Kainate Currents Research Articles

Xenon modulates the GABA and glutamate responses at genuine synaptic levels in rat spinal neurons

Effects of xenon (Xe) on whole-cell currents induced by glutamate (Glu), its three ionotropic subtypes, and GABA, as well as on the fast synaptic glutamatergic and GABAergic transmissions, were studied in the mechanically dissociated “synapse bouton preparation” of rat spinal sacral dorsal commissural nucleus (SDCN) neurons. This technique evaluates pure single or multi-synapse responses from native functional nerve endings and enables us to quantify how Xe influences pre- and postsynaptic transmissions accurately. Effects of Xe on glutamate (Glu)-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, kainate (KA)- and N-methyl-d-aspartate (NMDA)- and GABAA receptor-mediated whole-cell currents were investigated by the conventional whole-cell patch configuration. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were measured as spontaneous (s) and evoked (e) EPSCs and IPSCs. Evoked synaptic currents were elicited by paired-pulse focal electric stimulation. Xe decreased Glu, AMPA, KA, and NMDA receptor-mediated whole-cell currents but did not change GABAA receptor-mediated whole-cell currents. Xe decreased the frequency and amplitude but did not affect the 1/e decay time of the glutamatergic sEPSCs. Xe decreased the frequency without affecting the amplitude and 1/e decay time of GABAergic sIPSCs. Xe decreased the amplitude and increased the failure rate (Rf) and paired-pulse ratio (PPR) without altering the 1/e decay time of both eEPSC and eIPSC, suggesting that Xe acts on the presynaptic side of the synapse. The presynaptic inhibition was greater in eEPSCs than in eIPSCs. We conclude that Xe decreases glutamatergic and GABAergic spontaneous and evoked transmissions at the presynaptic level. The glutamatergic presynaptic responses are the main target of anesthesia-induced neuronal responses. In contrast, GABAergic responses minimally contribute to Xe anesthesia.

Sevoflurane activates hippocampal CA3 kainate receptors (Gluk2) to induce hyperactivity during induction and recovery in a mouse model

BackgroundIn addition to general anaesthetic effects, sevoflurane can also induce hyperactive behaviours during induction and recovery, which may contribute to neurotoxicity; however, the mechanism of such effects is unclear. Volatile anaesthetics including isoflurane have been found to activate the kainate (GluK2) receptor. We developed a novel mouse model and further explored the involvement of kainate (GluK2) receptors in sevoflurane-induced hyperactivity. MethodsMaximal speed, mean speed, total movement distance and resting percentage of C57BL/6 mice were quantitatively measured using behavioural tracking software before and after sevoflurane anaesthesia. Age dependence of this model was also analysed and sevoflurane-induced hyperactivity was evaluated after intracerebral injection of the GluK2 receptor blocker NS-102. Neurones from the hippocampal CA3 region were used to undertake in vitro electrophysiological measurement of kainate currents and miniature excitatory postsynaptic potential (mEPSP). ResultsSevoflurane induced significant hyperactivities in mice under sevoflurane 1% anaesthesia and during the recovery period, characterized as increased movement speed and total distance. The hyperactivity was significantly increased in young mice compared with adults (P<0.01) and pre-injection of NS-102 significantly prevented this sevoflurane-induced hyperactivity. In electrophysiological experiments, sevoflurane significantly increased the frequency of mEPSP at low concentrations and evoked kainate currents at high concentrations. ConclusionsWe developed a behavioural model in mice that enabled characterization of sevoflurane-induced hyperactivity. The kainate (GluK2) receptor antagonist attenuated these sevoflurane-induced hyperactivities in vivo, suggesting that kainate receptors might be the underlying therapeutic targets for sevoflurane-induced hyperactivities in general anaesthesia.

Critical roles of Src family tyrosine kinases in excitatory neuronal differentiation of cultured embryonic stem cells

Embryonic stem (ES) cells have been tested for potential cell transplantation therapy for CNS disorders. Understanding their differentiation mechanism and identifying factors involved in driving excitatory and inhibitory neuron lineages should enhance the efficacy and efficiency of the cell transplantation therapy. We tested the hypothesis that selective expression of Src family tyrosine kinases is required for phenotype-specific differentiation and functional maturation of ES cell derived neurons. Cultured mouse pluripotent ES cells were treated with retinoic acid (RA) to induce neural differentiation. After RA induction, neurons derived from ES cells showed significant neurite growth, increased expression of Src, Fyn and Lck and an extension of Src kinase expression from cell body to neurite processes. ES cell derived neuron-like cells expressed neurofilament, synaptophysin, glutamate receptors, NMDA and kainate currents, became vulnerable to excitotoxicity and formed functional excitatory synapses. These developmental events were blocked or attenuated when cells were grown in the presence of Src family kinase inhibitor PP2. However, there was no change in the expression of GABAergic-specific protein GAD67 during PP2 treatment. Our data suggest that Src tyrosine kinases are involved in the terminal differentiation of excitatory neuronal phenotype during ES cell neural differentiation after RA induction.

Mechanism of Action of Galantamine onN-Methyl-d-Aspartate Receptors in Rat Cortical Neurons

Galantamine, a new Alzheimer's drug approved in the United States, is known to inhibit acetylcholinesterase and potentiate acetylcholine-induced currents in brain neurons. However, because both cholinergic and N-methyl-D-aspartate (NMDA) systems are down-regulated in the brain of Alzheimer's patients, we studied the effects of galantamine on NMDA receptors. NMDA-induced whole-cell currents were recorded from the rat multipolar cortical neurons in primary culture. NMDA currents recorded in Mg2+-free media without addition of glycine were reversibly potentiated by bath and U-tube applications of galantamine at 10 to 10,000 nM, showing a bell-shaped dose-response relationship. However, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate currents were not affected by galantamine. The maximum potentiation of NMDA currents to approximately 130% of the control was obtained at 1 microM galantamine. The potentiation was due to a shift of the NMDA dose-response curve in the direction of lower NMDA concentrations. Glycine at 1 to 3000 nM enhanced NMDA currents, and potentiation by 1 microM galantamine and 1 to 300 nM glycine was additive. The glycine site antagonist 7-chlorokynurenic acid did not prevent the galantamine action. These results suggested that galantamine did not interact with the glycine binding site. Experiments with various concentrations of Mg2+ indicated that galantamine did not affect the Mg2+ blocking site of the NMDA receptor. PKC was involved in galantamine potentiation of NMDA currents, but protein kinase A, Gi/Go proteins, and Gs proteins were not involved. Potentiation of the activity of NMDA receptors is deemed partially responsible for the improvement of cognition, learning, and memory in Alzheimer's patients.

Glutamate receptor subtypes in human retinal horizontal cells

Glutamate receptor currents were examined in horizontal cells from cultured human retina using whole-cell recording procedures. Horizontal cells possess both AMPA and kainate receptors and both produce significant sustained currents. The kainate-induced current did not show significant desensitization and was not enhanced by concanavalin A. The sustained AMPA current was smaller than the kainate current, but the difference was almost entirely due to pronounced desensitization. The horizontal cell AMPA current was enhanced by cyclothiazide but not by PEPA, indicating the presence of the flip receptor variant. GYKI-52466 blocked the AMPA response (IC50 = 5 microM against 100 microM AMPA) but also blocked the kainate response (IC50 = 45 microM against 100 microM kainate). The diversity of glutamate receptors in human horizontal cells suggests that synaptic input to these neurons may be multiplexed through both kainate and AMPA channels.

Kainate-triggered currents in Xenopus oocytes injected with chick retinal membrane fragments: effect of guanine nucleotides.

To electrophysiologically characterize alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors in chick retinal membrane fragments, incorporated into Xenopus oocytes by direct microinjection. A 6-day retinal membrane suspension was injected into Xenopus oocytes by use of an electronic nanoliter injector. Fifteen to 40 hours after injection, the oocytes were assayed for kainate-elicited inward currents, under voltage-clamp conditions (membrane potential held at -70 mV). The structural incorporation of the retinal membrane fragments into the oocyte membrane was verified by specific immunofluorescent staining. Chick retinal membrane fragments were efficiently grafted onto Xenopus oocytes after microinjection, with 22.9% +/- 7.6% of the oocyte membrane being stained with anti-chick retina antibody. Part of the retinal material was seen as patches of relatively uniform size (292.1 +/- 72.3 microm(2)). Bath-applied kainate induced dose-dependent (EC(50): 64 +/- 7 microM), nondesensitizing inward currents (15-90 nA) in the chimeric Xenopus oocytes. Sham-injected oocytes did not respond to kainate. Kainate-driven currents were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 1-(4-aminopropyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), but not by gamma-D-glutamylaminomethyl sulfonic acid (GAMS) or aminophosphonoheptanoate (AP7), suggesting the involvement of AMPA receptors in the observed responses. Guanine nucleotides (GNs) also blocked kainate currents in a concentration-dependent manner. An alternative oocyte microinjection technique to analyze the electrophysiological properties of glutamate receptors in chick retinal membranes is described. The results show the functional activity of putative AMPA-preferring receptors from chick retina and confirm, in the chick retinal model, the antagonistic behavior of guanine nucleotides toward glutamate receptors and their potential role as neuroprotective agents under excitotoxic conditions.

AMPA receptors on developing medial septum/diagonal band neurons are sensitive to early postnatal binge-like ethanol exposure

The impact of binge-like, early postnatal ethanol treatment on AMPA or kainate whole cell currents was examined in acutely isolated medial septum/diagonal band (MS/DB) neurons. AMPA (10 or 100 μM) current was inhibited by GYKI 52466, a selective AMPA receptor (AMPAR) antagonist, in all neurons isolated on postnatal day (PD) 5–8, PD 12–15 or PD 32–35. Cyclothiazide, a selective inhibitor of AMPAR desensitization, also effectively potentiated AMPA currents. This suggests that non-NMDA, ionotropic glutamate receptors on immature MS/DB neuron are predominantly AMPARs. Concentration-dependent kainate (10–1000 μM) application evoked nondesensitizing currents that exhibited an increase in the maximum response by the end of first postnatal month, consistent with developmental regulation of AMPAR function. Acute 3 s ethanol application (100 mM) consistently blunted AMPA- and kainate currents ∼20–30% across age groups. Inhibition was sustained during continuous ethanol superfusion lasting 10–12 min without evidence of acute tolerance. Repeated oral intubation of rat pups with ethanol (5.25 g/kg/day on PD 4–9), which models third trimester human binge drinking, resulted in peak blood ethanol levels of ∼350 mg/dl (measured 90 min after PD 6 dosing). AMPA or kainate currents were upregulated in neurons isolated on PD 32–35 by earlier ethanol intubation suggesting that binge-like intoxication augments developing AMPAR function. Despite this augmentation of AMPAR function, no significant changes were found in the sensitivity of AMPA currents to GYKI 52466, cyclothiazide or acute ethanol (100 mM) sensitivity or in the levels of GluR1/GluR2 subunit proteins from MS/DB tissue. These results indicate that non-NMDA ionotrophic glutamate receptors on immature MS/DB neurons, which are largely of the AMPAR subtype, are moderately sensitive to immediate inhibition by ethanol. Repeating this inhibition during early postnatal binge-like intoxication can augment normal development of AMPAR function.

Subunit-Dependent Modulation of Kainate Receptors by Extracellular Protons and Polyamines

Synaptic activity causes significant fluctuations in proton concentrations in the brain. Changes in pH can affect neuronal excitability by acting on ligand-gated channels, including those gated by glutamate. We show here a subunit-dependent regulation of native and recombinant kainate receptors by physiologically relevant proton concentrations. The effect of protons on kainate receptors is voltage-independent and subunit dependent, with GluR5(Q), GluR6(Q), GluR6(R), and GluR6(R)/KA2 receptors being inhibited and GluR6(R)/KA1 receptors being potentiated. Mutation of two acidic residues (E396 and E397) to neutral amino acids significantly reduces the proton sensitivity of the GluR6(Q) receptor, suggesting that these residues influence proton inhibition. The endogenous polyamine spermine potentiated GluR6(R) kainate currents in a pH-dependent manner, producing an acidic shift in the IC(50) for proton inhibition. Spermine potentiation of GluR6(R) is voltage independent, does not affect receptor desensitization, and only slightly shifts the agonist affinity of the receptor. These results suggest that, similar to its action on NMDA receptors, spermine potentiates kainate receptors by relieving proton inhibition of the receptor. Furthermore, they suggest that fluctuations in brain pH during both normal and pathological processes could regulate synaptic transmission and plasticity mediated by kainate receptors.

Kainate-induced excitotoxicity is dependent upon extracellular potassium concentrations that regulate the activity of AMPA/KA type glutamate receptors.

In addition to well-known N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, recent studies suggest that non-NMDA type ionotropic glutamate receptors are also important mediators of excitotoxic neuronal death, and that their functional expression can be regulated by the cellular environment. In this study, we used cerebellar granule cells (CGCs) in culture to investigate kainate (KA)-induced excitotoxicity. Although previous reports indicated that KA induces apoptosis of CGCs in culture, no KA-induced excitotoxic cell death was observed in CGCs treated with KA when cells were maintained in high potassium media (24 mm K+). In contrast, when mature CGCs were shifted into low potassium media (3 mm K+), KA produced significant excitotoxicity. In electrophysiological studies, the KA-induced inward current density was significantly elevated in CGCs shifted into low K+ media compared with those maintained in high K+ media. Non-desensitizing aspects of KA currents observed in this study suggest that these responses were mediated by AMPA rather than KA receptors. In immunofluorescence studies, the surface expression of GluR1 subunits increased when mature CGCs were shifted into a low K+ environment. This study suggests that KA-induced excitotoxicity in mature CGCs is dependent upon the extracellular potassium concentration, which modulates functional expression and excitability of AMPA/KA receptors.

Expression of human epileptic temporal lobe neurotransmitter receptors in Xenopus oocytes: An innovative approach to study epilepsy.

Poly(A(+)) RNA was extracted from the temporal lobe (TL) of medically intractable epileptic patients which underwent surgical TL resection. Injection of this mRNA into Xenopus oocytes led to the expression of ionotropic receptors for gamma-aminobutyric acid (GABA), kainate (KAI) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Membrane currents elicited by GABA inverted polarity at -15 mV, close to the oocyte's chloride equilibrium potential, were inhibited by bicuculline, and were potentiated by pentobarbital and flunitrazepam. These basic characteristics were also displayed by GABA currents elicited in oocytes injected with mRNAs isolated from human TL glioma (TLG) or from mouse TL. However, the GABA receptors expressed by the epileptic TL mRNA exhibited some unusual properties, consisting in a rapid current run-down after repetitive GABA applications and a large EC(50) (125 microM). AMPA alone evoked very small or nil currents, whereas KAI induced larger currents. Nevertheless, upon cyclothiazide treatment, AMPA elicited substantial currents that, like the KAI currents, were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Furthermore, the glutamate receptor 5 (GluR5) agonist, ATPA, failed to evoke an obvious current although both RT-PCR and Western blot analyses showed GluR5 expression in the epileptic TL. Oocytes injected with mouse TL or human TLG mRNAs generated KAI and AMPA currents similar to those evoked in oocytes injected with epileptic TL mRNA but, in contrast to these, the mouse TL and human TLG oocytes were also responsive to ATPA. Our findings are in accord with the concept that both a depression of GABA inhibition and a dysfunction of the KAI-receptor system maintain a high neuronal excitability that results in epileptic seizures.

Novel potent AMPA/kainate receptor antagonists: synthesis and anticonvulsant activity of a series of 2-[(4-alkylsemicarbazono)-(4-amino-phenyl)methyl]-4,5-methylenedioxyphenylacetic acid alkyl esters.

In this paper we describe the synthesis of a series of novel 2-[(4-alkylsemicarbazono)-(4-aminophenyl)-methyl]-4,5-methylenedioxyphenylacetic acid alkyl esters (10-19) carrying an alkylsemicarbazono moiety at a benzylic site. The influence of this group on the biological activity was evaluated by testing the corresponding derivatives 20-22 in which the 4-alkylsemicarbazono moiety was removed (compound 20) or its alkylureido portion shifted at position 1 (compounds 21-22). Furthermore, the involvement of the 4-aminobenzyl moiety in the anticonvulsant activity was evaluated by testing derivative 23. The anticonvulsant activity of all compounds was assayed against audiogenic seizures induced in DBA/2 mice. Within this series of derivatives, 2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4,5-methylenedioxyphenylacetic acid methyl ester (10) proved to be the most active compound. It displayed a potency 5-fold higher than that shown by 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptor antagonist. Compound 10 was also effective in suppressing seizures induced in Swiss mice by maximal electroshock (MES) or pentylenetetrazole (PTZ). Furthermore, it antagonized in vivo seizures induced by icv administration of AMPA or kainate (KA). Using the patch-clamp technique in primary cultures of granule neurons we tested compounds 10 and 21 for their ability to modulate currents evoked by KA and 2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA). These two derivatives reduced KA and ATPA currents to a larger extent than that shown by reference compound 1. Compounds 10 and 21 were also able to reduce neuronal cell death induced by the application of KA (100 microM).

Developmental profiles of glutamate receptors and synaptic transmission at a single synapse in the mouse auditory brainstem.

Using whole-cell recordings from presynaptic terminals and postsynaptic principal neurons in the mouse medial nucleus of the trapezoid body (MNTB), we have characterized properties of the calyx of Held synapse during the first three postnatal weeks. We observed that evoked excitatory postsynaptic currents (EPSCs) mediated by NMDA receptors (NMDAR) increased until postnatal day 11/12 (P11/12) after which they declined to very low or undetectable levels at P16. Meanwhile, EPSCs mediated by AMPA receptors (AMPAR) showed an approximate three-fold increase in amplitude. These changes were paralleled by NMDAR and AMPAR currents evoked by exogenous NMDA and kainate to MNTB neurons except that whole-cell kainate currents remained constant after P7/8 while AMPAR-EPSCs continued to increase. We found that the decay time constant tau for NMDAR-EPSCs and AMPAR-EPSCs declined by about 30 % and 70 %, respectively. Analyses of NMDAR-EPSCs with subunit-specific pharmacological agents including ifenprodil, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), zinc and Mg(2+) revealed subtle developmental changes in subunit composition. As maturation progressed, this synapse displayed a reduction in the number of presynaptic spike failures and the extent of synaptic depression in response to trains of stimuli (50-300 Hz) while the recovery rate from depression accelerated. These results demonstrate profound changes in the size and kinetics of postsynaptic glutamate receptors and in the spike-firing capability of presynaptic terminals at the calyx of Held-MNTB synapse during early development. We suggest that these concurrent presynaptic and postsynaptic adaptations represent important steps for synapse consolidation and refinement and ultimately for the development of fast high-fidelity transmission at this synapse.

Acute Effects of Ethanol on Kainate Receptors in Cultured Hippocampal Neurons

Kainate receptors are a subclass of ionotropic glutamate receptors that regulate excitability and mediate synaptic transmission and plasticity in the hippocampus. The acute effects of ethanol on these receptors are not completely understood. The acute effects of ethanol on pharmacologically isolated kainate receptor-mediated currents were studied in cultured hippocampal neurons obtained from neonatal rats. Whole-cell patch-clamp electrophysiological techniques were used for these studies. LY303070 (GYKI-53784), a potent AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor-selective noncompetitive antagonist, was used to isolate kainate currents. Kainate receptor-mediated currents corresponded to 7% of the total non-N-methyl-D-aspartate (non-NMDA) currents in these neurons and were reduced to 24% of control values in the presence of 15 microM lanthanum. These kainate receptor-mediated currents were significantly inhibited by ethanol concentrations of 50 mM or more. Under our recording conditions, ethanol inhibited non-NMDA receptor- and NMDA receptor-mediated currents to a similar extent as kainate receptor-mediated currents. Western blot analysis indicated that glutamate receptor-5 and -6/7 subunits, and kainic acid-2 subunits are expressed in these cultured hippocampal neurons. The present results suggest that kainate receptors are important targets for the actions of ethanol in the central nervous system.

Cellular actions of topiramate: blockade of kainate-evoked inward currents in cultured hippocampal neurons.

This study was undertaken to evaluate the effects of topiramate (TPM) on excitatory amino acid-evoked currents. Kainate and N-methyl-D-aspartate (NMDA) were applied to cultured rat hippocampal neurons by using a concentration-clamp apparatus to selectively activate the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)/kainate and NMDA receptor subtypes, respectively. The evoked membrane currents were recorded by using perforated-patch whole-cell voltage-clamp techniques. TPM partially blocked kainate-evoked currents with an early-onset reversible phase (phase I) and a late-onset phase (phase II) that occurred after a 10- to 20-min delay and did not reverse during a 2-h washout period. Application of dibutyryl cyclic adenosine monophosphate (cAMP; 2 mM) during washout after phase II block enhanced reversal, with the kainate current amplitude being restored by approximately 50%. Phase II but not phase I block was prevented by prior application of okadaic acid (1 microM), a broad-spectrum phosphatase inhibitor, suggesting that phase II block may be mediated through interactions with intracellular intermediaries that alter the phosphorylation state of kainate-activated channels. Topiramate at 100 microM blocked kainate-evoked currents by 90% during phase II, but had no effect on NMDA-evoked currents. The median inhibitory concentration (IC50) values for phase I and II block of kainate currents were 1.6 and 4.8 microM, respectively, which are within the range of free serum levels of TPM in patients. The specific blockade of the kainate-induced excitatory conductance is consistent with the ability of TPM to reduce neuronal excitability and could contribute to the anticonvulsant efficacy of this drug.

The open channel blocking drug, IEM-1460, reveals functionally distinct α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors in rat brain neurons

The properties of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors were examined in various cell types isolated from young rat hippocampus, striatum and cerebellum using patch-clamp and fast application techniques. A dicationic adamantane derivative, IEM-1460, reversibly inhibited kainate-induced currents. In the presence of 100 μM IEM-1460, kainate currents in striatal giant cholinergic interneurons and hippocampal non-pyramidal neurons were inhibited by 95% and 81%, respectively, at V h=−70 mV. Striatal GABAergic principal cells, hippocampal pyramidal neurons and cerebellar Purkinje cells had low sensitivity to IEM-1460 (inhibition by 4–15%). Analysis of averaged data from the cell types studied revealed a highly significant positive correlation ( r=0.93, P<0.01) between percentage inhibition by 100 μM IEM-1460 and relative calcium permeability of AMPA receptors, P Ca/ P Na. Also, within each brain structure, the sensitivity of IEM-1460 block was lower the stronger the outward rectification of kainate currents. Some hippocampal neurons exhibited intermediate sensitivity to IEM-1460. Kainate currents were suppressed by 40% in the presence of 100 μM IEM-1460. Meanwhile, AMPA receptors in this cell type had low calcium permeability ( P Ca/ P Na=0.13) and demonstrated outwardly rectifying kainate currents. The interrelation of different properties of AMPA receptors considering their assembly is discussed. The data obtained suggest that IEM-1460 may be a convenient and promising marker of native AMPA receptor assembly: it selectively inhibits Ca 2+-permeable, GluR2-lacking AMPA receptors.

Ethanol inhibition of synaptically evoked kainate responses in rat hippocampal CA3 pyramidal neurons.

Many studies have demonstrated that intoxicating concentrations of ethanol (10-100 mM) can selectively inhibit the component of glutamatergic synaptic transmission mediated by N-methyl-D-aspartate (NMDA) receptors while having little or no effect on excitatory synaptic transmission mediated by non-NMDA receptors [i.e., alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and/or kainate (KA) receptors]. However, until the recent development of highly selective AMPA receptor antagonists, it was not possible to assess the relative contribution of AMPA and KA receptors to non-NMDA receptor-mediated synaptic transmission or to determine whether these glutamate receptor subtypes differed in their sensitivity to ethanol. In the present experiments, we used the highly selective AMPA receptor antagonist LY 303070 to pharmacologically isolate KA receptor-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal CA3 pyramidal neurons and tested their sensitivity to ethanol. Concentrations of ethanol as low as 20 mM significantly and reversibly depressed KA EPSCs. Ethanol also inhibited KA currents evoked by direct pressure application of KA in the presence of LY 303070, suggesting that this inhibition was mediated by a postsynaptic action. In contrast, ethanol had no effect on AMPA EPSCs in these cells, even at the highest concentration tested (80 mM). Ethanol significantly inhibited NMDA EPSCs in these neurons, but these responses were less sensitive to ethanol than KA EPSCs. These results suggest that in addition to its well-described depressant effect on NMDA receptor-mediated synaptic transmission, ethanol has an even greater inhibitory effect on glutamatergic synaptic transmission mediated by KA receptors in rat hippocampal CA3 pyramidal neurons.

Activation and Desensitization of Hippocampal Kainate Receptors

We have used whole-cell recordings and rapid agonist applications to characterize the physiological properties of kainate receptors expressed by rat hippocampal neurons in dissociated cell culture. Activation of NMDA and AMPA receptors was prevented by inclusion of the noncompetitive antagonists MK-801 (2 microM) and GYKI 53655 (100 microM), respectively. In the presence of these inhibitors, both kainate (EC50 = 23 microM) and glutamate (EC50 = 310 microM) evoked desensitizing currents. Maximal peak currents for kainate with GYKI 53655 were 15 +/- 3% as large as in control solutions without GYKI. In contrast to currents mediated by AMPA receptors, kainate currents recorded in GYKI were blocked potently by lanthanum (IC50 = 2 microM) and were desensitized by 1 microM 2S,4R-4-methylglutamate (SYM 2081). Coapplication of either 5 microM AMPA or 500 microM aspartate had little effect on responses to kainate, although AMPA alone elicited current at 1 mM. In most cells, the currents evoked by kainate, glutamate, and SYM 2081 varied linearly with membrane potential and reversed near 0 mV. Kainate elicited substantial current at steady state (approximately 30% of peak), whereas responses to glutamate and SYM 2081 desensitized almost completely within 0.2-2 sec. Inhibition produced by a 10 sec desensitizing prepulse was half-maximal at 0.22 microM for SYM 2081 and 13 microM for glutamate. Recovery from desensitization to kainate and glutamate was >80% complete within 60 sec but was three- to fourfold slower after exposure to SYM 2081. Exposure to Concanavalin A blocked desensitization of the currents but also reduced the peak current amplitudes. Collectively, these results confirm that kainate-preferring receptors underlie the currents evoked by kainate, glutamate, or SYM-2081 in the presence of GYKI 53655; they are not mediated by electrogenic transport or by AMPA-preferring receptors that are insensitive to GYKI. In contrast to previous work on embryonic hippocampal neurons, our results show that the properties of kainate receptors expressed by cells from older animals are distinct from those displayed by homomeric assemblies of the GluR6 subunit.

Regulation of voltage- and ligand-gated currents in rat hippocampal progenitor cells in vitro.

Growth medium and substrate regulate the proliferation and differentiation of neural progenitor cells in vitro and the expression of cell type-specific histochemical markers. An important question is whether neural progenitor cells exhibit voltage- and ligand-gated currents, features characteristic of neurons, and whether these currents are regulated differentially by growth conditions. Another issue of interest is whether passaged progenitor cells, after expansion with basic fibroblast growth factor (FGF-2), exhibit the same degree of plasticity as their primary counterparts, or whether they are more committed to a particular phenotype. In primary cultures of embryonic rat hippocampal progenitor cells, growth in proliferative conditions (FGF-2) was associated with low levels of sodium, calcium, N-methyl-D-aspartate (NMDA), and kainate currents compared with other growth conditions. After multiple passages in the continued presence of FGF-2, sodium, calcium, and NMDA, responses declined further; interestingly, kainate and gamma-aminobutyric acid (GABA) responses remained substantial. Moreover, the expression of functional channels and receptors in primary cultures of progenitor cells is up-regulated strongly by growth factors such as BDNF, and NT-3, whereas sodium and calcium currents in passaged cultures respond to such growth conditions to a lesser extent. Kainate and GABA responses were present to a significant extent in passaged cultures, independent of growth condition. We conclude that environmental cues regulate different channels and receptors in distinct ways in neural progenitor cells.

Nitric oxide and cGMP modulate retinal glutamate receptors.

1. In the retina, as in other regions of the vertebrate central nervous system, glutamate receptors mediate excitatory chemical synaptic transmission and are a critical site for the regulation of cellular communication. In this study, retinal horizontal cells from the hybrid less were dissociated in cell culture, voltage clamped by the whole cell recording technique, and the currents evoked by application of excitatory amino acids recorded. 2. Responses to glutamate and its agonist kainate were reduced by approximately 50% in the presence of the nitric oxide (NO) donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine. The effect of these compounds was blocked by the NO scavenger hemoglobin. 3. This effect of NO donors on kainate currents could be mimicked by the application of a membrane permeable guanosine 3',5'-cyclic monophosphate (cGMP) analogue, 8-Br-cGMP. The NO effect was also blocked by application of the guanylate cyclase inhibitor LY-83583, and by a protein kinase G inhibitor peptide. 4. In H1-type horizontal cells, stimulation of endogenous nitric oxide synthase with L-arginine reduced kainate responses, whereas application of D-arginine had no effect. 5. This receptor modulation mechanism may act in concert with other pre- and postsynaptic mechanisms to modify horizontal cell synaptic function according to the adaptational state of the retina and also may protect horizontal cells from glutamate excitotoxicity.

Characterization of YM90K, a selective and potent antagonist of AMPA receptors, in rat cortical mRNA-injected Xenopus oocytes

The inhibitory potencies of 6-(1 H-imidazol-1-yl)-7-nitro-2,3(1 H,4 H)-quinoxalinedione hydrochloride (YM90K), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo( F)quinoxaline (NBQX) and 1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5 H-2,3-benzodiazepine (GYKI 52466) at excitatory amino acid receptors were examined in rat cortical mRNA-injected Xenopus oocytes using a two-electrode voltage clamp. Schild analysis of YM90K and NBQX inhibition of kainate currents yielded pA 2 values of 6.83 ± 0.01 and 7.24 ± 0.01, respectively. GYKI 52466 reduced the maximum kainate response and increased the kainate EC 50 in a dose-dependent manner, suggesting that the antagonism of AMPA receptors by GYKI 52466 is mixed competitive and non-competitive for kainate. Schild analysis of YM90K and NBQX inhibition of kainate currents in the presence of 30 μM cyclothiazide yielded pA 2 values of 6.62 ± 0.03 (slope: 1.02 ± 0.01) and 7.10 ± 0.02 (slope: 1.00 ± 0.02), respectively, consistent with competitive antagonism. Cyclothiazide potentiated the AMPA response as well as the kainate response and increased the apparent Hill coefficients in a concentration-dependent manner. The potency of YM90K to inhibit AMPA-induced currents could be reduced by increasing the concentration of cyclothiazide. We showed that YM90K is a potent and competitive antagonist for AMPA receptors and the apparent affinity of competitive antagonists was reduced by cyclothiazide. Cyclothiazide can affect the interaction between receptors and both agonists and antagonists, suggesting that it might allosterically alter the affinity of agonists and competitive antagonists for their binding site on the AMPA receptor complex.