mV. Under voltage clamp conditions, a series of excitatory amino acid agonists evoked inward currents associated with little or no increase in baseline current noise. The order of potency of the excitatory amino acid agonists was quisqualate greater than kainate greater than glutamate greater than aspartate, while N-methyl-D-aspartic acid (NMDA) was inactive. Inward currents evoked by glutamate, as well as by kainate and quisqualate were attenuated reversibly by 1 mM kynurenic acid (KYN). In contrast, glutamate-evoked currents were not affected by 100 microM D(-)-2-amino-5-phosphonovaleric acid (APV), a selective NMDA antagonist. Spontaneously occurring and stimulus-evoked excitatory postsynaptic events were antagonized reversibly by 1 mM KYN. At this concentration, KYN had no effect on membrane potential, input resistance, or excitability of the cells. In contrast, excitatory postsynaptic currents were unaffected by APV. It is concluded that both glutamate responses and excitatory synaptic transmission in lamprey Müller neurons are mediated by non-NMDA-type receptors and that these receptors are associated with ionic channels with a low elementary conductance. The combined pharmacological and biophysical characteristics of these responses are therefore different from those previously reported in other preparations. Spontaneous (but not stimulus-evoked) inhibitory synaptic events in Müller neurons were blocked reversibly by 1 mM KYN but not by 100 microM APV, suggesting that excitation of interneurons inhibitory to Müller cells was also mediated by non-NMDA receptors.     

Dopaminergic modulation at the olfactory nerve synapse

Dopamine can change the membrane potential, regulate cyclic nucleotides, and modulate transmitter release in central neurons. In the olfactory bulb (OB), the dopamine synthetic enzyme, tyrosine hydroxylase, is largely confined to neurons in the glomerular layer. After demonstrating dopamine D2 receptors in the glomerular and olfactory nerve (ON) layers, Nickell et al. [W.T. Nickell, A.B. Norman, L.M. Wyatt, M.T. Shipley, Olfactory bulb DA receptors may be located on terminals of the olfactory nerve, NeuroReport, 2 (1991) 9-12.] proposed that these receptors may reduce transmitter release due to their localization to ON presynaptic boutons. We have previously demonstrated that olfactory receptor neurons use glutamate to excite OB neurons through activation of glutamate receptors subtypes, NMDA and AMPA/kainate [D.A. Berkowicz, P.Q. Trombley, G.M. Shepherd, Evidence for glutamate as the olfactory receptor cell neurotransmitter. J. Neurophysiol., 71 (1994) 2557-2561]. Here, we used a hemisected turtle OB preparation and patch-clamp recording techniques to assess dopamine modulation of the ON/OB neuron synapse. We found that dopamine (10-300 microM) reversibly decreased the excitatory postsynaptic response to ON stimulation. This effect could be overcome by recruiting additional nerve fibers by increasing the intensity of ON stimulation. Quinpirole (10 microM), a D2 agonist, mimicked the effects of dopamine. Conversely, sulpiride (300 microM), a D2 antagonist, prevented the inhibitory effects of dopamine on synaptic transmission. Whereas dopamine appeared to equally affect the NMDA and AMPA/kainate receptor-mediated components of the synaptically evoked response, it had no direct effect on membrane currents evoked by exogenous glutamate, kainate or NMDA applied to cultured OB neurons. Our data, therefore, support the notion that dopamine modulates synaptic transmission between olfactory receptor neurons and OB neurons via a presynaptic mechanism involving D2 receptor activation. Our abstract (Berkowicz et al. (1994) Neuroscience Abs. 20:328) is the first report of these results.     

Receptor types mediating the excitatory actions of exogenousl-aspartate andl-glutamate in rat olfactory cortex

Changes in potential between the pial and cut surfaces of rat olfactory cortex slices evoked by N-methyl-d-aspartate (NMDA), quisqualate, kainate,l-glutamate andl-aspartate and also by γ-aminobutyric acid (GABA) have been monitored using extracellular electrodes. All agonists produced a pial-negative potential response when superfused onto the pial surface, GABA,l-aspartate andl-glutamate being less potent than the others. Repeated applications of NMDA, but not of the other agonists, led to a progressive reduction in response to approximately 30% of the initial depolarization. The responses to NMDA (100 μM) were selectively abolished by(±)2-amino-5-phosphonopentanoic acid (APP; 100 μM) while depolarizations evoked byl-glutamate andl-aspartate (both at 10 mM) were only antagonized by21 ± 2 (n = 12) and36 ± 3 (n = 12) percent respectively (means ± S.E.M.). γ-d-Glutamylglycine (γ-DGG; 1 mM) and(±)cis-2,3-piperidine dicarboxylate (cis-PDA; 2 and 5 mM), in addition to antagonizing responses to NMDA, also partially blocked quisqualate- and kainate-evoked depolarizations. When a mixture of APP (100 μM), γ-DGG (1 mM) and cis-PDA (5 mM) was applied to preparations, although NMDA receptors were completely blocked and responses to both quisqualate and kainate antagonized by approximately 80%,l-glutamate andl-aspartate evoked depolarizations were only reduced by51 ± 7 (n = 4) and 49 ± 4 (n = 4) percent respectively (means ± S.E.M.). The results are discussed in terms of the contributions made by NMDA, quisqualate and kainate receptors to the composite responses evoked byl-aspartate andl-glutamate.     

Acetylcholine release from the rabbit retina mediated by kainate receptors.

The cholinergic amacrine cells of the rabbit retina may be labeled with 3H-choline (3H-Ch), and the activity of the cholinergic population may be monitored by following the release of 3H-ACh. Glutamate analogs caused massive ACh release, up to 50 times the basal efflux, with the following rank order of potency: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) greater than quisqualate (QQ) = kainate (KA) much greater than NMDA (in magnesium-free medium) much greater than glutamate greater than aspartate. In contrast, the release of 3H-Ch was unchanged. Submaximal doses of each agonist were used to establish the specifity of glutamate antagonists. Kynurenic acid was selective for KA much greater than QQ, and 6,7-dinitroquinoxaline-2,3-dione (DNQX) was selective for KA greater than QQ much greater than NMDA. At low doses, which selectively blocked the response to KA, both antagonists blocked the light-evoked release of ACh. These results suggest that ACh release may be produced via several glutamate receptors, but the physiological input to the cholinergic amacrine cells is mediated by KA receptors. Because these cells receive direct input from cone bipolar cells, this work supports previous evidence that the bipolar cell transmitter is glutamate.     

Kainate-induced lesion in the optic tectum: dependency upon optic nerve afferents or glutamate

Kainic acid is known to induce characteristic lesions in neurons receiving an intact input with presumed glutamate-mediated neurotransmission. There are indications for glutamate as a transmitter of retinal afferent terminals in the pigeon optic tectum. After tectal injection of kainic acid (0.5–2.0 μg in 0.5 μl) the optic tectum was studied by light and electron microscopy and the following changes were observed: (a) within 1–48 h important neuropil vacuolization predominantly in lower part of layer 5. Such vacuoles were sometimes postsynaptic to identified retinal afferent terminals: (b) within 1 h to 21 days progressive neuronal cell loss throughout the tectal layers. These toxic effects were not observed 2–12 weeks after contralateral retinal ablation but could partially be restored by combined glutamate (0.2 mg) and kainate injection. Thus in the pigeon tectum, kainic acid neurotoxicity is dependent upon an intact retinal input, a finding consistent with a special role for glutamate — possibly as a transmitter — in retinal terminals.     

Stimulatory effects of centrally injected kainate and N-methyl- -aspartate on gastric acid secretion in anesthetized rats

The effects of N-methyl- -aspartate (NMDA), kainate and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), ionotropic glutamate agonists, on gastric acid secretion were investigated in the continuously perfused stomach of anesthetized rats. The lateral ventricular (LV) injection of kainate (0.01–1 μg) or NMDA (0.3–3 μg) dose-dependently stimulated gastric acid secretion. AMPA (3–10 μg) also stimulated gastric acid secretion but the effect was very weak. Repeated injections of kainate (0.1 μg) or NMDA (1 μg), at least twice, stimulated gastric acid secretion to a similar degree. The effect of kainate (0.1 μg) was blocked by the kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione disodium (3 μg, LV) and -γ-glutamylaminomethanesulfonic acid (30 μg, LV), but not by NMDA receptor antagonists. The effect of NMDA (10 μg) was blocked by (±)-3-(2-carboxypiperazin-4-yl)-1-propylphosphonic acid (10 μg, LV), a competitive NMDA receptor antagonist, and (+)-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine hydrogen maleate (10 μg, LV), a non-competitive NMDA receptor antagonist, but not by kainate receptor antagonists. Moreover, the gastric acid secretion stimulated by kainate and NMDA were completely blocked by systemic atropine injection (1 mg/kg, i.v.) and vagotomy. These findings suggest that kainate and NMDA receptor mechanisms are independently involved in the central nervous system to control gastric acid secretion through vagus cholinergic activation.     

Pharmacological characterization of the glutamate receptor in cultured astrocytes

Cultured astrocytes from neonatal rat cerebral hemispheres are depolarized by the excitatory neurotransmitter glutamate. In this study we have used selective agonists of different neuronal glutamate receptor subtypes, namely, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate type, to characterize pharmacologically the glutamate receptor in astrocytes. The agonists of the neuronal quisqualate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) and quisqualate, depolarized the membrane. Kainate, an agonist of the neuronal kainate receptor, depolarized astrocytes more effectively than quisqualate. Combined application of kainate and quisqualate depolarized astrocytes to a level which was intermediate to that evoked by quisqualate and kainate individually. Agonists activating the neuronal NMDA receptor, namely NMDA and quinolinate, were ineffective. Application of NMDA did not alter the membrane potential even in combination with glycine or in Mg2+-free solution, conditions under which neuronal NMDA receptor activation is facilitated. The nonselective agonists L-cysteate, L-homocysteate, and beta-N-oxalylamino-L-alanine (BOAA) mimicked the effect of glutamate. Dihydrokainate, a blocker of glutamate uptake, did not, and several antagonists of neuronal glutamate receptors only slightly affect the glutamate response. These findings suggest that astrocytes express one type of glutamate receptor which is activated by both kainate and quisqualate, lending further support to the notion that cultured astrocytes express excitatory amino acid receptors which have some pharmacological similarities to their neuronal counterparts.     

Excitatory Amino Acid Pathway from the Hippocampus to the Prefrontal Cortex. Contribution of AMPA Receptors in Hippocampo-prefrontal Cortex Transmission

Previous experiments in the rat have demonstrated that field CA1 and the subiculum project to the prefrontal cortex and that this direct unilateral pathway is excitatory. In the present study, anatomical and electrophysiological approaches were used to determine the transmitter mediating the excitatory responses in prefrontal cortex neurons to low-frequency stimulation of the hippocampus. The method of selective retrograde d-[3H]aspartate labelling was used to identify putative glutamatergic and/or aspartatergic hippocampal afferent fibres to the prefrontal cortex. Unilateral microinjection of d-[3H]aspartate into the prelimbic area of the prefrontal cortex resulted in the retrograde labelling of a fraction of hippocampal neurons. Some labelled cell bodies were distributed in field CA1 and the subiculum but larger numbers of neurons were detected in the ventral and intermediary subiculum. In a second series of experiments, the excitatory transmission from the hippocampus to the prefrontal cortex was pharmacologically analysed to provide further evidence for the involvement of glutamate and/or aspartate in the pathway. All prefrontal cortex neurons responding to the stimulation of the hippocampus were activated by selective agonists of the glutamate receptor subtypes alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-d-aspartate (NMDA), and these effects were selectively antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (APV) respectively. Most of the excitatory responses of prefrontal cortex neurons to single and paired-pulse stimulation of the hippocampus were antagonized by CNQX. APV only affected the excitatory response in a few cells. These results suggest that the hippocampal input to the prefrontal cortex utilizes glutamate and/or aspartate as a transmitter. Even though prefrontal cortex neurons responding to the stimulation of the hippocampus appear to have both AMPA and NMDA receptors, low-frequency stimulation of the hippocampo-prefrontal cortex pathway activates cortical neurons mostly through AMPA receptors.     

Functional evidence for D-serine inhibition of non-N-methyl-D-aspartate ionotropic glutamate receptors in retinal neurons

D-Serine is an important signaling molecule throughout the central nervous system, acting as an N-methyl-D-aspartate (NMDA) receptor coagonist. This study investigated the D-serine modulation of non-NMDA ionotropic glutamate receptors expressed by inner retinal neurons. We first identified that the degradation of endogenous retinal D-serine, by application of D-amino acid oxidase, caused an enhancement of kainate- and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor-mediated calcium responses from the ganglion cell layer of the isolated rat retina and light-evoked responses obtained by multi-electrode array recordings from the guinea pig retina. Approximately 30-45% of cells were endogenously inhibited by D-serine, as suggested by the effect of D-amino acid oxidase. Conversely, bath application of D-serine caused a reduction in multi-electrode array recorded responses and decreased kainate, but not potassium-induced calcium responses, in a concentration-dependent manner (IC(50), 280 μm). Using cultured retinal ganglion cells to reduce network influences, D-serine reduced kainate-induced calcium responses and AMPA induced whole-cell currents. Finally, the inhibitory effect of D-serine on the kainate-induced calcium response was abolished by IEM 1460, thereby identifying calcium-permeable AMPA receptors as a potential target for D-serine. To our knowledge, this is the first study to address specifically the effect of D-serine on AMPA/kainate receptors in intact central nervous system tissue, to identify its effect on calcium permeable AMPA receptors and to report the endogenous inhibition of AMPA/kainate receptors.     

Intracellular acidification of the leech giant glial cell evoked by glutamate and aspartate

Glutamate is an excitatory receptor agonist in both neurones and glial cells, and, in addition, glutamate is also a substrate for glutamate transporter in glial cells. We have measured intracellular and extracellular pH changes induced by bath application of glutamate, its receptor agonist kainate, and its transporter agonist aspartate, in the giant neuropile glial cell in the central nervous system of the leech Hirudo medicinalis, using double-barrelled pH-sensitive microelectrodes. The giant glial cells responded to glutamate and aspartate (100–500 μM), and kainate (5–20 μM) with a membrane depolarization or an inward current, and with a distinct intracellular acidification. Glutamate and aspartate (both 500 μM) evoked a decrease in intracellular pH (pH_i) by 0.187 ± 0.081 (n = 88) and 0.198 ± 0.067 (n = 86) pH units, respectively. With a resting pH_i of 7.1 or 80 nM H⁺, these acidifications correspond to a mean increase of the intracellular H⁺ activity by 42 nM and 45 nM. Kainate caused a decrease of pH_i by 0.1 − 0.35 pH units (n = 15). The glutamate/aspartate-induced decrease in pH_i was not significantly affected by the glutamate receptor blockers kynurenic acid (1 mM) and 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX, 50–100 μM), which greatly reduced the kainate-induced change in pH_i. Extracellular alkalinizations produced by glutamate and aspartate were not affected by CNQX. Reduction of the external Na⁺ concentration gradually decreased the intracellular pH change induced by glutamate/aspartate, indicating half maximal activation of the acidifying process at 5–10 mM external Na⁺ concentration. When all external Na⁺ was replaced by NMDG⁺, the pH_iresponses were completely suppressed (glutamate) or reduced to 10% (aspartate). When Na⁺ was replaced by Li⁺, the glutamate- and aspartate-evoked pH_i responses were reduced to 18% and 14%, respectively. Removal of external Ca²⁺ reduced the glutamate- and aspartate-induced pH_i responses to 93 and 72%, respectively. The glutamate/aspartate-induced intracellular acidifications were not affected by the putative glutamate uptake inhibitor amino-adipidic acid (1 mM). DL-aspartate-β-hydroxamate (1 mM), and dihydrokainate (2 mM), which caused some pH_i decrease on its own, reduced the glutamate/aspartate-induced pH_i responses by 40 and 69%, respectively. The putative uptake inhibitor DL-threo-β-hydroxyaspartate (THA, 1 mM) induced a prominent intracellular acidification (0.36 ± 0.05 pH units, n = 9), and the pH_i change evoked by glutamate or aspartate in the presence of THA was reduced to less than 10%. The results indicate that glutamate, aspartate, and kainate produce substantial intracellular acidifications, which are mediated by at least two independent mechanisms: 1) via activation of non-NMDA glutamate receptors and 2) via uptake of the excitatory amino acids into the leech glial cell. © 1997 Wiley-Liss Inc.