Crocin antagonizes ethanol inhibition of NMDA receptor-mediated responses in rat hippocampal neurons

We have previously found that crocin (crocetin di-gentiobiose ester) antagonizes the inhibitory effect of ethanol on long-term potentiation in the rat hippocampus in vivo and in vitro. To explore mechanisms underlying the antagonism of crocin against ethanol, we investigated the effects of ethanol and crocin on synaptic potentials mediated by N-methyl-d-aspartate (NMDA) receptors in the dentate gyrus of rat hippocampal slices. Synaptic potential mediated by non-NMDA receptors was recorded in normal medium (1.3 mM Mg²⁺), while NMDA receptor-mediated synaptic potential was isolated in low (0.13 mM) Mg²⁺ medium containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 μM). Crocin (10 μM) alone did not affect synaptic potentials mediated by non-NMDA nor NMDA receptors. Non-NMDA response was slightly inhibited by 100 mM ethanol, while NMDA response was selectively inhibited by lower concentrations (10–50 mM) of ethanol. Crocin (10 μM) did not affect the inhibition of non-NMDA response by 100 mM ethanol, but significantly blocked the inhibition of NMDA response by 10–50 mM ethanol. In addition, we performed whole-cell patch recording with primary cultured rat hippocampal neurons, and confirmed that crocin blocked ethanol inhibition of inward currents evoked by application of NMDA. These results suggest that crocin specifically antagonizes the inhibitory effect of ethanol on NMDA receptor-mediated responses in hippocampal neurons.     

Mg dependence of membrane resistance increases evoked by NMDA in hippocampal neurones

The response of granule cells and CA1 pyramidal neurones to NMDA was studied in the presence of and absence of Mg²⁺ using an in vitro slice preparation. In the absence of Mg²⁺ the depolarizing response of hippocampal neurones to NMDA is accompanied by a decrease in input resistance. In the presence of Mg²⁺ ions, however, the response to NMDA is always associated with an apparent increase in input resistance. These results indicate that the action of NMDA is by a classical mechanism of conductance increase and are in agreement with the suggestion that the apparent increase in input resistance associated with NMDA depolarizations is the result of voltage-dependent channel block by Mg²⁺ of the NMDA evoked current.     

Postsynaptic blockade of inhibitory postsynaptic currents by plasmin in CA1 pyramidal cells of rat hippocampus

We have shown previously that plasmin facilitated the generation of long-term potentiation (LTP) in CA1 and dentate region of rat hippocampus. In the present study, we investigated the effects of plasmin on postsynaptic currents in CA1 pyramidal neurons of rat hippocampal slices. Plasmin (100 nM) had no effect on NMDA nor on non-NMDA receptor-mediated excitatory postsynaptic currents. However, plasmin significantly decreased GABA_A receptor-mediated inhibitory postsynaptic currents. This effect of plasmin disappeared when intracellular Ca²⁺ was strongly chelated with BAPTA. Furthermore, plasmin attenuated the GABA-induced currents in CA1 pyramidal cells. These results suggest that the STP-enhancing effect of plasmin is due to a blockade of postsynaptic GABA_A responses and that an increase in intracellular Ca²⁺ by plasmin may be involved in its mechanism.     

NMDA receptor-mediated synaptic excitation selectively inhibited by ethanol in hippocampal slice from adult rat

The effect of ethanol (EtOH) on synaptic transmission mediated by N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors was investigated in slices from adult rat hippocampus. Synaptic responses were elicited by stimulation of stratum radiatum and were recorded in CA1 stratum radiatum or stratum pyramidale. Population EPSPs (pEPSPs) mediated by NMDA receptor activation were isolated by application of a solution containing the kainate/quisqualate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione and either low (0.1 mM) Mg2+ or 100 microM bicuculline. Increasing concentrations of EtOH produced increasing inhibition of NMDA receptor-mediated pEPSPs with EtOH concentrations between 1 and 50 mM. At a concentration of 50 mM, EtOH inhibited NMDA receptor-mediated pEPSPS by 43%; the inhibition by 100 mM EtOH was not significantly different from that produced by 50 mM. Methanol and 1-butanol also inhibited the NMDA receptor-mediated pEPSPs; the potency of the alcohols for inhibition of NMDA receptor-mediated pEPSPs was 1-butanol greater than ethanol greater than methanol. pEPSPs mediated by non-NMDA glutamate receptors were isolated by the application of the NMDA receptor antagonist d,1-2-amino-5-phosphonovaleric acid in the presence of 1.5 mM Mg2+. These pEPSPs were not significantly affected by 50 mM EtOH, whereas 100 mM EtOH reduced the amplitude of these pEPSPs by 9%. The observations indicate that synaptic excitation mediated by NMDA receptors in tissue from adult rat is inhibited by intoxicating concentrations of EtOH. The data are consistent with the hypothesis that EtOH-induced inhibition of EPSPs mediated NMDA receptors may contribute to the intoxicating effects of EtOH.     

Evidence forN-methyl-d-aspartic acid receptor-mediated modulation of the commissural input to central vestibular neurons of the frog

We have investigated the role ofN-methyl-d-asparte (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg²⁺ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-appliedd-2-amino-5-phosphonovaleric acid (D-APV, 25–50 μM), a selective NMDA antagonist. In a low Mg²⁺ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and had a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of the latter was reduced by D-APV (25–50 μM) to about 65% of the control value in the presence of 1 mM Mg²⁺. During bath application of NMDA (100 μM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg²⁺ concentration. Bath application of NMDA (0.1–1 μM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were stimulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these stimulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.     

Cytotoxic actions and effects on intracellular Ca2+ and cGMP concentrations of sulphur-containing excitatory amino acids in cultured cerebral cortical neurons

Effects of the sulphur-containing acidic amino acids (SAAs) cysteic acid (CA), homocysteic acid (HCA), cysteine sulphinic acid (CSA), homocysteine sulphinic acid (HCSA), and S-sulphocysteine (SC) on intracellular concentrations of Ca²⁺ ([Ca²⁺]_i) and cGMP ([cGMP]_i) as well as their cytotoxic actions were investigated in cultured cerebral cortical neurons. The glutamate receptor subtype selective antagonists APV (D-(?)-2-amino-5-phosphonopentanoate) acting on N-methyl-D-aspartate (NMDA) receptors and DNQX (6,7-dinitroquinoxaline-2,3-dione) acting on non-NMDA receptors were employed to obtain information about the involvement of glutamate receptor subtypes in these actions of the SAAs. It was found that all SAAs exerted a cytotoxic action on the neurons. The ED₅₀ values for CSA, CA, HCSA, and HCA were around 30 to 50 μM and that for SC was about 150 μM. The glutamate transport blocker L-aspartate-β-hydroxamate increased the efficacy of CSA and CA but had no effect on the cytotoxic actions of the remaining SAAs. In case of CA, HCA, and SC the cytotoxicity could be prevented by APV alone and for HCSA, DNQX could block the toxic action. DNQX reduced the toxicity of HCA somewhat but the presence of APV was required for complete protection. CSA toxicity could only be blocked by the combination of APV and DNQX. All SAAs induced an increase in [cGMP]_i and [Ca²⁺]_i and with regard to [Ca²⁺]_i SC was the most potent and CA the least potent SAA. The effect of all SAAs on [cGMP]_i could be blocked by APV alone whereas DNQX had no effect except in the case of HCSA where the response was blocked completely and HCA where the response was inhibited by 75%. The SAA-induced increase in [Ca²⁺]_i could in all cases be significantly reduced by 0.6 mM Mg²⁺ and in the presence of Mg²⁺, APV dose dependently blocked the remaining SAA induced increase in [Ca²⁺]_i completely. Under these conditions DNQX was also found to block the SAA-induced increase in [Ca²⁺]_i dose dependently. In the absence of Mg²⁺, DNQX (25 μM) inhibited the response of the SAAs only by 65–75%. Under these conditions all SAA responses except that to SC could be fully antagonized by 300 μM APV. The SC-induced increase in [Ca²⁺]_i was inhibited by 60% by APV. The results show that no simple correlation exists between SAA-induced cytotoxicity and their ability to increase intracellular levels of Ca²⁺ and cGMP. However, when both NMDA and non-NMDA receptors were antagonized no toxicity or changes in calcium or cGMP were observed. © 1993 Wiley-Liss, Inc.     

Developmental increase in the sensitivity to magnesium of NMDA receptors on CA1 hippocampal pyramidal cells

The N-methyl-D-aspartate (NMDA) receptor is involved in processes, such as associative learning, that are particularly important during early postnatal development. It has been suggested that the activity and regulation of this receptor changes during development. Activation of the NMDA receptor is normally limited by Mg2+ present in the extracellular fluid of brain. We have found that Mg2+ less potently antagonizes the depolarizing action of NMDA in developing rats than in adults. A grease-gap method was used to record depolarizations evoked in CA1 hippocampal pyramidal cells by the excitants NMDA and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate). In the adult CA1 area, Mg2+ shifted the NMDA concentration-response curve to the right in a manner consistent with voltage-dependent open channel block (uncompetitive antagonism) in a preparation with significant receptor reserve. The potency of Mg2+ increased during development; a greater than two-fold change in the EC50 for Mg2+ was observed between 10-15 days of age and adulthood. A concentration of 10 mM reduced the maximum response of CA1 pyramidal cells to NMDA in adult rats, but not in developing rats. In addition, Mg2+ often enhanced the maximum depolarizations evoked by NMDA in 10- to 15-day-old rats, but very seldom in adults. No significant developmental changes in AMPA-induced depolarizations were observed in the presence or absence of Mg2+. These results suggest that synaptically released glutamate will readily activate NMDA receptors during early development and that its ability to do this declines with the maturation of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

NBQX is a selective non-NMDA receptor antagonist in rat hippocampal slice°

The effects of NBQX and DNQX on synaptic transmission in rat hippocampal slice were investigated. Both agents produced dosedependent blockade of field potentials evoked by low frequency stimulation of Schaffer collateral-commissural fibers recorded in medium containing 4 mM Mg²⁺ (non-NMDA. mediated transmission), with half-maximal effects at about 0.15μM for NBQX and 1.0μM for DNQX. When the studies were conducted in Mg²⁺-free medium (predominantly NMDA mediated transmission), 100 μM NBQX failed to block transmission; however, the response could be completely blocked by the addition of 10μM of the competitive NMDA antagonist CPP. In contrast, 47 μM DNQX completely blocked secondary field potentials recorded in Mg²⁺-free medium and this effect could be reversed by the addition of 200 μM of the glycine agonist D-serine. Thus, NBQX exhibited selective blockade of non-NMDA mediated synaptic transmission whereas DNQX had effects at both non-NMDA and NMDA receptor sites, the latter effect via an interaction with the glycine site on the NMDA receptor complex.     

Extracellular potassium, glial and neuronal potentials in the solitary complex of rat brainstem slices

Extracellular K⁺ activities (aK_e) and neuronal and glial membrane potentials were recorded in the nucleus tractus solitarius (NTS) and in the dorsal vagal motor nucleus (DVMN) of rat brainstem slices after orthodromic stimulation of the tractus solitarius (TS). In glial cells, repetitive stimulation of the TS induced depolarizations of up to 30 mV followed by repolarizations which were fitted by exponential curves with a time constant of 1.6–5 s. Similar stimulations induced elevations ofaK_e of up to 8 mM, the recovery of which was fitted by single exponential curves with a time constant ranging between 1.6 and 4 s. These elevations inaK_e were reduced by 75% during blockage of synaptic transmission in low Ca²⁺, high Mg²⁺ solution, and by 24% with application of 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 50 μM). Perfusion with a low Mg²⁺ solution increased theaK_e response to stimulation of the TS, an effect that was reduced by the addition of 2-amino-5-phosphono-valeric acid (AP7, 50 μM) to the bath. No significant change inaK_e and glial potential was seen when superfusing high concentrations of the C-terminal octapeptide of cholecystokinin (CCK₈, 1–5 μM) and C-terminal tetrapeptide (CCK₄, 50–100 μM). The effect of TS stimulations on solitary complex neurons suggests that extracellular K⁺ concentration is increased during synaptic activation of non-NMDA or NMDA ionotropic receptors. Conversely, slow depolarizations elicited by repetitive afferent activity or excitation by CCK agonists develop in neurons in the absence of measurable extracellular K⁺ fluctuations or glial depolarization.     

A new type of Ca channel blocker, NC-1100, inhibits the low- and high-threshold Ca currents in the rat CNS neurons

The effect of a new type of organic Ca²⁺ channel blocker, NC-1100 [(±)-1-(3,4-dimethoxyphenyl)-2-(4-diphenylmethylpiperazinyl)ethanol dihydrochloride], on both low- and high-threshold Ca²⁺ currents was studied in the whole-cell mode of the pyramidal neurons freshly dissociated from rat hippocampal CA1 region under voltage-clamp condition. The NC-1100 reversibly reduced the high-threshold Ca²⁺ current (HVAI_Ca) in a concentration-dependent manner without affecting the current-voltage relationship. The values of half-inhibition (IC₅₀) were 1.3 × 10⁻⁵ and 9.1 × 10⁻⁶M in external solution containing 10 and 2.5 mM Ca²⁺, respectively. The NC-1100 also decreased the low-threshold Ca²⁺ current (LVAI_Ca) in a concentration-dependent manner. The inhibitory potency was augmented by increasing the stimulation frequency and / or decreasing the extracellular Ca²⁺ concentration to a physiological range (2.5 mM). The IC₅₀ value decreased to 7.7 × 10⁻⁷M in external solution containing 2.5 mM Ca²⁺ at a stimulation frequency of 1 Hz. The NC-1100 delayed the reactivation of LVA Ca²⁺ channel and enhanced voltage-dependently the steady-state inactivation, suggesting that this drug bound not only the resting LVA Ca²⁺ channel but also the inactivated one.     

Single-channel and whole-cell analysis of ethanol inhibition of NMDA-activated currents in cultured mouse cortical and hippocampal neurons

The effects of 0.1 to 500 mM ethanol on NMDA-activated currents were studied in primary cultures of mouse cortical and hippocampal neurons. In whole-cell recordings the IC₅₀S for inhibition of NMDA-activated currents by ethanol were 129 mM ± 20 mM in hippocampal neurons and 126 ± 18 mM in cortical neurons. In single-channel recordings from excised outside-out patches of cortical neurons, ethanol inhibited total charge per minute with an IC₅₀ of 174 ± 23 mM, which was not significantly different from the IC₅₀S for inhibition of whole-cell current. The reduction in mean open channel lifetime by ethanol was fit by the logistic equation with an apparent IC₅₀ of 340 ± 28 mM. Analysis of single-channel data indicated that ethanol inhibition of NMDA currents did not involve substantial changes in fast closed state kinetics, changes in open channel conductance, or block of the open channel. At the whole-cell IC₅₀, of ethanol, mean open channel lifetime would decrease by 28% and frequency of opening would decline by 31% to account for the reduction in current. Single-channel data were consistent with ethanol being an allosteric modulator of gating which reduces agonist efficacy.     

A quantitative description of excitatory amino acid neurotransmitter responses on cultured embryonic Xenopus spinal neurons

We have performed a quantitative analysis of excitatory amino acid neurotransmitter receptors on cultured embryonic Xenopus spinal neurons using the whole-cell patch-clamp technique. Neuroblasts and underlying mesodermal cells isolated from spinal regions of neural plate-stage embryos were placed into dissociated cell culture, and responses were studied soon after the appearance of neurites on embryonic neurons. Glutamate (Glu) receptors were separated into two general classes based on responses to the characteristic agonists quisqualate (Quis), kainate (Ka) and N-methyl-d-aspartate (NMDA); these were NMDA receptors (those activated by NMDA) and non-NMDA receptors (those activated by Ka and Quis). Half-maximal responses to Glu and other agonists on NMDA and non-NMDA receptors were determined from Hill analysis of dose response relations. The order of sensitivities observed was: Glu_NMDA(ED₅₀ = 5.1 μM) >Glu_non-NMDA(ED₅₀ = 28 μM), and for Glu receptor agonists, Quis (ED₅₀ = 1.5 μM) >NMDA(ED₅₀ = 41 μM) >Ka(ED₅₀ = 58 μM). The order of response amplitudes recorded at concentrations near the appropriate ED₅₀s was Glu_NMDA > Glu_non-NMDA, and Ka > NMDA > Quis. A 10-fold decrease in external [Na⁺] shifted the reversal potentials for Glu_non-NMDA, Ka, and Quis to more negative voltages. Increasing external [Ca²⁺] shifted the reversal potential for NMDA responses to more positive potentials, an observation consistent with Ca²⁺ permeation of the embryonic NMDA-activated channel. NMDA-evoked currents could not be recorded in nominally glycine (Gly)-free media. Addition of Gly to external solutions potentiated NMDA responses (ED₅₀ = 644nM). NMDA responses were blocked by dl-2-amino-5-phosphonovaleric acid (APV;ED₅₀ = 1.9 μM) and by Mg²⁺ at negative potentials. In their sensitivities to agonists and antagonists, and ionic dependences, amino acid neurotransmitter responses on embryonic Xenopus neurons closely resembled those previously observed for mature Xenopus and mammalian central neurons. The Glu_NMDA receptors present on these immature neurons were sufficiently sensitive to be activated by endogenous concentrations of extracellular Glu, suggesting a possible role for receptor activation in modulating early neural development.     

Interactions of dextromethorphan with theN-methyl-d-aspartate receptor-channel complex: single channel recordings

The actions of dextromethorphan (DXM) on the 50 pS conductance state of theN-methyl-d-aspartate (NMDA) receptor-operated channel were studied using outside-out patches obtained from cultured rat hippocampal pyramidal neurons. DXM (5–50 μM) had no effect on the amplitudes of unitary currents but caused concentration-dependent reductions in channel mean open times and the frequency of channel openings. Channel open probability was reduced in a concentration-dependent manner by DXM and was one-half of the control value at a DXM concentration of 6 μM, with the patch potential held at −60 mV. An IC₅₀ value of 4 μM was obtained for the reduction by DXM of NMDA-evoked rises in [Ca²⁺]_i in cultured rat hippocampal pyramidal neurons loaded with Fura-2. The results were consistent with drug block of the open NMDA channel with an onward (blocking) rate constant of 7.7 × 10⁶ M⁻¹ · s⁻¹ (at −60 mV). The estimated unblocking rate constant was about 10 s⁻¹, a value considerably higher compared to the off-rate constant found for dizocilpine block of the NMDA channel.     

Sensitivity of postsynaptic GABAB receptors on hippocampal CA1 and CA3 pyramidal neurons to ethanol

Baclofen-induced hyperpolarization of hippocampal CA1 and CA3 pyramidal neurons was examined to assess the impact of ethanol on postsynaptic GABA_B receptors. These receptors activate outward K⁺ currents via a pertussis toxin-sensitive G protein cascade to reduce membrane potential during the slow inhibitory postsynaptic potential. This inhibitory action may play a role in ethanol intoxication and withdrawal excitability. In both types of pyramidal neurons, baclofen applied consecutively in increasing concentrations caused concentration dependent hyperpolarization. There were no significant differences in resting membrane potential, input resistance, maximum baclofen-induced hyperpolarization or EC₅₀ between CA1 and CA3 neurons, although slope values were significantly smaller in the former neurons. These parameters were not significantly changed in the presence of ethanol 10–100 mM. Chronic ethanol treatment (12 days) sufficient to induce physical dependence also did not shift sensitivity or maximum response to baclofen in CA1 neurons. These results suggest that GABA_B receptors in this model are essentially insensitive to ethanol and do not confirm our earlier preliminary observation of a possible down-regulation of postsynaptic GABA_B receptor function by chronic ethanol treatment.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

Full-size image

-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Mechanisms underlying the neuropathological consequences of epileptic activity in the rat hippocampus in vitro

Blockade of γ-aminobutyric acid (GABA)ergic synaptic transmission in mature hippocampal slice cultures for a period of 3 days with convulsants was shown previously to induce chronic epileptiform activity and to mimic many of the degenerative changes observed in the hippocampi of epileptic humans. The cellular mechanisms underlying the induction of this degeneration were examined in the present study by comparing the effects of GABA blockers with the effects produced by the K⁺ channel blocker tetraethylammonium (2 mM). Both types of convulsant caused a comparable decrease in the number of Nissl-stained pyramidal cells in areas CA1 and CA3. No significant cell loss was induced by tetraethylammonium when epileptiform discharge was reduced by simultaneous exposure of cultures to tetrodotoxin (0.5 μM) or to the anticonvulsants pentobarbital (50 μM) or tiagabine (50 μM). We conclude that this degeneration was mediated by convulsant-induced epileptiform discharge itself. The hypothesis that N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity underlies cell death in this model was tested by applying convulsants together with specific antagonists of glutamate receptors. Whereas coapplication of antagonists of both non-NMDA and NMDA receptors strongly reduced the degeneration induced by the convulsants, application of either class of antagonist alone did not. Application of exogenous NMDA produced potent cell death, and this degeneration was blocked by the NMDA receptor antagonist methyl-10,11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK-801). Convulsants also induced a loss of dendritic spines that could be partially prevented by NMDA or non-NMDA receptor antagonists. We conclude that NMDA receptor activation is not solely responsible for the neuronal pathology resulting as a consequence of epileptiform discharge. © 1996 Wiley-Liss, Inc.     

Effect of oxidative stress on the release of [H]GABA in cultured chick retina cells

The effect of ascorbate (1.5 mM)/Fe²⁺ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [³H]γ-aminobutyric acid ([³H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K⁺ increased the release of [³H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca²⁺, respectively. Lipid peroxidation increased the release of [³H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca²⁺-free or in Ca²⁺-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [³H]GABA due to K⁺-depolarization in the absence of Ca²⁺, but only 65% of the release occurring in the presence of Ca²⁺ in control and peroxidized cells. Under oxidative stress retina cells release more [³H]GABA than control cells, being the Ca²⁺-independent mechanism, mediated by the reversal of the Na⁺/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [³H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [³H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [³H]GABA release evoked by 50 mM K⁺-depolarization in control but not in peroxidized cells. The release of -[³H]aspartate (a non-metabolized analog of -glutamate) evoked by 50 mM K⁺, in the absence of Ca²⁺, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe²⁺ causes an excessive release of endogenous excitatory amino acids upon K⁺-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na⁺ concentration and consequently stimulating the release of [³H]GABA by reversal of the Na⁺/GABA carrier.     

Intracellular calcium elevation during plateau potentials mediated by extrasynaptic NMDA receptor activation in rat hippocampal CA1 pyramidal neurons is primarily due to calcium entry through voltage‐gated calcium channels

We reported previously that plateau potentials mediated by extrasynaptic N‐methyl‐d ‐aspartate receptors (NMDARs) can be induced either by synaptic stimulation in the presence of glutamate transporter antagonist or by iontophoresis of NMDA in rat hippocampal CA1 pyramidal neurons. To examine whether the plateau potentials are accompanied by an elevation of intracellular Ca²⁺ and to determine the source of Ca²⁺ elevation, we performed Ca²⁺ imaging during the plateau potential. Neurons were loaded with Ca²⁺ indicator fluo‐4, and the plateau potentials were generated either synaptically in the presence of glutamate transporter antagonist or by iontophoretically applying NMDA. We have found that a transient elevation in intracellular Ca²⁺ accompanies the plateau potential. The synaptically induced plateau potential and the Ca²⁺ elevation were blocked by 5,7‐dichlorokynurenic acid (5,7‐dCK), an antagonist for the glycine‐binding sites of NMDAR. A mixture of Cd²⁺ and tetrodotoxin did not block NMDA‐induced plateau potentials, but completely abolished the accompanying Ca²⁺ elevation in both the presence and absence of Mg²⁺ ions in the bathing solution. The NMDA‐induced plateau potential was blocked by further adding 5,7‐dCK. Our results show that the NMDAR‐mediated plateau potential is accompanied by elevation of intracellular Ca²⁺ that is primarily caused by the influx of Ca²⁺ through voltage‐gated Ca²⁺ channels.     

Protection by ethanol of cortical neurons from N-methyl-d-aspartate-induced neurotoxicity is associated with blocking calcium influx

Effect of ethanol on N-emthyl-d-aspartate (NMDA)-induced neurotoxicity in rat dissociated cortical cells (8–12 day cultures) was studied. Treatment of cells with NMDA (50 and 500 μM) for 15 min caused cytotoxic effects on the cells, as examined by microscopic observations and lactate dehydrogenase release from cells 18 h after the treatment. Ca²⁺ is essential for these effects in medium during treatment. Presence of ethanol (50–300 mM) simultaneously with NMDA protected cells from the cytotoxicity depending on the concentration of ethanol. Calcium accumulation in cells on addition of NMDA, as monitored by fluorescence ratio (F₄₀₅/F₄₈₅) of Indo-1-preloaded cortical cells, was also decreased depending on the concentration pf added ethanol. APV (200 μM) and ketamine (100 μM) blocked both the cytotoxicity and cellular calcium accumulation due to NMDA. These results suggest that ethanol effects its protection of neurons from NMDA-induced cytotoxicity by blocking the receptor-mediated calcium influx.     

Functional characterization of a kindling-like model of ethanol withdrawal in cortical cultured neurons after chronic intermittent ethanol exposure

Chronic ethanol exposure has been reported to alter NMDA and GABA_A receptor function and gene expression in brain regions of animals and mammalian cultured cortical neurons. In the present study, we investigated the effects of another model of chronic, but intermittent, ethanol treatment (CIE) on GABA_A and NMDA receptor systems in cortical neurons. CIE (50 mM ethanol, 12 h exposure/12 h withdrawal, 5 cycles) exposure produced increased [³H]MK-801 binding and diazepam insensitive binding sites as measured by [³H]Ro15-4513 binding to cortical cultured neuronal membranes, at 0 h following the last treatment cycle relative to control neurons. The NMDA mediated increase in intracellular calcium [Ca²⁺]_i was also increased following similar CIE treatment. CIE treatment also increased the ability of pentylenetetrazol (PTZ) to inhibit GABA mediated ³⁶Cl⁻ influx relative to control neurons. These effects were not reversible following 1 week ethanol withdrawal, implying enhanced sensitivity of PTZ to inhibit GABA_A receptor mediated inhibition, and an increased NMDA receptor function in CIE treated cortical neurons. These alterations are consistent with the behavioral studies in animals, and suggest that both GABA_A and NMDA receptors play an important role in ethanol withdrawal following either chronic or CIE exposure. Furthermore, this provides a feasible in vitro model for further biochemical and molecular studies of the mechanism underlying the CIE induced kindling-like phenomenon observed in humans.