The mechanism by which NBQX enhances NMDA currents in retinal ganglion cells

When the quinoxaline NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline), a KA/AMPA antagonist, is bath applied to the tiger salamander retina, a paradoxical action is evident in the light-evoked synaptic responses of ganglion cells: NBQX enhances excitatory synaptic currents at light onset observed under whole-cell voltage-clamp conditions in a perfused retinal slice preparation. This observation was surprising because synaptic inputs into ganglion cells that are mediated by KA/AMPA receptors are entirely blocked by NBQX. Thus, the NBQX-enhanced current is entirely mediated by NMDA receptors. The purpose of this study was to determine the mechanism(s) by which blocking KA/AMPA receptors appears to enhance NMDA currents. Using hyperosmotic sucrose stimulation to activate neurotransmitter release from the inner retina, we observed that NBQX augmented the sucrose-evoked response, suggesting that at least a component of this enhancement may reside in the inner retina. NBQX does not enhance NMDA currents activated by bath applied NMDA, demonstrating that the NBQX-induced enhancement does not result from modulation of NMDA receptors. Voltage-clamp studies, carried out at the appropriate holding potential, indicate that NBQX enhances glutamatergic transmission and reduces inhibitory inputs onto ganglion cells. In the presence of strychnine and picrotoxin, the NBQX-induced enhancement of NMDA currents is eliminated, suggesting that NBQX facilitates the expression of NMDA currents by a selective and partial reduction of inhibitory mechanisms. Additional studies suggest that part of the NMDA enhancement by NBQX is evident at the postsynaptic level, but a presynaptic component probably also participates, perhaps at the level of bipolar cell terminals. One way to account for this observation is to assume that a subpopulation of inhibitory amacrine cells requires KA/AMPA receptors exclusively for their synaptic activation: previous studies of sustained amacrine cells support this interpretation. Thus the NBQX-induced enhancement phenomenon may reflect a network-selective distribution of NMDA and KA/AMPA receptors among third-order neurons.     

Ionotropic glutamate receptors during the development of the chick retina.

Glutamate is the main neurotransmitter of photoreceptors, bipolar cells, and ganglion cells of the vertebrate retina. Three main classes of ionotropic glutamate receptors comprising different subunits can be distinguished: AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionate), KA (kainate), and NMDA (N-methyl-D-aspartate). This study was undertaken to characterize the AMPA (GluR1, GluR2/3, and GluR4), KA (GluR5/6/7), and NMDA (NR1) ionotropic glutamate receptor subunits and to determine their distribution during the development of the chick retina by Western blotting and immunohistochemistry. Western blotting analysis at 1 day after hatching indicated that the antibodies against GluR1, 2/3, 4, and 5/6/7 and NR1 recognized specifically a single band of 100-110 kDa. In turn, immunohistochemistry at P1 showed that all subunits were expressed in cells of the inner nuclear and ganglion cell layers of the chick retina, mostly amacrine and ganglion cells, and their processes in the inner plexiform layer. In addition, stained processes in the outer plexiform layer were observed with the antibodies against GluR2/3, GluR4, and GluR5/6/7. Although all subunits appeared around E5-E6 in the prospective ganglion cell layer, and later in the prospective inner nuclear layer, the distribution of cells containing these glutamate receptor subunits revealed distinct ontogenetic patterns. This multiplicity of glutamate receptors may contribute to different processes that occur in the chick retina during development.     

Functional activation of glutamate ionotropic receptors in the developing mouse retina

Ionotropic glutamate receptors have been associated with early development of the visual process by regulating cell differentiation, cell motility, and synaptic contacts. We determined the expression of functional ionotropic glutamate receptors during development of the mouse retina by assessing 1-amino-4-guanidobutane (agmatine; AGB) immunolabelling after application of a range of glutamate analogs. Colocalization of AGB with calretinin and islet-1 allowed the identification of functional receptors in neurochemically defined neurons. Activation with kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA) resulted in AGB entry into neurons consistent with that found previous receptor subunit localization studies in the developing retina. Temporal analysis revealed that application of 50 microM KA activated receptors as early as embryonic day 18 in the ventricular zone and in the ganglion cell layer, whereas 30 muM AMPA activated cells predominantly in the ganglion cell layer. Cholinergic amacrine cells showed functional KA and AMPA receptors upon their insertion into the conventional amacrine cell layer from postnatal day 1 (P1). OFF cone bipolar cells showed functional KA receptors from P6, at a developmental age when they are known to make contact with ganglion cells. NMDA activation led to diffuse AGB labeling at birth among cells in the ganglion cell layer, whereas, at P1, regularly spaced cholinergic amacrine cells in the conventional amacrine cell layer started to be responsive to NMDA. Non-NMDA receptors were first to show functional activation in the developing retina, and cholinergic amacrine cells displayed functional ionotropic glutamate receptors after reaching their final destination.     

Stimulation of N-methyl-D-aspartate receptors, AMPA receptors or metabotropic glutamate receptors leads to rapid internalization of AMPA receptors in cultured nucleus accumbens neurons

In hippocampus and other regions, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are inserted into synapses during long-term potentiation and removed during long-term depression. However, little is known about regulation of AMPA receptor trafficking in the nucleus accumbens (NAc), despite growing evidence that glutamate-dependent forms of plasticity in the NAc contribute to drug addiction. Using postnatal rat NAc cultures and an immunocytochemical method that selectively detects newly internalized GluR1, we studied the regulation of AMPA receptor internalization in NAc neurons by glutamate agonists. Newly internalized GluR1 was detected during 15 or 30 min of incubation at room temperature, indicating a basal rate of GluR1 turnover. The rate of GluR1 internalization was increased by glutamate (50 microM) within 5 min of its addition. Glutamate-induced GluR1 internalization was partially blocked by either an AMPA receptor antagonist (CNQX; 20 microM) or an N-methyl-D-aspartate (NMDA) receptor antagonist (APV; 50 microM). Both NMDA (50 microM) and AMPA (50 microM) increased GluR1 internalization in a Ca(2+)-dependent manner. The NMDA effect was blocked by APV while the AMPA effect was blocked by APV or CNQX. We interpret these findings to suggest that NMDA and AMPA ultimately trigger GluR1 internalization through the same NMDA receptor-dependent pathway. The effect of glutamate was also partially blocked by the group 1 metabotropic glutamate receptor antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 50 microM), while the group 1 agonist 3,5-dihydroxyphenylglycine (DHPG; 50 microM) stimulated GluR1 internalization. These data suggest that AMPA receptors on NAc neurons may be subject to rapid regulation of their surface expression in response to changes in the activity of glutamate inputs from cortical and limbic regions.     

The role of AMPA and metabotropic glutamate receptors on morphine withdrawal in infant rats

Glutamate receptors, especially N-methyl-d-aspartate (NMDA) receptors, are hypothesized to play key roles in opiate tolerance and withdrawal. There is also accumulating evidence that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists and group II metabotropic glutamate receptor (mGluR) agonists attenuate opiate withdrawal. However, most existing data are derived from adult animal models. Glutamate receptor types undergo dramatic developmental changes during early life. Thus, the pharmacological effects on opiate withdrawal of NMDA receptor, AMPA receptor, and mGluR antagonists in the developing organism may not be comparable to those in the adult. Indeed, NMDA receptor antagonists do not block morphine tolerance or withdrawal in the 7-day-old rat, but are partially effective in the 14-day-old, and fully effective in the 21-day-old. Thus, there is a transition period around the second post-natal week for NMDA receptor antagonists to suppress opiate tolerance and withdrawal. A combination of in vivo and in vitro assays was used in the present studies to test the effect of drugs acting on AMPA and group II mGlu receptors on morphine withdrawal in rats at 7, 14, and 21 days of age. These ages represent the critical periods when various glutamate receptor subunits undergo differential change. In contrast to NMDA receptor antagonists' early ineffectiveness in suppressing morphine withdrawal, the AMPA receptor antagonist and the group II mGluR agonists were effective at all ages tested. Thus, for the human infant patient, pharmacotherapies to reduce opiate tolerance and withdrawal should focus on non-NMDA ionotropic and metabotropic receptors.     

NMDA receptors of the vestibular nuclei neurones

Cloning and pharmacological studies have shown that glutamatergic receptors can be divided in two classes (refer to Table 1): ionotropic receptors including N-methyl- -aspartate (NMDA) and non-NMDA subtypes, and the G-protein-coupled metabotropic receptors (glutamate metabotropic receptor). There are two types of non-NMDA receptors: the AMPA/low-affinity kainate receptor type (the AMPA receptors) activated by a specific agonist, the alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionate (AMPA), and the high affinity kainate receptors. The vestibular nuclei neurones are endowed with all these types of glutamatergic receptors, which fits well with the fact that various afferents, including the primary vestibular afferents, most probably use glutamate or aspartate as a neurotransmitter. This article is aimed at summarising several past studies of our group and some more recent data obtained in the in vitro wholebrain preparation concerning the NMDA receptors of the central vestibular neurones. In that process, we will detail also many valuable studies of other groups that had been devoted to the same topic.     

Development of excitatory synapses in cultured neurons dissociated from the cortices of rat embryos and rat pups at birth

We studied the development of excitatory synapses in cultured neurons dissociated from the cortices of rat embryos at the 18th day of gestation (E18) and rat pups at birth (P0). Between 7 and 14 days in vitro (DIV), large increases in the amplitudes and frequencies of the spontaneous excitatory postsynaptic currents (EPSCs) of both cultured E18 and P0 neurons were observed. The EPSCs of E18 neurons were mediated primarily by alpha-amino-3-hydroxy-5-methyl-4-iso-xazole-propionic acid (AMPA) receptors at 7 DIV and by both N-methyl-D-aspartate (NMDA) and AMPA receptors at 14 DIV. Consistently, immunostaining indicated significant increases in the proportion of the clusters of NR1, an NMDA receptor subunit, which were associated with the accumulation of synaptophysin, a presynaptic marker, in cultured E18 neurons between 7 and 14 DIV. The proportion of NR1 clusters residing in synaptic regions and the proportion of synapses that colocalized with NR1 clusters in 7-day-old P0 neurons were not different statistically from those found in 7-day-old E18 neurons. However, cultured P0 neurons at 7 DIV displayed clear EPSCs mediated by NMDA receptors. Our results suggest that the targeting of NMDA receptors to synaptic regions lag behind the synaptic clustering of AMPA receptors during the in vitro development of cultured rat E18 cortical neurons. The results further suggest that the cortical neurons at P0 differ from those at E19 in certain cellular properties; as a result, the currents mediated by the synaptic NMDA receptors in 7-day-old P0 neurons are larger than those mediated by the synaptic NMDA receptors in 7-day-old E18 neurons.     

NBQX, an improved non-NMDA antagonist studied in retinal ganglion cells

The quinoxaline derivative, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline (NBQX), significantly reduced the currents evoked by exogenous application of quisqualate (QQ), kainate (KA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) when applied to ganglion cells, using whole-cell recording in a slice preparation of the tiger salamander retina. A comparison between NBQX and CNQX indicates that NBQX is more effective in blocking AMPA receptors. Also, at up to 10 μM, NBQX has no effect on NMDA-induced currents. Thus at this concentration, NBQX shows no affinity for the glycine binding site of NMDA receptors. For this reason, NBQX is preferred over CNQX for a more effective and selective antagonism toward non-NMDA receptors.     

Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats

The effects of phosphatidylserine (PS) on the binding properties of the AMPA (-amino-3-hydroxy-5-methylisoxazolepropionic acid) and NMDA ( N-methyl-d-aspartate) subtypes of glutamate receptors were analyzed by quantitative autoradiography of [³H]AMPA, [³H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [³H]glutamate binding on at brain tissue sections. Preincubation of brain sections with PS produced an increase in [³H]AMPA binding without modifying the binding properties of [3H]CNQX, an antagonist of AMPA receptors. This effect of PS appeared to be specific for the AMPA subtype of glutamate receptors as the same treatment did not modify [³H]glutamate binding to the NMDA receptors. Furthermore, the PS-induced increase in [3H]AMPA binding was different in various brain structures, being larger in the molecular layer of the cerebellum and almost absent in the striatum. Preincubation with calcium also augmented [³H]AMPA binding, and the lack of additivity of the effects of calcium and PS on [³H]AMPA binding strongly suggests that both treatments share a common mechanism(s) for producing increased agonist binding. Finally, the effect of PS on AMPA receptor properties was markedly reduced in rat brain sections prepared from neonatal rats at a developmental stage that is normally characterized by the absence of LTP expression in certain brain regions. The present data are consistent with the hypothesis that alteration in the lipid composition of synaptic membranes may be an important mechanism for regulating AMPA receptor properties. which could be involved in producing long-lasting changes in synaptic operation.     

Expression of AMPA/kainate receptors during development of chick embryo retina cells: in vitro versus in vivo studies

The activity and the subunit expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate ionotropic glutamate receptors were studied in retina cells developing in chick embryos and in retina cells cultured as retinospheroids, at the same stages of development. In the retinospheroids, the activity of the AMPA/kainate receptors was monitored by following the changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to AMPA, kainate or to L-glutamate, and the expression of the receptor subunits GluR1, GluR2/3, GluR4 and GluR6/7 was determined in the retinospheroids and in chick retinas by immunodetection using polyclonal antibodies. The changes in [Ca(2+)](i) in response to 400 microM kainate increased from 5h in vitro to 3 days, and remained constant until day 14, whereas the [Ca(2+)](i) in response to 500 microM L-glutamate or 400 microM AMPA increased from 5h in vitro to 3 days, and thereafter decreased slightly until day 14. The [Ca(2+)](i) responses to kainate are mainly due to AMPA receptor stimulation, since the signals were abolished by LY303070, the AMPA receptor antagonist, and were not affected by MK-801, the NMDA receptor antagonist. In retinospheroids, the levels of expression of GluR1 subunit increased from 5h in vitro until day 7, then decreased until day 14. The levels of expression of GluR2/3 and GluR4 subunits increased from 5h in vitro until day 10, and remained constant until day 14. The levels of kainate receptor subunits GluR6/7 increased from 5h in vitro until day 3, and thereafter decreased slightly until day 14. In the retinas, the expression of GluR1 and GluR6/7 subunits increased from day 8 until day 15, and then decreased until day 22 (post-natal 1). The subunits GluR2/3 and GluR4 increased from day 8 until day 18, and remained constant until day 22. The results suggest that AMPA/kainate receptors are expressed at early embryonic stages, although at low levels and before synapse formation (E12). However, the AMPA receptors are not completely functional at the first stage studied since they do not respond to the agonist AMPA. Also, the patterns of AMPA/kainate receptor subunit expression in retinospheroids of chick embryo retina cells cultured in vitro and in retina cells developing in the embryo (in vivo) were similar, indicating that the AMPA/kainate receptor subunits expression in these primary cultures mimics their expression in the developing chick retina.     

Emergence of cellular markers and functional ionotropic glutamate receptors on tangentially dispersed cells in the developing mouse retina

Tangential cell dispersion in the retina is a spacing mechanism that establishes a regular mosaic organization among cell types and contributes to their final positioning. The present study has used the X-inactivation transgenic mouse expressing the lacZ reporter gene on one X chromosome. Due to X chromosome inactivation, 50% of early progenitor cells express beta-galactosidase (beta-Gal); therefore, all cells derived from a particular beta-Gal-expressing progenitor cell can be identified in labeled columns. The radial segregation of clonally related beta-Gal-positive and beta-Gal-negative cells can be used to determine whether single cells transgress a clonal boundary in the retina. We investigated the extent to which particular cell classes tangentially disperse by analyzing the placement of labeled cells expressing particular markers at several ages and quantifying their tangential displacement. Retinal neurons expressing cell markers at postnatal day (P) 1 have a greater degree of tangential dispersion compared with amacrine and bipolar cells at P5-6. We also studied whether there is a functional correlation with these dispersion patterns by investigating the emergence of functional ionotropic glutamate receptors. To determine the degree of functional glutamate receptor activation, agmatine (AGB) was used in combination with cell-specific labeling. AGB permeates functional glutamate receptor channels following activation with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate or N-methyl-D-aspartate (NMDA). Within these receptor groups, high concentrations of AMPA, kainate, and NMDA are associated with a high degree of tangential dispersion in the adult. Developmentally, functional kainate and AMPA receptors were detected by P1 and were associated with tangentially dispersed cells. Functional NMDA receptors were not detected as early as kainate and AMPA receptors. These results indicate that cells generated early during development are more likely to disperse tangentially compared with those generated later in development. Therefore, functional AMPA and kainate receptors may play a critical role in tangentially displacing cell types.     

The network-selective actions of quinoxalines on the neurocircuitry operations of the rabbit retina

We examined the contribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxalole-4-propionic acid (AMPA)/kainate (KA) receptors to the light-responses of rabbit retinal neurons. In the outer retina, bath application of the AMPA/KA receptor antagonists 6,7-dinitro-quinoxaline-2,3-dione (DNQX) and 2,3,dihydroxy-6-nitro-7-sulfamoyl-benzo-f-quinoxaline (NBQX) blocked the light-responses of horizontal cells. Application of quinoxalines enhanced ON-bipolar cell light-responses, and was associated with a hyperpolarization of their resting potentials. In the inner retina, application of both AMPA/KA and NMDA antagonists to AII amacrine-like cells only partially blocked their light-responses. Their residual responses may reflect electrical coupling to neighboring ON-center cone bipolar cells, and can inhibit OFF-center ganglion cells. ON-sustained ganglion cells were highly sensitive to the quinoxalines, which reduced their light-evoked firing, while the firing of ON-transient cells remained as NMDA-mediated light-responses. Quinoxalines had differential effects on the firing rates of ON- and OFF-center ganglion cells: ON-cells were reduced, while OFF-cells were increased. In contrast, firing rates of ON-OFF ganglion cells were not excited by NBQX, and showed a recovered light-response mediated by NMDA receptors. The receptive field surround was lost in ganglion cells. For comparison, NMDA antagonists had only moderate effects on all ganglion cell light-responses. Our results indicate that NMDA and AMPA/KA receptors both contribute to ganglion cell light-responses. However, AMPA/KA receptors also significantly effect the light-response of neurons presynaptic to retinal ganglion cells, altering the observed pharmacology at the ganglion cell level.     

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.     

Development of excitatory amino acid induced cytotoxicity in cultured neurons

The neurotoxicity of the excitatory amino acids (EAAs) L-glutamate (L-glu), L-aspartate (L-asp), N-methyl-D-aspartate (NMDA), kainate (KA), quisqualate (QA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) was followed as a function of development in primary cultures of cerebral cortex neurons and cerebellar granule cells. These two types of neurons express, respectively, glutamate receptor subtypes with sensitivity to all of these excitatory amino acids or only to glutamate and aspartate. None of the EAAs were toxic in cerebral cortex neurons at 2 days in culture, whereas at culture day 4 the neurons became sensitive to glutamate, at day 5 to KA followed by sensitivity to QA at day 6, and finally to NMDA, L-asp and AMPA at day 7. The rank order of potency of the EAAs was in cerebral cortex neurons cultured for 12 days: L-asp (ED50 = 0.5 microM) = L-glu (ED50 = 1 microM) greater than AMPA (ED50 = 10 microM) greater than NMDA (ED50 = 65 microM) greater than QA = KA (ED50 = 100 microM). Cerebellar granule cells were insensitive to all of the EAAs at 3 and 5 days in culture but at day 8 the cells became sensitive to toxicity induced by L-glu (ED50 = 70 microM) and L-asp (ED50 = 30 microM). In order to determine ED50 values for L-asp and L-glu accurately, media in these experiments also contained 500 microM of the glutamate uptake inhibitor L-aspartate-beta-hydroxamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC(50) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 microM). In low GluR5 selective concentrations (0.3-3 microM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration-response curves with calculation of EC(50) values for glutamate receptor agonists are possible, as well as comparison of inhibition data for glutamate receptor antagonists. The observation that the slice cultures respond with more in vivo-like patterns of excitotoxicity than primary neuronal cultures, suggests that slice cultures are the best model of choice for a number of glutamate agonist and antagonist studies.     

Comparing long-term depression with pharmacologically induced synaptic attenuations in young rat hippocampi

Field excitatory postsynaptic potentials (EPSPs) were recorded in the CA1 region of hippocampal slices from 12–18-day-old rats. The isolated N-methyl-D-aspartate (NMDA) receptor mediated field EPSP as well as the composite field EPSP of both NMDA and α-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor mediated components were obtained in low Mg²⁺ solutions with 10 μM or 1 μM of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. The isolated AMPA receptor mediated field EPSP was obtained either in normal Mg²⁺ solution or in a low Mg²⁺ solution in the presence of the NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid. The waveforms of the field EPSPs were studied and the effect of long-term depression (LTD) on these waveforms was compared with the effects of several pharmacological agents that attenuate the synaptic efficacy. It was shown that LTD occurred without changes in the waveforms of isolated AMPA and NMDA EPSPs. Reducing the number of release sites by lowering the stimulus strength or reducing the probability of transmitter release by an adenosine agonist N⁶-cyclohexyl-adenosine both mimicked the LTD-induced changes. Partial blockade of the AMPA receptors was also without effect on the waveforms of isolated AMPA EPSPs. In contrast, partial blockade of the NMDA receptors in several different ways resulted in waveform changes. A similar result could be inferred from experiments using composite field EPSPs. The synaptic attenuation caused by a partial blockade of NMDA receptors therefore appears to differ mechanistically from that involved in LTD, arguing against a postsynaptic locus of the modification involved in LTD. However, directly testing for alterations in transmitter release using the open channel blocker of NMDA receptors MK-801 failed in revealing such presynaptic changes during LTD. Our results therefore suggest that LTD might be due to a coordinated pre- and postsynaptic change instead of distinct pre- or postsynaptic modifications. Synapse 26:329–340, 1997. © 1997 Wiley-Liss Inc.     

Subcellular and subsynaptic distribution of the NR1 subunit of the NMDA receptor in the neostriatum and globus pallidus of the rat: co-localization at synapses with the GluR2/3 subunit of the AMPA receptor

Glutamatergic neurotransmission in the neostriatum and the globus pallidus is mediated through NMDA-type as well as other glutamate receptors and is critical in the expression of basal ganglia function. In order to characterize the cellular, subcellular and subsynaptic localization of NMDA receptors in the neostriatum and globus pallidus, multiple immunocytochemical techniques were applied using antibodies that recognize the NR1 subunit of the NMDA receptor. In order to determine the spatial relationship between NMDA receptors and AMPA receptors, double labelling was performed with the NR1 antibodies and an antibody that recognizes the GluR2 and 3 subunits of the AMPA receptor. In the neostriatum all neurons with characteristics of spiny projection neurons, some interneurons and many dendrites and spines were immunoreactive for NR1. In the globus pallidus most perikarya and many dendritic processes were immunopositive. Immunogold methods revealed that most NR1 labelling is associated with asymmetrical synapses and, like the labelling for GluR2/3, is evenly spread across the synapse. Double immunolabelling revealed that in neostriatum, over 80% of NR1-positive axospinous synapses are also positive for GluR2/3. In the globus pallidus most NR1-positive synapses are positive for GluR2/3. In both regions many synapses labelled only for GluR2/3 were also detected. These results, together with previous data, suggest that NMDA and AMPA receptor subunits are expressed by the same neurons in the neostriatum and globus pallidus and that NMDA and AMPA receptors are, at least in part, colocalized at individual asymmetrical synapses. The synaptic responses to glutamate in these regions are thus likely be mediated by both AMPA and NMDA receptors at the level of individual synapses.     

Spatiotemporal patterning of glutamate receptors in developing ferret striate cortex

We have studied glutamate receptor levels during very early phases of cortical formation by using quantitative in vitro autoradiography to map the expression of NMDA, AMPA and kainate receptors in the developing primary visual cortex of the ferret. NMDA and non-NMDA receptors exhibit very different developmental profiles in primary visual cortex. NMDA receptor density is low at birth and increases throughout the first 2 postnatal months, rising between threefold (layers II/III) and ninefold (layer VI). In contrast, AMPA receptors are abundant at birth and their density remains constant for the first postnatal month, before rising by a maximum of 1.7-fold (layer I) at around the time of eye-opening (postnatal day 32). Kainate receptors are also present in high levels at birth and their expression levels rise in the early postnatal period by between 1. 5-fold (layer I) and threefold (layers V/VI) to a peak just after eye-opening. The proportion of the total ionotropic glutamate receptor binding contributed by NMDA receptors thus rises from 5% at birth to a maximum of 22% at 2 months of age, while the AMPA receptor contribution falls from 87% to 72% over the same period. Below cortex, all three glutamate receptor subtypes are expressed in the subplate region for the first 3 postnatal weeks. These developmental patterns, combined with the fact that AMPA receptors are densely expressed in the proliferative zones underlying presumptive area 17, indicate that non-NMDA receptor expression levels in primary visual cortex are mostly specified much earlier than those of NMDA receptors.     

AMPA-preferring receptors with high Ca2+ permeability mediate dendritic plasticity of retinal horizontal cells

The synaptic complex formed by the cone photoreceptor pedicles and the dendrites of horizontal cells in the teleost retina undergoes structural changes during light adaptation. Numerous spinules are formed by the terminal dendrites, and they are subsequently retracted during dark adaptation. In a retina kept under continuous illumination, the retraction process can be initiated by analogues of the neurotransmitter glutamate acting at AMPA/kainate receptors. On the other hand, the retraction process depends on calcium influx and the subsequent activation of CaMkII. We show here that the retraction of spinules induced by AMPA or kainate is not impaired in the presence of cobalt, making an involvement of voltage-gated calcium channels unlikely. Using calcium imaging techniques with isolated horizontal cells, we demonstrate that AMPA and kainate, but not NMDA, increase [Ca2+]i in the presence of nicardipine, caffeine and thapsigargin. The increase of [Ca2+]i under these conditions depends on [Ca2+]o and on the agonist in a dose-dependent manner, suggesting that the increase of [Ca2+]i is largely due to calcium influx through the agonist-gated channel. Pharmacological studies were performed to determine whether AMPA- and/or kainate-preferring receptors mediate the calcium influx. The AMPA-preferring receptor antagonist LY303070 blocked glutamate- and kainate-evoked increases of [Ca2+]i in a concentration-dependent manner, indicating that kainate-preferring receptors contributed little or nothing to the observed [Ca2+]i increase. This was supported by experiments where cyclothiazide (which blocks the desensitization of AMPA receptors) and concanavalin A (which potentiates responses mediated by kainate receptors) were applied. In all cases, LY303070 blocked the agonist-evoked increase of [Ca2+]i. The presence of AMPA-preferring receptors with high Ca2+ permeability on horizontal cells was also supported by measuring agonist-induced currents using whole-cell recording techniques. Furthermore, LY303070 was able to impair the retraction of spinules during dark adaption in the in vivo situation.     

Glutamate receptors at bipolar synapses in the inner plexiform layer of primate retina: light microscopic analysis

At least 10 different types of bipolar cells have been distinguished in the primate retina. The axon terminals of these cells stratify in distinct strata in the inner plexiform layer and are involved in parallel pathways to distinct types of ganglion cells. Ionotropic glutamate receptor (GluR) subunits also show a stratified distribution in the inner plexiform layer. Here, we investigated whether different types of bipolar cells are associated with different types of ionotropic glutamate receptors in the inner retina of a New World primate, the common marmoset Callithrix jacchus. Vertical cryostat sections through central retina were double labeled with immunohistochemical markers for bipolar cell types and with antibodies to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1 to 4, kainate receptor subunits GluR6/7, and the NR1C2' subunit of the N-methyl-D-aspartate (NMDA) receptor. The axon terminals of bipolar cell types were reconstructed from confocal sections, and the colocalized immunoreactive puncta were quantified. For all bipolar cell types, immunoreactive puncta for the AMPA receptor subunits GluR2, 2/3, and 4 were colocalized at highest densities, whereas GluR1-immunoreactive puncta were expressed at very low densities. The kainate receptor subunits GluR6/7 were predominantly associated with diffuse bipolar (DB6) and rod bipolar cells. The NMDA receptor subunit NR1C2' was specifically colocalized with flat midget and DB3 axons. These findings suggest that rod and cone bipolar cell types contribute to multiple but distinct glutamate receptor pathways in primate retina.