NMDA and GABA B receptors are involved in controlling nematocyst discharge in hydra

The role of chemical neurotransmission in nematocyst discharge was investigated by stimulating the cnidocils of nematocysts in ablated tentacles of Hydra vulgaris with a piezoelectrically-driven glass probe, in the presence of selected neurotransmitters. Acetylcholine, dopamine, epinephrine, glycine, and serotonin (10^− 4, 10^− 6, 10^− 8 M) per se, did not alter stenotele and desmoneme discharge. γ-Amino-butyric acid (GABA) significantly increased desmoneme discharge when the cnidocil of another desmoneme in the same or adjacent battery cell complex was stimulated without affecting the discharge rates of the directly stimulated desmonemes or stenoteles. Baclofen (GABA_B agonist) mimicked the increase; its antagonist, phaclofen, counteracted it. GABA_A agonists and antagonists did not alter discharge rates. Glutamate caused a dose-dependent increase in the discharge rate of directly stimulated stenoteles; distant stenotele and desmoneme discharge rates were unaffected. Kainate, AMPA, and NMDA, per se, did not alter discharge rates. Co-administration of NMDA and kainate mimicked glutamate's effects. AMPA plus NMDA increased discharge rates. DAP-5 (NMDA antagonist) and CNQX, (kainate/AMPA antagonist) counteracted the increase. The findings suggest that metabotropic GABA is involved in recruiting desmonemes by disinhibiting those previously inhibited, and that the NMDA/kainate–AMPA mechanism regulating Ca⁺⁺ entry in higher neuroeffector systems is an early-evolved process, which, in hydra, modulates nematocyst discharge.     

N-Methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4- isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks

Glutamate, an excitatory amino acid, acts at several glutamate receptor subtypes. Recently, we reported that central administration of glutathione induced hypnosis under stressful conditions in neonatal chicks. Glutathione appears to bind to the N-methyl-d-aspartate (NMDA) receptor. To clarify the involvement of each glutamate receptor subtype during stressful conditions, intracerebroventricular (i.c.v.) injection of several glutamate receptor agonists was given to chicks under social separation stress. Glutamate dose-dependently induced a hypnotic effect. NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate are characterized as ionotropic glutamate receptors (iGluRs). Although NMDA also induced sleep-like behavior or sedative effects, the potency of NMDA was less than that of glutamate. AMPA tended to decrease distress vocalizations induced by acute stress and brought about a sedative effect. Kainate and (S)-3, 5-dehydroxyphenylglycine, which is a metabotropic glutamate receptor agonist, had no influence on chick behavior. Thus, it is suggested that the iGluRs, NMDA and AMPA, are important in inducing hypnosis and sedation under acute stress in chicks.     

Role of Glutamate Receptors and Glutamate Transporters in the Regulation of the Glutamate-Glutamine Cycle in the Awake Rat

In the present study we investigate the effects of a specific glutamate reuptake blocker, L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC), on extracellular concentrations of glutamine and glutamate in the striatum of the freely moving rat. Intracerebral infusions of PDC (1, 2 and 4 mM) produced a dose-related increase in extracellular concentrations of glutamate and a dose-related decrease in extracellular concentrations of glutamine. These increases in extracellular glutamate and decreases in extracellular glutamine were significantly correlated. To investigate the involvement of ionotropic glutamate receptors in the decreases of extracellular glutamine produced by PDC, N-methyl-D-aspartate (NMDA) receptor antagonist and -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist were used. Perfusion of the NMDA receptor antagonist blocked the decrease of extracellular glutamine but had no effect on the increase of extracellular glutamate, both produced by PDC. Perfusion of the AMPA/kainate receptor antagonist attenuated the increase of extracellular glutamate and not only blocked the decrease of extracellular glutamine but also produced a significant increase of extracellular glutamine. The results reported in this study suggest that both NMDA and AMPA/kainate glutamatergic receptors are involved in the regulation of extracellular glutamine.     

Presynaptic kainate and NMDA receptors are implicated in the modulation of GABA release from cortical and hippocampal nerve terminals

One of the pathways implicated in a fine-tuning control of synaptic transmission is activation of the receptors located at the presynaptic terminal. Here we investigated the intracellular events in rat brain cortical and hippocampal nerve terminals occurring under the activation of presynaptic glutamate receptors by exogenous glutamate and specific agonists of ionotropic receptors, NMDA and kainate. Involvement of synaptic vesicles in exocytotic process was assessed using [³H]GABA and pH-sensitive fluorescent dye acridine orange (AO). Glutamate as well as NMDA and kainate were revealed to induce [³H]GABA release that was not blocked by NO-711, a selective blocker of GABA transporters. AO-loaded nerve terminals responded to glutamate application by the development of a two-phase process. The first phase, a fluorescence transient completed in ∼1 min, was similar to the response to high K⁺. It was highly sensitive to extracellular Ca²⁺ and was decreased in the presence of the NMDA receptor antagonist, MK-801. The second phase, a long-lasting process, was absolutely dependent on extracellular Na⁺ and attenuated in the presence of CNQX, the kainate receptor antagonist. NMDA as well as kainate per se caused a rapid and abrupt neurosecretory process confirming that both glutamate receptors, NMDA and kainate, are involved in the control of neurotransmitter release. It could be suggested that at least two types ionotropic receptor are attributed to glutamate-induced two-phase process, which appears to reflect a rapid synchronous and a more prolonged asynchronous vesicle fusion.     

Immunocytochemical evidence for an NMDA1 receptor subunit in dissociated cells of<Emphasis Type="Italic"> Hydra vulgaris</Emphasis>

We have previously reported immunocytochemical, biochemical, behavioral, and electrophysiological evidence for glutamatergic transmission through (±)--amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)/kainate receptors in hydra. We now report specific localization of the N-Methyl-D-aspartic acid receptor subunit 1 (NMDAR1) in epithelial, nerve, nematocytes, and interstitial cells of hydra. Macerates of tentacle/hypostome pieces of Hydra vulgaris were prepared on agar-coated slides, fixed with buffered formaldehyde/glutaraldehyde, and fluorescently labeled with monoclonal antibodies against mammalian NMDAR1. Negative controls omitted primary antibody. Digital images were recorded and analyzed. Specific localized and intense labeling was found in ectodermal battery cells, other epithelial cells, nematocytes, interstitial cells, and sensory and ganglionic nerve cells, and in battery cells was associated with enclosed nematocytes and neurons. The labeling of myonemes was more diffuse and less intense. In nerve and sensory cells, punctate labeling was prominent on cell bodies. These results are consistent with our earlier evidence for glutamatergic neurotransmission and kainate/NMDA regulation of stenotele discharge. They support other behavioral and biochemical evidence for a D-serine-sensitive, strychnine-insensitive, glycine receptor in hydra and suggest that the glutamatergic AMPA/kainate-NMDA system is an early evolved, phylogenetically old, behavioral control mechanism.     

Non-NMDA excitatory amino acid receptors in a subcellular fraction enriched in cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl^–/Ca²⁺ independent [³H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl^– dependent/Ca²⁺ activated [³H]glutamate binding sites were more abundant and exhibited a single K _d in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 andtrans-ACPD inhibited [³H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere.Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [³H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [³H]AMPA binding. The density of the high affinity [³H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli. High-affinity [³H]kainate binding sites are more abundant in fraction G than in fraction T; the same, but with smaller differences, occurs for the distribution of the low affinity [³H]kainate binding sites. The density of the latter sites is close to that of the high affinity [³H]AMPA binding sites confirming the presence of quisqualate/kainate receptors on granule cells, as previously hypothesized (for review, see Gallo et al., 1990). Taken together, these results indicate a segregation of the glutamate binding sites types at specialized synapses or neuronal cell types in the cerebellar network.Abbreviations AMPA (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid - DL-AP4 dl-2-amino-4-phosphonobutyric acid - D-AP5 d-2-amino-5-phosphonovaleric acid - EAA excitatory amino acid - EGTA ethylene glycol-bis(-aminoethyle ether) N,N,N,N-tetracetic acid - NMDA N-methyl-D-aspartate - Quisqualate -[3,5-dioxo-1,2,4-oxadiazolidin-2-yl]-L-alanine - trans-ACPD trans-1-amino-cyclopentyl-1,3-dicarboxylic acid     

Differential Contribution of L-, N-, and P/Q-type Calcium Channels to [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> Changes Evoked by Kainate in Hippocampal Neurons

We investigated the contribution of L-, N- and P/Q-type Ca²⁺ channels to the [Ca²⁺]_i changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca²⁺ imaging. Selective Ca²⁺ channel blockers, namely nitrendipine, ω-Conotoxin GVIA (ω-GVIA) and ω-Agatoxin IVA (ω-AgaIVA) were used. The [Ca²⁺]_i changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-d-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca²⁺ channel blocker caused differential inhibitory effects on [Ca²⁺]_i responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by ω-GVIA or ω-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of ω-GVIA or ω-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q- type Ca²⁺ channels activated by stimulation of the AMPA/kainate receptors. Special issue article in honor of Dr. Ricardo Tapia.     

N-Methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks

Glutamate, an excitatory amino acid, acts at several glutamate receptor subtypes. Recently, we reported that central administration of glutathione induced hypnosis under stressful conditions in neonatal chicks. Glutathione appears to bind to the N-methyl-d-aspartate (NMDA) receptor. To clarify the involvement of each glutamate receptor subtype during stressful conditions, intracerebroventricular (i.c.v.) injection of several glutamate receptor agonists was given to chicks under social separation stress. Glutamate dose-dependently induced a hypnotic effect. NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate are characterized as ionotropic glutamate receptors (iGluRs). Although NMDA also induced sleep-like behavior or sedative effects, the potency of NMDA was less than that of glutamate. AMPA tended to decrease distress vocalizations induced by acute stress and brought about a sedative effect. Kainate and (S)-3, 5-dehydroxyphenylglycine, which is a metabotropic glutamate receptor agonist, had no influence on chick behavior. Thus, it is suggested that the iGluRs, NMDA and AMPA, are important in inducing hypnosis and sedation under acute stress in chicks.     

Early activation of Ca(2+)-permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons.

Calcium entry through Ca(2+)-permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca(2+)-indicator Calcium Green 1-AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca(2+)] in embryonic chick retina from day 6 (E6) onwards. This Ca(2+) increase is due to entry through AMPA-preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N-methyl-D-aspartic acid (NMDA) receptor antagonist AP5, the voltage-gated Ca(2+) channel blockers diltiazem or nifedipine, or by the substitution of Na+ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca(2+) influx through L-type voltage-gated Ca(2+) channels with diltiazem and nifedipine prevented the effect of 10-100 microM kainate but not that of 500 microM kainate. In addition, joro spider toxin-3, a blocker of Ca(2+)-conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells.     

Ionotropic glutamate receptors

In the brain, most fast excitatory synaptic transmission is mediated through L-glutamate acting on postsynaptic ionotropic glutamate receptors. These receptors are of two kinds—the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (non-NMDA) and theN-methyl-D-aspartate (NMDA) receptors, which are thought to be colocalized onto the same postsynaptic elements. This excitatory transmission can be modulated both upward and downward, long-term potentiation (LTP) and long-term depression (LTD), respectively. Whether the expression of LTP/LTD is pre-or postsynaptically located (or both) remains an enigma. This article will focus on what postsynaptic modifications of the ionotropic glutamate receptors may possibly underly long-term potentiation/depression. It will discuss the character of LTP/LTD with respect to the temporal characteristics and to the type of changes that appears in the non-NMDA and NMDA receptor-mediated synaptic currents, and what constraints these findings put on the possible expression mechanism(s) for LTP/LTD. It will be submitted that if a modification of the glutamate receptors does underly LTP/LTD, an increase/decrease in the number of functional receptors is the most plausible alternative. This change in receptor number will have to include a coordinated change of both the non-NMDA and the NMDA receptors.     

鸡视网膜谷氨酸受体和GABA受体在两栖类卵母细胞中的表达

本工作利用两栖类卵母细胞作为功能表达系统,对鸡视网膜中的谷氨酸受体和ＧＡＢＡ受体的类型和基本性质进行了研究。在注射鸡视网膜ｍＲＮＡ的卵母细胞上,谷氨酸受体有明显的表达。Ｌ－Ｇｌｕ及其类似物ＫＡ,ＡＭＰＡ,ＱＡ都毫无例外地能诱导卵母细胞产生快速平滑的去极化电流,而ＮＭＤＡ,Ｌ－ＡＰ４,ＡＣＰＤ以及天冬氨酸不能诱导明显的电流反应。并且ＡＭＰＡ,ＱＡ对ＫＡ反应存在一定的抑制作用,提示ＡＭＰＡ,ＱＡ可能与ＫＡ作用于同一受体。抑制性氨基酸ＧＡＢＡ的受体被证明大部分为ＧＡＢＡＡ亚型,但有小部分的ＧＡＢＡ反应不能为荷包牡丹碱（ｂｉｃｕｃｕｌｌｉｎｅ）所阻断。     

Long-Term GABA Treatment Elicits Supersensitivity of Quisqualate-Preferring Metabotropic Glutamate Receptor in Cultured Rat Cerebellar Neurons

Abstract: In primary cultures of rat cerebellar granule neurons, GABA treatment (50 μ M , 7 days) caused a withdrawal supersensitivity selective for the metabotropic glutamate receptors that mainly prefer l -glutamate, quisqua- late and, to a lesser extent, kainate. The withdrawal supersensitivity was absent when 10 μ M SR-95531 was coadministered with GABA during the treatment period, an event that suggests the GABA_A receptors primarily produced the GABA treatment effect. This was supported further by the inability of baclofen treatment to mimic completely the treatment effect of GABA. Withdrawal from 7 days of baclofen treatment only produced a slight increase in the metabotropic effect of l -glutamate and carbachol. In addition, in untreated neurons, baclofen had no acute effect, whereas GABA inhibited the effect of l -glutamate and carbachol. The inhibitory effect of GABA was reversed by SR-95531 and was absent in neurons treated with GABA. These observations suggest the involvement of GABA_A receptors and the apparent development of tolerance to GABA, respectively. Also, dependence on GABA may have occurred; the metabotropic effects of glutamate, kainate, and quisqualate were not altered in neurons maintained with GABA treatment.     

Early activation of Ca2+‐permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons

Calcium entry through Ca²⁺‐permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca²⁺‐indicator Calcium Green 1‐AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca²⁺] in embryonic chick retina from day 6 (E6) onwards. This Ca²⁺ increase is due to entry through AMPA‐preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N‐methyl‐D ‐aspartic acid (NMDA) receptor antagonist AP5, the voltage‐gated Ca²⁺ channel blockers diltiazem or nifedipine, or by the substitution of Na⁺ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca²⁺ influx through L‐type voltage‐gated Ca²⁺ channels with diltiazem and nifedipine prevented the effect of 10–100 μM kainate but not that of 500 μM kainate. In addition, joro spider toxin‐3, a blocker of Ca²⁺‐conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 200–211, 2001     

GABA and glutamate specifically induce contractions in the sponge <Emphasis Type="Italic">Tethya wilhelma</Emphasis>

Sponges (Porifera) are nerve- and muscleless. Nevertheless, they react to external stimuli in a coordinated way, by body contraction, oscule closure or stopping pumping activity. The underlying mechanisms are still unknown, but evidence has been found for chemical messenger-based systems. We used the sponge Tethya wilhelma to test the effect of γ-aminobutyric acid (GABA) and glutamate (l-Glu) on its contraction behaviour. Minimal activating concentrations were found to be 0.5 μM (GABA) and 50 μM (l-Glu), respectively. Taking maximum relative contraction speed and minimal relative projected body area as a measure of the sponge’s response, a comparison of the dose–response curves indicated a higher sensitivity of the contractile tissue for GABA than for l-Glu. The concentrations eliciting the same contractile response differ by about 100-fold more than the entire concentration range tested. In addition, desensitising effects and spasm-like reactions were observed. Presumably, a GABA/l-Glu metabotropic receptor-based system is involved in the regulation of contraction in T. wilhelma. We discuss a coordination system for sponges based on hypothetical chemical messenger pathways. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. K. Ellwanger and A. Eich contributed equally and designed and performed experiments, analysed data and revised the paper, M. Nickel designed the study and experiments, analysed data, prepared the figures, wrote and revised the paper.     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [³H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [³H]GABA release and the release evoked by a high K⁺ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [³H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [³H]GABA release. The presence of glycine and the absence of Mg²⁺ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K⁺ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [³H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [³H]GABA release but not the NMDA-stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

鲫鱼脑氨基酸类神经递质受体在两栖类卵母细胞中的表达

两栖类卵母细胞表达系统经注射鲫鱼脑ｍＲＮＡ后可表达多种神经递质受体和某些离子通道。本工作利用电压箝方法结合药理学手段对ＧＡＢＡ受体和谷氨酸离子型受体作了较详细的研究。结果表明,由ＧＡＢＡ诱发的电流反应中,约９０％由ＧＡＢＡＡ受体介导,乘余约１０％的成分对ＧＡＢＡＡ受体的专一性拮抗剂Ｂｉｃｕｃｕｌｌｉｎｅ不敏感,而ＧＡＢＡＢ受体的专一性激动剂Ｂａｃｌｏｆｅｎ不能引进电流反应,因此这部分受体特性与ＧＡ     

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg²⁺-containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca²⁺ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg²⁺ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.     

Modulation of adenosine-induced cAMP accumulation via metabotropic glutamate receptors in chick optic tectum

Changes on cyclic adenosine monophosphate (cAMP) levels in response to adenosine and glutamate and the subtype of glutamate receptors involved in this interaction were studied in slices of optic tectum from 3-day-old chicks. cAMP accumulation mediated by adenosine (100 M) was abolished by 8-phenyltheophylline (15 uM). Glutamate and the glutamatergic agonists kainate or trans-d,l-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) did not evoke cAMP accumulation. Glutamate blocked the adenosine response in a dose-dependent manner. At 100 M, glutamate did not inhibit the effect of adenosine. The 1 mM and 10 mM doses of glutamate inhibited adenosine-induced cAMP accumulation by 55% and 100%, respectively. When glutamatergic antagonists were used, this inhibitory effect was not affected by 200 M 6,7-dihydroxy-2,3,dinitroquinoxaline (DNQX), an ionotropic antagonist, and was partially antagonized by 1 mM (rs)-alpha-methyl-4-carboxyphenylglycine [(rs)M-CPG], a metabotropic, antagonist, while 1 mMl-2-amino-3-phosphonopropionate (l-AP3) alone, another metabotropic antagonist, presented the same inhibitory effect of glutamate. Kainate (10 mM) and trans-ACPD (100 M and 1 mM) partially blocked the adenosine response. This study indicates the involvement of metabotropic glutamate receptors in adenylate cyclase inhibition induced by glutamate and its agonists trans-ACPD and kainate.Abbreviations ADO adenosine - DNQX 6,7-dihydroxy-2,3-dinitro-quinoxaline - KA kainate - l-AP3 l-2-amino-3-phosphonopropionate - mGluRs metabotropic glutamate receptors - P-THEO 8-phenyltheophylline - (rs)M-CPG (rs)-alpha-methyl-4-carboxyphenyl-glycine - trans-ACPD trans-d,l-1-aminocyclopentane-1,3-dicarboxyho acid     

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain‐derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole‐cell patch‐clamp recordings, l ‐glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration‐response plots of differentiated NPCs yielded an EC₅₀ of 2.2 μM for glutamate and an EC₅₀ of 36 μM for NMDA. Glutamate‐induced currents were markedly inhibited by memantine in contrast to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) suggesting a higher density of functional NMDA than alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptors. NMDA‐evoked currents and calcium signals were blocked by the NR2B‐subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage‐gated calcium channels by verapamil abolished AMPA‐induced calcium responses but only partially reduced NMDA‐evoked transients suggesting the expression of calcium‐impermeable, GluR2‐containing AMPA receptors. Quantitative real‐time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 μM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase‐immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.     

GABAA- and AMPA-like receptors modulate the activity of an identified neuron within the central pattern generator of the pond snail Lymnaea stagnalis

To examine the neurochemistry underlying the firing of the RPeD1 neuron in the respiratory central pattern generator of the pond snail, Lymnaea stagnalis, we examined electrophysiologically and pharmacologically either “active” or “silent” preparations by intracellular recording and pharmacology. GABA inhibited electrical firing by hyperpolarizing RPeD1, while picrotoxin, an antagonist of GABA_A receptors, excited silent cells and reversed GABA-induced inhibition. Action potential activity was terminated by 1 mM glutamate (Glu) while silent cells were depolarized by the GluR agonists, AMPA, and NMDA. Kainate exerted a complex triphasic effect on membrane potential. However, only bath application of AMPA desensitized the firing. These data indicate that GABA inhibits RPeD1 via activation of GABA_A receptors, while Glu stimulates the neuron by activating AMPA-sensitive GluRs.