Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Germany
(Received 21 June 2002; first review notified 3 September 2002; in revised form 28 March 2003; accepted 20 May 2003)
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ABSTRACT |
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INTRODUCTION |
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Electrophysiological studies have indicated that ethanol can depress AMPA/kainate-evoked currents in different neuronal preparations using patch-clamp techniques, but these receptors seem to be less sensitive to ethanol than the N-methyl-D-aspartic acid (NMDA) subtype (Lovinger et al., 1989; Weight et al., 1992
; Valenzuela et al., 1998
). These data conflict somewhat with recent findings showing that AMPA or kainate-activated currents are also strongly inhibited by pharmacologically relevant concentrations of ethanol in cultured hippocampal or medial septum/diagonal band neurons of the rat (Costa et al., 2000
; Frye and Fincher, 2000
). On the other hand, measurements of AMPA-induced increase in free intracellular Ca2+ concentration ([Ca2+]i) using fura-2 imaging have shown that acute ethanol application may reduce the amplitude of Ca2+ signals in cultured rat cerebellar Purkinje neurons (Gruol et al., 1997
). However, the effect of ethanol on AMPA receptor-mediated Ca2+ signaling in cortical or hippocampal neurons has hitherto not been investigated.
In the present study we examined the inhibition of (S)-AMPA-induced Ca2+ influx by acutely applied ethanol in cultured rat cortical nonpyramidal neurons using single-cell fura-2 microfluorimetry. The AMPA receptor-mediated Ca2+ responses in these neurons, identified as GABAergic interneuron-like cells by immunocytochemistry, have been characterized in detail elsewere (Fischer et al., 2002).
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MATERIALS AND METHODS |
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Fura-2 microfluorimetry
Following loading with fura-2/AM (5 µm; see Drugs) in culture medium (37°C, 30 min), the cells were washed in physiological saline (composition in mm: NaCl 133, KCl 4.8, KH2PO4 1.2, MgCl2 1, CaCl2 1.3, HEPES 10, D(+)-glucose 10; pH 7.4; room temperature, 30 min) to remove extracellular traces of the dye. The coverslips were then mounted cell-side up in the bottom of a perfusion chamber (250 µl), placed on the stage of an inverted microscope with epifluorescence optics (Diaphot 200; Nikon, Kanagawa, Japan). Throughout the experiments, the cells were continuously superfused at 0.8 ml/min by means of a roller pump with drug-free or drug-containing solution. In a first series of experiments, the glutamate receptor agonists (AMPA, NMDA or kainate) were applied three times (S1, S2, S3) for 60 s every 10 min by using the roller pump. Ethanol was superfused 10 min before and during S2. In an additional series of experiments, AMPA was applied directly to single cells by pressure for 10 s every 10 min using a DAD12 fast application system (Adams and List, Westbury, NY, USA). Ethanol, tetrodotoxin/bicuculline or nifedipine were superfused 10 min before and during the next application of AMPA (in combination with the drug tested) by pressure application. The tissue bath was continuously superfused with drug-free or drug-containing solution (see above), and the solution was removed from the bath with a vacuum pump.
Fura-2 fluorescence (over the cell somata of medium-sized multipolar nonpyramidal neurons), excited alternatively at 340 and 380 nm, was measured at 510/520 nm by a microscope photometer attached to a photomultiplier detection system (Ratiomaster System; PTI, Lawrenceville, NJ, USA). The agonist-induced rise of [Ca2+]i was defined as the peak increase in the fluorescence ratio (i.e. the fluorescence ratio 340/380 nm in response to the agonist minus the basal fluorescence ratio) (details of fura-2 microfluorimetry can be found in Fischer et al., 2002
). Data acquisition and analysis were performed computer-controlled by using commercially available software (FeliX, Vers.1.1; PTI). Calibration of [Ca2+]i was performed according to Grynkiewicz et al. (1985)
: Ca2+-saturated fura-2 signals (Rmax) were determined in the presence of 10 µm ionomycin (Mg2+-free buffer), and Ca2+-free signals (Rmin) with 25 mm EGTA (Ca2+-free buffer).
GABA immunochemistry
Sister cell cultures were briefly washed with phosphate-buffered saline (0.1 m, pH 7.4), fixed in 4% paraformaldehyde/ 0.25% glutaraldehyde (4°C, 20 min), treated with 1% H2O2 to inactivate endogenous peroxidase activity and preincubated with a blocking solution (10% normal horse serum and 0.1% Triton X-100 in Tris buffered saline) before incubation (4°C, overnight) with the primary antibody (mouse monoclonal anti-GABA antibody, clone 5A9, 1:1000; ICN Biomedicals, Aurora, OH, USA). The preparations were then washed in Tris buffered saline and incubated with the secondary antibody, biotinylated horse anti-mouse immunoglobulin G (1:65; 20°C, 60 min). Following treatment with avidin-biotin complex (ABC Elite Kit, 1:50; 60 min; both Vector Laboratories, Burlingame, CA, USA), the cells were visualized by 3,3'-diaminobenzidine (DAB-Ni/Co; Sigma-Aldrich, Taufkirchen, Germany). The stained cultures were dehydrated in a series of graded ethanol and covered with entellan. Control experiments were carried out without the primary antibody. In some cultures representative fields with living neurons were photographed using an inverted phase-contrast microscope (CK2-TR; Olympus, Hamburg, Germany) before starting GABA-immunocytochemistry. GABA-stained preparations were investigated and photographed using a light microscope (Axioskop 50; Carl-Zeiss Jena, Germany).
Drugs
The following drugs were used: (S)--amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid, NMDA, (+)bicuculline, EGTA, fura-2 acetoxymethyl ester (Fura-2/AM), ionomycin, nifedipine, tetrodotoxin citrate (TTX) (all from Sigma-Aldrich, Taufkirchen); ethanol absolute (Mallinckrodt Baker; Deventer, The Netherlands); 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-4,5-dihydro-3-methyl-carbamoyl-2,3-benzodiazepine (GYKI 5365; gift of Dr L. Harsing, Institute for Drug Research, Budapest). All other chemicals for the superfusion solutions were obtained from Sigma-Aldrich.
Statistics
The data were calculated as means ± SEM of n determinations (individual cells). In all cells included in statistical analysis, the control response to the tested agonist (AMPA, NMDA or kainate) fully recovered following the 10-min-washout period with drug-free solution. The difference between the control response and the response after ethanol superfusion (absolute values) for each cell was compared with the paired Students t-test after passing the normality test. Comparison of the inhibitory effects by 100 mm ethanol on AMPA- versus NMDA-induced response (normalized values) was performed by using two-way analysis of variance (ANOVA) with doses as within-subject factor and drug treatment as the between-subject factor. Pairwise multiple comparisons of control response with different drug-modulated response (absolute values) were performed by one-way ANOVA followed by post-hoc StudentNewmanKeuls test. The statistics software used were SigmaStat (Ver. 2.03; SPSS, Erkrath, Germany). A probability level of <0.05 was considered to be statistically significant.
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RESULTS |
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In the previous microfluorimetric investigations, the cells were superfused with the glutamate receptor agonists (AMPA, NMDA, kainate) for 60 s, every 10 min at a slow rate. In an additional series of experiments, AMPA was applied directly to single cells by fast pressure-application for 10 s, every 10 min. AMPA (30 µm) induced a rapidly rising and declining Ca2+ signal, which was reproducible upon repetitive stimulations. First tests with ethanol at 100 mm (superfused 10 min before and during the following AMPA (plus ethanol 100 mm)-pressure application) reduced the peak amplitude by 19 ± 3% (n = 9; P < 0.05), well comparable to the value measured previously with the slow superfusion system.
To address the question whether GABA itself induces a possible increase in [Ca2+]i in developing neurons of our cell cultures, the effect of pressure applied GABA (1, 10 and 100 µm) on single cells was investigated. The lowest GABA concentration failed to produce any significant change in fluorescence intensity, but 10 and 100 µm of this agonist induced a rise in Ca2+ response depending on the age of the cultures. In younger cultures (1012 days in vitro) 17 from 24 (71%) of the neurons tested showed a marked Ca2+ signal. The means of fluorescence ratios evoked by GABA (100 µm) and AMPA (30 µm) were 0.78 ± 0.12 and 2.14 ± 0.25 (n = 17 each), respectively. In older cultures (1315 days in vitro), only in 5 from 21 (24%) neurons a transient Ca2+ signal could be observed. The GABA (100 µm)-induced Ca2+ signals were inhibited by nifedipine (10 µm; 70 ± 3% inhibition, n = 8; P < 0.001) and bicuculline (10 µm; >90% inhibition, only three neurons tested).
However, the superfusion of the GABAA receptor antagonist bicuculline (10 µm) together with the Na+ channel blocker tetrodotoxin (0.5 µm), to rule out possible effects of spontaneously released GABA and synaptic spike activity in the cell culture, did not significantly modulate the peak amplitude of the AMPA-induced Ca2+ signal as well as the inhibitory effects of ethanol on AMPA response (Fig. 3a,c). In these experiments, ethanol inhibited the AMPA response by 20 ± 4% (n = 7; P < 0.05) and the AMPA response following tetrodotoxin/bicuculline superfusion by 23 ± 4% (n = 7; P < 0.05). Noteworthy, in some separate tests, the superfusion of bicuculline (10 µm) alone practically did not alter the AMPA-induced response (105 ± 3%, n = 16; P > 0.05).
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DISCUSSION |
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Previous studies of the inhibitory effect by ethanol on non-NMDA receptor-mediated currents in various neuronal preparations yielded controversial data. For example, in brain slices from rat hippocampus or nucleus accumbens, intoxicating concentrations of ethanol (50100 mm) exhibit little or no effects on excitatory synaptic transmission mediated by AMPA receptors (Lovinger et al., 1990; Nie et al., 1994
; Weiner et al., 1999
). Weiner et al. (1999)
reported that in rat CA3 pyramidal neurons ethanol depressed pharmacologically isolated kainate and NMDA receptor-mediated excitatory postsynaptic currents (EPSCs), whereas EPSCs mediated by AMPA receptors were not significantly affected even at the highest ethanol concentration tested (80 mm). In cultured rat neuronal cell populations, however, ethanol can significantly depress AMPA/kainate activated currents (Valenzuela et al., 1998
; Costa et al., 2000
; Frye and Fincher, 2000
). Thus, Frye and Fincher (2000; see their fig. 2)
observed at 100 mm ethanol about 2030% inhibition of AMPA (100 µm)- or kainate- (100 µm) activated currents in acutely isolated medial septum/diagonal band neurons of the rat, well comparable with our values concerning AMPA-induced Ca2+ signaling. Inhibition by ethanol (25200 mm) of AMPA and kainate receptor-mediated depolarizations of the hippocampal CA1 area in brain slices was also reported (Martin et al., 1995
).
As suggested by various patch-clamp studies, the potency of ethanol to inhibit the NMDA receptor-function appears to be higher than its potency to inhibit AMPA/kainate receptors (Lovinger, 1993; Valenzuela et al., 1998
). However, the potency with which ethanol inhibits NMDA receptors also greatly varies between native neurons. Various factors, such as differences in experimental conditions (e.g. buffered medium, temperature, intact or dialysed intracellular milieu) and different neuronal cell preparations (special brain regions, brain slices, cultured neurons with or without glial bed, developing and mature neurons) may contribute to the differences in the inhibitory potency of ethanol for different glutamate receptor-mediated responses (for discussion see Lovinger et al., 1990
).
On the other hand, there is very limited information about the inhibitory action by ethanol on AMPA receptor-mediated Ca2+ signaling. In one study, non-NMDA (i.e. quisqualate, kainate)-mediated increases in cytosolic Ca2+ were not affected by a high concentration (100 mm) ethanol in acutely dissociated brain cells from newborn rats (Dildy-Mayfield et al., 1991). In contrast, some non-NMDA and NMDA responses were inhibited by ethanol to a similar extent in oocytes expressing rat hippocampal mRNA (Dildy-Mayfield and Harris, 1992
). In another report, ethanol was shown to reduce the amplitude of Ca2+ signals evoked by AMPA in cultured rat cerebellar Purkinje cells (Gruol et al., 1997
). A pharmacological characterization of the AMPA receptors involved in the mediation of Ca2+ signals was not performed in this study. However, Gruol and Curry (1995)
reported also an enhancement of quisqualate-induced Ca2+ signals following acute ethanol application in cerebellar Purkinje and granule cells.
In the present investigation, we provide evidence that the AMPA receptor-induced increase in [Ca2+]i is sensitive to ethanol in cultured rat cortical GABAergic interneuron-like cells. However, the findings indicate that only high concentrations of ethanol inhibit Ca2+ signalling. Our observation that AMPA- and NMDA-induced Ca2+ responses were reduced by ethanol (100 mm) to an almost similar extent, suggested a lack of selectivity of the depressant effects of ethanol on Ca2+ signaling in these cells. However, it is possible that NMDA receptors may be more sensitive than AMPA receptors to concentrations of ethanol lower than 100 mm, which were not tested with NMDA in the present study. Previous findings, however, argued against such an idea. Thus, Bhave et al. (1996) found in rat cortical neuronal cell cultures a threshold concentration of ethanol at about 30 mm for inhibiting the NMDA (100 µm)-induced increase in [Ca2+]i; the inhibition by ethanol was at 50 mm about 15% and at 100 mm about 35%. Wirkner et al. (1999)
measured higher values (~20 and ~40% inhibition at 30 and 100 mm ethanol, respectively) for the inhibition of NMDA (20 µm)-induced Ca2+ signals. Further studies in our laboratory under similar methodical condition as used in the first series of experiments in the present work found for the NMDA (30 µm)-induced Ca2+ response no significant inhibition by 30 mm ethanol, ~20% at 50 mm and ~30% at 100 mm ethanol (A. Kronfeld and C. Allgaier, unpublished results). All these data, when supplemented with our present results indicate a comparable sensitivity of NMDA and AMPA receptor-induced Ca2+ signaling to both high and low concentrations of ethanol.
In agreement with the recently reported effects of ethanol on AMPA-induced currents (Costa et al., 2000; Frye and Fincher, 2000
), it can be suggested that both AMPA and NMDA glutamate receptor subtypes are targets of ethanol in diverse cell populations. Moreover, our data are also consistent with the findings of previous whole-cell voltage-clamp experiments showing that ethanol inhibits NMDA and AMPA receptor function by a similar and noncompetitive mechanism (Wirkner et al., 2000
). It is of note that the lipophilic trichloroethanol, the main metabolite of chloral hydrate, which more potently (in the low millimolar range) inhibits Ca2+ responses to both AMPA and NMDA than ethanol, also causes similar inhibition of Ca2+ signals to both types of excitatory amino acid agonists (Scheibler et al., 1999
; Fischer et al., 2000
).
In the first series of experiments, the studies were performed in the absence of tetrodotoxin and the GABAA receptor antagonist bicuculline. Since GABA can function as an excitatory neurotransmitter during early neuronal development, inducing membrane depolarization via GABAA receptor-activation, and an increase in [Ca2+]i as well as firing of action potentials (Owens et al., 1996; Rego et al., 2001
; Ben-Ari, 2002
), it seemed of interest to carry out new experiments in the presence of tetrodotoxin and bicuculline. However, these drugs did not alter the effect of ethanol in the 1015-day-old cortical cultures used, although in separate studies, GABA (10100 µm) induced a transient increase of [Ca2+]i in most neurons of younger cell cultures (1012 days in vitro). Therefore, it is concluded, that neither a possible GABA release in our cell cultures enriched with GABA-immunopositive neurons, nor the additional block of spike activity seems to be of major significance for the Ca2+ signals elicited by AMPA as well as for the inhibitory effects of ethanol.
There was a small component of AMPA receptor-mediated response which could be mediated by voltage-gated Ca2+ channels. The possibility that the inhibitory effects of ethanol may be related to the inhibition of these Ca2+ channel-dependent component, could, however, be excluded by the experiments with nifedipine and ethanol alone and in combination, in which additive inhibitory effects were demonstrated.
It is well established that interneurons have powerful regulatory roles in controlling the activity of complex neuronal circuits in diverse brain structures (Ross and Soltesz, 2001). As a cellular consequence of acute ethanol exposure (e.g. a decreased AMPA and NMDA receptor function), the excitability of interneurons can be reduced and consequently the inhibitory control function to pyramidal neurons is diminished. However, detailed studies have shown that various neuronal cell populations as well as multiple ligand- or voltage-gated ion channels are affected by ethanol and may contribute to the known hypnotic or intoxicating effects of this substance at higher concentrations (Faingold et al., 1998
; Little, 1999
). Therefore, the significance of the observed decrease in Ca2+ transients for neuronal function is still a matter of debate. Finally, although there is now compelling evidence for the fact that excitatory amino acid receptors are involved in major actions of ethanol, the key question for a specific site of interaction at the NMDA or AMPA/kainate receptor channels remains hitherto unanswered (Woodward, 1999
).
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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