Mood and cognition in pregnant workers

We investigated the role of hypothalamic glutamate receptors in mediating the stimulatory effect of low glucose (< 5 mM) on somatostatin release. We also studied whether alteration in glutamate release might contribute to the reduced hypothalamic somatostatin response to low glucose observed in diabetic (Goto-Kakizaki) rat hypothalami. Hypothalamic somatostatin release in response to incubation with 1 mM D-glucose was inhibited by the ionotropic glutamate receptor antagonists MK801, D-AP5 and DNQX but not by the metabotropic antagonists L-AP3 or MCPG. The release of somatostatin was increased by the ionotropic agonists NMDA, AMPA and kainate but not by metabotropic agonists t-ACPD or L-AP4. Basal and peak glutamate release in response to incubation with 1 mM glucose, were significantly lower from GK hypothalami There were no significant differences in the basal or stimulated release of serine and GABA. These data indicate that ionotropic NMDA/AMPA/kainate receptors and not metabotropic receptors mediate the effects of glucose on rat hypothalamic somatostatin release. Reduced hypothalamic somatostatin release in response to low glucose in diabetic (Goto-Kakizaki) rats may well be secondary, at least in part, to reduced glutamate release.     

Strong nuclear factor-kappaB-DNA binding parallels cyclooxygenase-2 gene transcription in aging and in sporadic Alzheimer''s disease superior temporal lobe neocortex

This study investigated the putative role of non-NMDA excitatory amino acid (EAA) receptors in the ventral tegmental area (VTA) for the increase in dopamine (DA) release in the nucleus accumbens (NAC) and behavioral stimulation induced by systemically administered dizocilpine (MK-801). Microdialysis was utilized in freely moving rats implanted with probes in the VTA and NAC. Dialysates from the NAC were analyzed with high-performance liquid chromatography for DA and its metabolites. The VTA was perfused with the AMPA and kainate receptor antagonist CNQX (0.3 or 1 mM) or vehicle. Forty min after onset of CNQX or vehicle perfusion of the VTA, MK-801 (0.1 mg/kg) was injected subcutaneously. Subsequently, typical MK-801 induced behaviors were also assessed in the same animals by direct observation. MK-801 induced hyperlocomotion was associated with a 50% increase of DA levels in NAC dialysates. Both the MK-801 evoked hyperlocomotion and DA release in the NAC was antagonized by CNQX perfusion of the VTA in a concentration-dependent manner. None of the other rated MK-801 evoked behaviors, e.g. head weaving or sniffing, were affected by CNQX perfusion of the VTA. By itself the CNQX or vehicle perfusion of the VTA alone did not affect DA levels in NAC or any of the rated behaviors. These results indicate that MK-801 induced hyperlocomotion and DA release in the NAC are largely elicited within the VTA via activation of non-NMDA EAA receptors, tentatively caused by increased EAA release. Thus, the locomotor stimulation induced by psychotomimetic NMDA receptor antagonists may not only reflect impaired NMDA receptor function, but also enhanced AMPA and/or kainate receptor activation in brain, e.g., in the VTA. In view of their capacity to largely antagonize the behavioral stimulation induced by psychotomimetic drugs, such as MK-801, AMPA, and/or kainate receptor antagonists may possess antipsychotic efficacy.     

Acute effects of ethanol on pharmacologically isolated kainate receptors in cerebellar granule neurons: comparison with NMDA and AMPA receptors

Comparisons of acute ethanol's effects on individual members of the three major families of ionotropic glutamate receptors (kainate, AMPA, and NMDA) have been performed only with recombinant receptors. However, no study has compared the acute effects of ethanol on individual members of each one of these receptor families in the same neuron. We accomplished this task by using cultured cerebellar granule neurons and LY303070 (GYKI-53784), a noncompetitive and selective AMPA receptor antagonist. Ethanol concentrations of 25, 50, 75, and 100 mM decreased the amplitude of pharmacologically isolated kainate-activated currents by 3 +/- 1, 9 +/- 2, 14 +/- 2, and 22 +/- 3% (n = 8), respectively. The magnitude of the ethanol-induced inhibition of nonselective kainate-activated currents, i.e., in the absence of LY303070, and currents activated by submaximal AMPA concentrations was not significantly different from that obtained with isolated kainate currents. However, the magnitude of the ethanol-induced inhibition of NMDA receptor-activated currents was about twofold greater than that of kainate and/or AMPA receptors.     

Longitudinal involvement of the spinal cord in a patient with lupus related transverse myelitis

Arachidonic acid is suggested to play a role in the expression of long-term potentiation (LTP), a synaptic analog of memory and learning. However, it is unknown whether this free fatty acid is actually released during LTP or not. To address this question, we assayed arachidonic acid with an HPLC system using 9-anthryldiazomethane (ADAM) as a fluorescent probe. High frequency stimulation (tetanic stimulation) to a hippocampal slice from the guinea pig brain caused a huge increase in the release of glutamate from presynaptic terminals and in turn, a gradual increase in the release of arachidonic acid. A similar increase in the release of arachidonic acid was induced by application of glutamate and the increase was inhibited by either the selective AMPA/kainate receptor antagonist, DNQX, or to a lesser extent by the selective NMDA receptor antagonist, APV. These findings suggest that arachidonic acid is produced by activation of ionotropic glutamate receptors involving expression of LTP. Arachidonic acid exerted a long-lasting facilitatory action on synaptic transmission in the CA1 region of rat hippocampal slices and the facilitation occluded the tetanic LTP. Arachidonic acid, thus, appears to be a significant factor for the expression of LTP.     

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-D-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-iso-xalone 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 whole-brain 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.     

Surfactant nebulization versus instillation during high frequency ventilation in surfactant-deficient rabbits

Glutamate-mediated excitotoxicity plays an important role in the degeneration of nigrostriatal dopamine (DA) neurons induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), although the role of the N-methyl D-aspartate (NMDA) receptor subtype in this process is still uncertain. We studied glutamate receptor subtype agonist-induced ionic currents in acutely dissociated DAergic neurons from the rat substantia nigra zona compacta (SNc) using the nystatin-perforated patch-clamp whole-cell recording technique. The results fall into four main categories. First, single neurons, freshly isolated from SNc, exhibited a large soma and multipolar morphology, responded to DA, and stained positively for tyrosine hydroxylase (TH). Second, rapid application of L-glutamate (> 10(-5) M) induced an inward current with minimal desensitization at a clamp voltage of -60 mV. Third, kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-isoxazole (AMPA) induced an inward current that was similar to the glutamate-induced current while, in the same neuron, NMDA (10(-4) M) failed to induce any current response in Mg2+-free solution that contained 10(-5) M glycine at a clamp voltage of -60 mV. Under the same experimental conditions, NMDA induced a clear current response in isolated substantia nigra reticulata (SNr) neurons. Fourth, the specific NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV, 10(-4) M) failed to block 10(-4) M glutamate-induced inward current, while the specific KA/AMPA receptor antagonist 6-cyano-7-nitroguinoxaline-2, 3-dione (CNQX, 10(-5) M) completely blocked the glutamate-induced current. These results indicate that in single SNc DAergic neurons of 2-week-old rats, L-glutamate-induced inward current is mediated by non-NMDA receptors rather than by NMDA receptors.     

Protection from neuronal damage induced by combined oxygen and glucose deprivation in organotypic hippocampal cultures by glutamate receptor antagonists

Organotypic hippocampal cultures were exposed to defined periods (30 and 60 min) of combined oxygen and glucose deprivation, mimicking transient ischemic conditions. The involvement of different glutamate receptors in individual hippocampal subfields (CA1, CA3 and dentate gyrus) was studied using antagonists of NMDA (dizocilpine) and AMPA/kainate receptors (CNQX and GYKI 52466). Staining with the fluorescent dye propidium iodide (PI) allowed detection of damaged cells. For quantitative determination of neuronal damage, fluorescence intensity was measured after a 22 h recovery period and was related to maximal fluorescence intensity measured after fixation and PI restaining of the cultures at the end of the experiment. Dizocilpine (10 microM), CNQX (100 microM) and GYKI 52466 (100 microM) provided complete protection in CA1, CA3 and dentate gyrus following the moderate ischemic insult, when the antagonists were present permanently. This indicates that none of the ionotropic glutamate receptor subtypes dominated toxicity in the most sensitive subpopulation of neurons. When applied only during the recovery period protection with dizocilpine (10 microM) or CNQX (100 microM) was drastically reduced by about 60% in the most sensitive area (CA1), but only slightly by 15% in CA3. Therefore the onset of irreversible damage seems to occur earlier in CA1 than in CA3. Blockade of AMPA/kainate receptors by GYKI 52466 (100 microM) offered no neuroprotection if the compound was applied only during the recovery period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between metabotropic and ionotropic glutamate receptor agonists in the rat spinal cord in vivo

Microelectrophoretic application of the non-selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] and the group I selective mGluR agonist (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG] potentiated the responses of rat spinal neurones to the cyclically-ejected ionotropic excitatory amino acid (EAA) agonists NMDA, AMPA and kainate in vivo. Potentiation was not selective between the three ionotropic responses and was paralleled by an enhancement of background activity in spontaneously active cells. "Correcting" spike count data for this increase in background activity showed that the EAA responses were not potentiated beyond the apparent enhancement of cell excitability. Neither mGluR agonist produced potentiation when NMDA/AMPA cycling was superimposed on background discharge held constant with kainate. It is concluded that potentiation produced by both (1S,3R)-ACPD and (RS)-3,5-DHPG is secondary to an enhancement of cell excitability rather than being due to a specific interaction with ionotropic EAA receptors. The mechanism of excitability enhancement cannot be determined by extracellular recording, but group I mGluRs are most likely to be responsible.     

AMPA and NMDA receptors expressed by differentiating Xenopus spinal neurons

N-methyl--aspartate (NMDA) receptors are often the first ionotropic glutamate receptors expressed at early stages of development and appear to influence neuronal differentiation by mediating Ca2+ influx. Although less well studied, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors also can generate Ca2+ elevations and may have developmental roles. We document the presence of AMPA and NMDA class receptors and the absence of kainate class receptors with whole cell voltage-clamp recordings from Xenopus embryonic spinal neurons differentiated in vitro. Reversal potential measurements indicate that AMPA receptors are permeable to Ca2+ both in differentiated neurons and at the time they first are expressed. The PCa/Pmonocation of 1.9 is close to that of cloned Ca2+-permeable AMPA receptors expressed in heterologous systems. Ca2+ imaging reveals that Ca2+ elevations are elicited by AMPA or NMDA in the absence of Mg2+. The amplitudes and durations of these agonist-induced Ca2+ elevations are similar to those of spontaneous Ca2+ transients known to act as differentiation signals in these cells. Two sources of Ca2+ amplify AMPA- and NMDA-induced Ca2+ elevations. Activation of voltage-gated Ca2+ channels by AMPA- or NMDA-mediated depolarization contributes approximately 15 or 30% of cytosolic Ca2+ elevations, respectively. Activation of either class of receptor produces elevations of Ca2+ that elicit further release of Ca2+ from thapsigargin-sensitive but ryanodine-insensitive stores, contributing an additional approximately 30% of Ca2+ elevations. Voltage-clamp recordings and Ca2+ imaging both show that these spinal neurons express functional AMPA receptors soon after neurite initiation and before expression of NMDA receptors. The Ca2+ permeability of AMPA receptors, their ability to generate significant elevations of [Ca2+]i, and their appearance before synapse formation position them to play roles in neural development. Spontaneous release of agonists from growth cones is detected with glutamate receptors in outside-out patches, suggesting that spinal neurons are early, nonsynaptic sources of glutamate that can influence neuronal differentiation in vivo.     

Characterization of ionotropic glutamate receptor-mediated nitric oxide production in vivo in rats

BACKGROUND AND PURPOSE: Glutamate receptor activation can stimulate nitric oxide (NO) production and possibly play a role in long-term potentiation and excitotoxic-mediated injury. We studied the differential effect of agonist-induced activation of ion channel-linked N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtypes on NO production in vivo in rat hippocampus. We also studied whether dantrolene, a ryanodine calcium channel inhibitor previously shown to attenuate metabotropic glutamate receptor stimulation of NO production, also attenuated ionotropic glutamate receptor-mediated stimulation of NO production. METHODS: Microdialysis probes were placed bilaterally into the CA3 region of the hippocampus of pentobarbital-anesthetized adult Sprague-Dawley rats and were perfused for 5 hours with artificial cerebrospinal fluid (CSF) containing 3 mumol/L [14C]L-arginine. Recovery of [14C]L-citrulline in the effluent was used as a marker of NO production. In 13 groups of rats, increases in [14C]L-citrulline recovery were compared between right- and left-sided probes perfused with no additional drugs versus combinations of NMDA, AMPA, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), the non-competitive glutamate receptor blocker MK-801, the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and dantrolene. RESULTS: Recovery of [14C]L-citrulline during perfusion with artificial CSF progressively increased to 272 +/- 73 fmol/min (+/-SEM) over 5 hours. Contralateral perfusion with 1 mmol/L L-NAME inhibited [14C]L-citrulline recovery. Perfusion with 1 mmol/L MK-801 or 1 mmol/L CNQX reduced [14C]L-citrulline recovery compared with contralateral perfusion with CSF alone. Perfusion with 1 mmol/L NMDA enhanced [14C]L-citrulline recovery, and this enhancement was attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. Perfusion with 1 mmol/L AMPA enhanced [14C]L-citrulline recovery, and this enhancement was also attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. CONCLUSIONS: Through an indirect method of assessing NO production in vivo, results with MK-801 and CNQX indicate that NMDA and AMPA receptor activation contribute to basal NO production in the rat hippocampus. Enhanced NO production with NMDA and AMPA agonists appears to involve a complex neuronal interaction because the effect of NMDA was attenuated by both MK-801 and CNQX and because the effect of AMPA was attenuated by both CNQX and MK-801. In contrast to metabotropic glutamate receptor activation, release of calcium from intracellular ryanodine calcium channels does not appear to be a prominent mediator of ionotropic glutamate receptor stimulation of NO production.     

Pharmacology of glutamate receptor antagonists in the kindling model of epilepsy

It is widely accepted that excitatory amino acid transmitters such as glutamate are involved in the initiation of seizures and their propagation. Most attention has been directed to synapses using NMDA receptors, but more recent evidence indicates potential roles for ionotropic non-NMDA (AMPA/kainate) and metabotropic glutamate receptors as well. Based on the role of glutamate in the development and expression of seizures, antagonism of glutamate receptors has long been thought to provide a rational strategy in the search for new, effective anticonvulsant drugs. Furthermore, because glutamate receptor antagonists, particularly those acting on NMDA receptors, protect effectively in the induction of kindling, it was suggested that they may have utility in epilepsy prophylaxis, for example, after head trauma. However, first clinical trials with competitive and uncompetitive NMDA receptor antagonists in patients with partial (focal) seizures, showed that these drugs lack convincing anticonvulsant activity but induce severe neurotoxic adverse effects in doses which were well tolerated in healthy volunteers. Interestingly, the only animal model which predicted the unfavorable clinical activity of competitive NMDA antagonists in patients with chronic epilepsy was the kindling model of temporal lobe epilepsy, indicating that this model should be used in the search for more effective and less toxic glutamate receptor antagonists. In this review, results from a large series of experiments on different categories of glutamate receptor antagonists in fully kindled rats are summarized and discussed. NMDA antagonists, irrespective whether they are competitive, high- or low-affinity uncompetitive, glycine site or polyamine site antagonists, do not counteract focal seizure activity and only weakly, if at all, attenuate propagation to secondarily generalized seizures in this model, indicating that once kindling is established, NMDA receptors are not critical for the expression of fully kindled seizures. In contrast, ionotropic non-NMDA receptor antagonists exert potent anticonvulsant effects on both initiation and propagation of kindled seizures. This effect can be markedly potentiated by combination with low doses of NMDA antagonists, suggesting that an optimal treatment of focal and secondarily generalized seizures may require combined use of both non-NMDA and NMDA antagonists. Given the promising results obtained with novel AMPA/kainate antagonists and glycine/NMDA partial agonists in the kindling model, the hope for soon having potentially useful glutamate antagonists for use in epileptic patients is increasing.     

Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo

Glutamatergic retinotectal inputs mediated principally by NMDA receptors can be recorded from optic tectal neurons early during their morphological development in Xenopus tadpoles. As tectal cell dendrites elaborate, retinotectal synaptic responses acquire an AMPA receptor-mediated synaptic component, in addition to the NMDA component. Here, we tested whether glutamatergic activity was required for the elaboration of dendritic arbors in Xenopus optic tectal neurons. In vivo time-lapse imaging of single DiI-labeled neurons shows that the NMDA receptor antagonist APV (100 microM) blocked the early development of the tectal cell dendritic arbor, whereas the AMPA receptor antagonist CNQX (20 microM) or the sodium channel blocker TTX (1 microM) did not. The decreased dendritic development is attributable to failure to add new branches and extend preexisting branches. These observations indicate that NMDA-type glutamatergic activity promotes the initial development of the dendritic arbor. At later stages of tectal neuron development when AMPA receptor-mediated synaptic transmission is strong, both APV and CNQX decrease dendritic arbor branch length, consistent with a role for glutamatergic synaptic transmission in maintaining dendritic arbor structure. These results indicate that AMPA and NMDA receptors can differentially influence dendritic growth at different stages of neuronal development, in correlation with changes in the relative contribution of the receptor subtype to synaptic transmission.     

Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness

The present study determines the proportions of unmyelinated cutaneous axons at the dermal-epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal-epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Adelta and Abeta, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive.     

The glutamate receptor/NO/cyclic GMP pathway in the hippocampus of freely moving rats: modulation by cyclothiazide, interaction with GABA and the behavioural consequences

Monitoring of extracellular cGMP during intracerebral microdialysis in freely moving rats permits the study of the functional changes occurring in the glutamate receptor/nitric oxide (NO) synthase/guanylyl cyclase pathway and the relationship of these changes to animal behaviour. When infused into the rat hippocampus in Mg2+-free medium, cyclothiazide, a blocker of desensitization of the AMPA-preferring receptor, increased cGMP levels. The effect of cyclothiazide (300 microM) was abolished by the NO synthase inhibitor L-NARG (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (100 microM). During cyclothiazide infusion the animals displayed a pre-convulsive behaviour characterized by frequent "wet dog shakes" (WDS). Neither L-NARG nor ODQ decreased the WDS episodes. Both cGMP and WDS responses elicited by cyclothiazide were prevented by blocking NMDA receptor function with the glutamate site antagonist CGS 19755 (100 microM), the channel antagonist MK-801 (30 microM) or Mg2+ ions (1 mM). The AMPA/kainate receptor antagonists DNQX (100 microM) and NBQX (100 microM) abolished the WDS episodes but could not inhibit the cyclothiazide-evoked cGMP response. DNQX or NBQX (but not MK-801) elevated, on their own, extracellular cGMP levels. The cGMP response elicited by the antagonists appears to be due to prevention of a glutamate-dependent inhibitory GABAergic tone, since infusion of bicuculline (50 microM) caused a strong cGMP response. The results suggest that (a) AMPA/kainate receptors linked to the NO/cGMP pathway in the hippocampus (but not NMDA receptors) are tonically activated and kept in a desensitized state by endogenous glutamate; (b) blockade of AMPA/kainate receptor desensitization by cyclothiazide leads to endogenous activation of NMDA receptors; (c) the hippocampal NO/cGMP system is under a GABAergic inhibitory tone driven by non-NMDA ionotropic receptors; (d) the pre-convulsive episodes observed depend on hippocampal NMDA receptor activation but not on NO and cGMP production.     

In-vitro characterization of YM872, a selective, potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL(-1) in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 +/- 0.0024 microM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 +/- 0.14 microM), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 microM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 microM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 +/- 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-microM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 +/- 0.031 microM, and blocked 300-microM kainate-induced neurotoxicity with an IC50 value of 1.02 microM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.     

Glutamatergic N2v cells are central pattern generator interneurons of the lymnaea feeding system: new model for rhythm generation

Electrophysiological and pharmacological methods were used to examine the role of glutamate in mediating the excitatory and inhibitory responses produced by the N2v rasp phase neurons on postsynaptic cells of the Lymnaea feeding network. The N2v --> B3 motor neuron excitatory synaptic response could be mimicked by focal or bath application of -glutamate at concentrations of >/=10(-3) M. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were potent agonists for the B3 excitatory glutamate receptor (10(-3) M), whereas kainate only produced very weak responses at the same concentration. This suggested that non-N-methyl--aspartate (NMDA), AMPA/quisqualate receptors were present on the B3 cell. The specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10(-5) M) blocked 85% of the excitatory effects on the B3 cell produced by focal application of glutamate (10(-3) M), confirming the presence of non-NMDA receptors. CNQX also blocked the major part of the excitatory postsynaptic potentials on the B3 cell produced by spontaneous or current-evoked bursts of spikes in the N2v cell. As with focal application of glutamate, a small delayed component remained that was CNQX insensitive. This provided direct evidence that glutamate acting via receptors of the non-NMDA, AMPA/quisqualate type were responsible for mediating the main N2v --> B3 cell excitatory response. NMDA at 10(-2) M also excited the B3 cell, but the effects were much more variable in size and absent in one-third of the 25 B3 cells tested. NMDA effects on B3 cells were not enhanced by bath application of glycine at 10(-4) M or reduction of Mg2+ concentration in the saline to zero, suggesting the absence of typical NMDA receptors. The variability of the B3 cell responses to NMDA suggested these receptors were unlikely to be the main receptor type involved with N2v --> B3 excitation. Quisqualate and AMPA at 10(-3) M also mimicked N2v inhibitory effects on the B7 and B8 feeding motor neurons and the modulatory slow oscillator (SO) interneuron, providing further evidence for the role of AMPA/quisqualate receptors. Similar effects were seen with glutamate at the same concentration. However, CNQX could not block either glutamate or N2v inhibitory postsynaptic responses on the B7, B8, or SO cells, suggesting a different glutamate receptor subtype for inhibitory responses compared with those responsible for N2v --> B3 excitation. We conclude that glutamate is a strong candidate transmitter for the N2v cells and that AMPA/quisquate receptors of different subtypes are likely to be responsible for the excitatory and inhibitory postsynaptic responses.     

NMDA and non-NMDA receptors mediate taste afferent inputs to cortical taste neurons in rats

Two main subclasses of ionotropic receptors for excitatory amino acids (EAAs), N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors, are involved in neurotransmission in the cortex of mammals. To examine whether EAAs are transmitters at the cortical taste area (CTA) in rats and to elucidate which types of the two ionotropic receptors operate at these synapses, we studied the effects of microiontophoretic administration of EAA antagonists on the responses of 64 taste cortical neurons to four basic taste stimuli in urethane-anesthetized rats. Both D-2-amino-5-phosphonovalerate (APV), a selective antagonist for NMDA receptors, and 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), a selective antagonist for non-NMDA receptors, suppressed most of the taste responses. The percentage of neurons suppressed by APV (70.3%) was almost the same as that suppressed by CNQX (64.1%). These suppressive effects were independent of the effects of background discharges during the prestimulus, water-rinsing period. The percentage of neurons suppressed by the antagonists did not differ between any pairs of taste stimuli. The number of neurons possessing both receptors was larger in the granular insular area (area GI), one of the two CTAs, than in the dysgranular insular area (area DI). In addition, taste responses were suppressed by CNQX or by both APV and CNQX in area GI in a significantly larger number of layer V neurons than in area DI. The present results indicate that normal excitatory transmission of taste afferents in the CTA in rats was mediated by both NMDA and non-NMDA receptors. The finding that a large fraction of neurons in the CTA in rats mediated taste information through NMDA receptors in normal transmission might be related to the higher potency of the plasticity observed in the CTA.     

Membrane currents evoked by ionotropic glutamate receptor agonists in rod bipolar cells in the rat retinal slice preparation

1. With the use of the whole cell voltage-clamp technique, I have recorded the current responses to ionotropic glutamate receptor agonists of rod bipolar cells in vertical slices of rat retina. Rod bipolar cells constitute a single population of cells and were visualized by infrared differential interference contrast video microscopy. They were targeted by the position of their cell bodies in the inner nuclear layer and, after recording, were visualized in their entirety by labeling with the fluorescent dye Lucifer yellow, which was included in the recording pipette. To study current-voltage relationships of evoked currents, voltage-gated potassium currents were blocked by including Cs+ and tetraethylammonium+ in the recording pipette. 2. Pressure application of the non-N-methyl-D-aspartate (non-NMDA) receptor agonists kainate and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) from puffer pipettes evoked a long-latency conductance increase selective for chloride ions. When the intracellular chloride concentration was increased, the reversal potential changed, corresponding to the change in equilibrium potential for chloride. The response was evoked in the presence of 5 mM Co2+ and nominally O mM Ca2+ in the extracellular solution, presumably blocking all external Ca2(+)-dependent release of neurotransmitter. 3. The long latency of kainate-evoked currents in bipolar cells contrasted with the short-latency currents evoked by gamma-aminobutyric acid (GABA) and glycine in rod bipolar cells and by kainate in amacrine cells. 4. Application of NMDA evoked no response in rod bipolar cells. 5. Coapplication of AMPA with cyclothiazide, a blocker of agonist-evoked desensitization of AMPA receptors, enhanced the conductance increase compared with application of AMPA alone. Coapplication of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the response to kainate and AMPA, indicating that the response was mediated by conventional ionotropic glutamate receptors. 6. The conductance increase evoked by non-NMDA receptor agonists could not be blocked by a combination of 100 microM picrotoxin and 10 microM strychnine. Application of the GABAC receptor antagonist 3-aminopropyl (methyl)phosphinic acid (3-APMPA) strongly reduced the response, and coapplication of 500 microM 3-APMPA and 100 microM picrotoxin completely blocked the response. These results suggested that the conductance increase evoked by non-NMDA receptor agonists was mediated by release of GABA and activation of GABAC receptors, and most likely also GABAA receptors, on rod bipolar cells. 7. Kainate responses like those described above could not be evoked in bipolar cells in which the axon had been cut somewhere along its passage to the inner plexiform layer during the slicing procedure. This suggests that the response was dependent on the integrity of the axon terminal in the inner plexiform layer, known to receive GABAergic synaptic input from amacrine cells. 8. The results indicate that ionotropic glutamate receptors are not involved in mediating synaptic input from photoreceptors to rod bipolar cells and that an unconventional mechanism of GABA release from amacrine cells might operate in the inner plexiform layer.     

NMDA and non-NMDA ionotropic glutamate receptors modulate striatal acetylcholine release via pre- and postsynaptic mechanisms

The effects of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on endogenous acetylcholine release from rat striatal slices and synaptosomes were investigated. Both agonists (1-300 microM) facilitated acetylcholine release from slices in a dose-dependent manner. NMDA (100-300 microM) and AMPA (30-300 microM), however, subsequently inhibited acetylcholine release. NMDA (100 microM)-induced facilitation was antagonized by 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and dizocilpine (both 1-10 microM), whereas the 10 microM AMPA effect was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1-30 microM). NMDA (100 microM)-induced inhibition was counteracted by CPP, but not dizocilpine, and by the nitric oxide synthase inhibitor L-nitroarginine (1-100 microM). Tetrodotoxin (0.5 microM) prevented the facilitatory effect of 3 microM NMDA and AMPA, but left unchanged that of 30 microM NMDA and 100 microM AMPA. Acetylcholine release from synaptosomes was stimulated by KCl (7.5-100 mM) in a dose-dependent manner. NMDA and AMPA maximally potentiated the 20 mM KCl effect at 1 microM and 0.01 microM, but were ineffective at 100 microM and 10 microM, respectively. Inhibition of acetylcholine release was never found in synaptosomes. The effects of 1 microM NMDA and 0.01 microM AMPA were antagonized by CPP (0.0001-1 microM) or dizocilpine (0.0001-10 microM) and by CNQX (0.001-1 microM), respectively. These data suggest that glutamatergic control of striatal acetylcholine release is mediated via both pre- and postsynaptic NMDA and non-NMDA ionotropic receptors.     

Excitatory amino acid receptors in glial progenitor cells: molecular and functional properties

We have analyzed the molecular and biophysical properties of glutamate-gated channels in cells of the oligodendrocyte lineage, using both the CG-4 primary cell line (Louis et al: J. Neurosci. Res. 31:193-204, 1992a) and oligodendrocyte progenitors purified from the rat cerebral cortex. CG-4 progenitor cells, as well as primary progenitors, were stained with a specific anti-GABA antibody. In whole-cell patch-clamp recordings, rapid perfusion of the agonists L-glutamate, kainate, and AMPA produced rapidly desensitizing currents in CG-4 cells. NMDA was ineffective. Both rapidly desensitizing and steady-state components of responses to kainate were inhibited by the kainate/AMPA receptor antagonist CNQX. Northern blot analysis of total mRNA isolated from CG-4 cells revealed co-expression of both AMPA- and kainate-preferring glutamate receptor subunits. The activation of glutamate receptors in CG-4 cells caused a rapid and transient elevation of mRNAs for the immediate early gene NGFI-A.