Introduction

Abstract Introduction Methods Results Discussion References

The major excitatory neurotransmitter in the brain is glutamate, which exerts its effects through two classes of receptors, ionotropic and metabotropic. Several subtypes of the iGluRs have been defined based on their selective activation by agonists, NMDA, AMPA and KA. Excessive activation of these receptors has been implicated in the neuropathology associated with certain disease states, such as Parkinson's disease, Huntington's disease, Alzheimer's disease and epilepsy (see Bennett and Huxlin, 1996; Whetsell, 1996 for review). It is also likely that overstimulation of iGluRs participates in the neuronal loss observed in alcoholics (Krill et al., 1995).

Chronic alcohol abuse has been shown to be associated with a selective neuronal loss in various regions of the brain including the mamillary bodies, cerebellum, hippocampus and cortex. Several studies have shown that CEE may sensitize central neurons to excitotoxic damage by glutamate (Ahern et al., 1994; Blevins et al., 1995; Chandler et al., 1993; Davidson et al., 1993). The NMDA subtype of glutamate receptor is a principal conduit for neuronal death given the high permeability of the NMDA-associated ion channel to calcium (Mayer and Westbrook, 1987). Furthermore, the NMDA receptor is also the most efficacious of the iGluRs in mediating cell death (Hartley et al., 1993; Tymianski et al., 1993). Hence, NMDA receptors likely play a major role in the neuronal toxicity observed with alcohol abuse or as a consequence of alcohol withdrawal (Lovinger, 1993).

The NMDA receptor is particularly sensitive to inhibition by acute ethanol exposure (Bhave et al., 1996; Lovinger et al., 1989) which may vary regionally in the brain (Randoll et al., 1996). Conversely, the chronic presence of ethanol appears to induce an adaptive increase in the function of NMDA receptors. CEE has been reported to enhance NMDA-mediated increases in [Ca⁺⁺]_i and excitotoxicity in cortical and cerebellar neurons (Ahern et al., 1994; Blevins et al., 1995; Iorio et al., 1992, 1993). However, whether this functional enhancement occurs in NMDA receptors on hippocampal neurons is unknown, despite the fact that the hippocampus is among the most severely affected brain regions after chronic ethanol treatment (see Walker et al., 1993 for review). Recent evidence has shown that non-NMDA receptors (i.e., AMPA/kainate) may be more sensitive to inhibition by ethanol than originally thought (Dildy-Mayfield and Harris, 1995; Martin et al., 1995). In addition, VSCCs are also inhibited by ethanol and have been implicated in the hyperexcitability after CEE (Leslie et al., 1990; Ripley et al., 1996). The sensitivity of VSCCs and iGluRs to CEE and the contribution of these channels to increases in intracellular calcium concentration and/or neurotoxicity after CEE in hippocampal neurons remain unclear.

In the present study, we have characterized the role in agonist-induced excitotoxicity after CEE of iGluRs and VSCCs in cultured hippocampal neurons. To assess whether an increase in receptor function underlies enhanced excitotoxicity, we measured agonist- or KCl-induced increases the level of intracellular calcium in individual hippocampal neurons. The specificity of CEE effects on NMDA receptors was assessed by examining calcium transients induced by non-NMDA iGluRs and KCl-induced depolarization after CEE. The results obtained in this study indicate selective CEE-induced changes in NMDA receptor function.

	Methods

Abstract Introduction Methods Results Discussion References

Neuronal cell culture. Primary hippocampal mixed neuronal/glial cultures were established according to the methods of Banker and Cowan (1977) with modifications described by Mattson et al. (1988). Hippocampi were excised from 18-day-old Sprague-Dawley rat fetuses, trypsinized (0.125% trypsin for 30 min), triturated with a narrow-bore pipette and the resulting dissociated cells distributed onto collagen/poly-D-lysine-coated substrates. Cultures used for intracellular calcium measurements were plated onto glass coverslips, whereas cultures used for excitotoxicity were plated onto multiwell plastic plates at a density of 2 × 10⁵ cells per well. The plating medium for these cells consisted of Eagle's MEM (adjusted to 320-330 mOsmol) supplemented with 10% fetal calf serum, 10% heat-inactivated horse serum and 2 mM L-glutamine. After 24 h, the plating medium was replaced with feeding medium which consisted of MEM supplemented with 5% heat-inactivated horse serum and 2 mM L-glutamine. Cultures were maintained at 37°C in a humidified 10% CO₂/90% air mixture. The mitotic inhibitor, 5-fluoro-2-deoxyuridine, was added to the medium after 7 DIV to retard the proliferation of non-neuronal cells. Culture medium was replenished every 7 days. All excitotoxicity treatments or measurements of intracellular calcium were performed on cultures at 14 DIV.

CEE. Hippocampal neurons in culture were exposed chronically to 100 mM ethanol for 7 DIV. Distilled, absolute ethanol purchased from Midwest Grain Products (Perkin, IL) was used in all experiments. Ethanol was added directly to the feeding medium and the culture was placed in a plastic container along with an isomolar concentration of ethanol in an open well. The container was equilibrated with 10% CO₂/90% air and sealed to prevent evaporative loss of ethanol. Control cultures were sealed likewise in plastic containers not containing ethanol. The method of ethanol addition, maintenance and preparation of control cultures was identical in all subsequent paradigms. The day at which CEE was begun and the duration of CEE varied in some paradigms. One- and two-day ethanol treatment paradigms were initiated in hippocampal cultures beginning on day 13 and day 12 in vitro, respectively. A 100 mM ethanol concentration was used for all 1- and 2-day ethanol treatment experiments.

Chronic APV and ethanol + APV paradigm. Chronic APV cultures were exposed to 70 µM APV for 7 days starting at 7 DIV. Control and APV-treated cultures were equilibrated with 10% CO₂/90% air and sealed in plastic containers not containing ethanol. In the chronic ethanol + APV paradigm, 70 µM APV and 100 mM ethanol were added to cultures at 7 DIV and maintained for 7 days. Chronic ethanol + APV cultures were equilibrated with 10% CO₂/90% air and sealed in plastic containers with an isomolar concentration of ethanol in an open well. Control cultures were sealed likewise in plastic containers not containing ethanol.

Ethanol determination. The determination of ethanol concentration was performed by an assay based on the ability of the enzyme alcohol dehydrogenase to catalyze the oxidation of alcohol to acetaldehyde with simultaneous reduction of nicotinamide adenine dinucleotide (NAD) to NADH, which was measured spectrophotometrically with absorbance at 340 nm. This assay was performed with a commercial kit (332-UV) supplied by Sigma Chemical Company, St Louis, MO.

Calcium determination. Fluorescence ratio imaging with the calcium indicator fura-2 was used to measure free intracellular calcium concentrations ([Ca⁺⁺]_i). Cells were loaded at 37°C for 20 min with 4-6 µM fura-2 AM (Molecular Probes Inc. Eugene, OR) dissolved in an external perfusion buffer of the following composition (in mM): NaCl, 150; KCl, 2.5; CaCl₂, 2.5; HEPES, 10; glucose, 10; tetrodotoxin, 0.001; and glycine, 0.003 (adjusted to 320-340 mOsmol with sucrose; pH 7.4). After fura-2 loading, cells were washed with perfusion buffer and allowed to incubate an additional 30 to 50 min before determinations of [Ca⁺⁺]_i were attempted.

Drug and solution application for calcium determination. A perfusion chamber was constructed by sandwiching the neurons between two coverslips separated by spacers. The volume of this chamber was 150 µl and allowed for the complete exchange of solutions within 5 s. Solution flow was gravity driven, and different solutions could be switched via a multiport distribution valve. All drug solutions for [Ca⁺⁺]_i determination were dissolved in external perfusion buffer. NMDA and APV were purchased from Research Biochemicals International, Natick, MA. Kainic acid, glycine and all other chemicals were purchased from Sigma Chemical Company, St. Louis, MO.

Excitotoxicity. Exposure to excitatory amino acids was similar to that described by Koh and Choi (1988). Before exposure to test solutions, cells were washed twice with a Tris-buffered CSS, consisting of the following (in mM): NaCl, 120; KCl, 5.4; CaCl₂, 1.8; Tris-HCl (pH 7.4), 25; and glucose, 15. Glycine (3 µM) was included in the test solutions of all experiments involving NMDA excitotoxicity, but not in excitotoxicity experiments involving other agents. In experiments involving the continued presence of ethanol, ethanol was included in the pretest wash and during the excitotoxic drug exposure. Test solutions of excitotoxic agents dissolved in CSS were applied to cells for 5 min at room temperature. After the 5-min exposure, the test solutions were washed out thoroughly. Feeding medium lacking serum replaced the CSS and the cells were returned to the incubator. Twenty to 24 h after exposure to the test solutions, the cells were exposed to 0.4% trypan blue (5 min) which stained nonviable cells. Neurons were identified by morphology. Cell death was determined by counting viable (phase bright, lacking staining) and nonviable (stained) neurons in three randomly selected, nonoverlapping fields in each well.

Statistical analysis. Sister cultures (control vs. experimental) from a single culture preparation were compared in all analyses with control for interculture variability within treatment paradigms. Data are reported as the mean values ± S.E.M. from the indicated number of cells and cultures. For multiple comparisons, data were analyzed by appropriate ANOVA procedures. Post hoc analysis was performed with the Scheffe test for multiple comparisons. For single comparisons, data were analyzed by Student's t test. A level of P < .05 was considered statistically significant. Statistical analysis was performed with Statview II (Abacus Concepts, Berkeley, CA) or SAS (Statistical Analysis System, Cary, NC).

	Results

Abstract Introduction Methods Results Discussion References

Exposure of hippocampal neurons to a 5-min application of NMDA produced a dose-dependent increase in percent cell death as measured by trypan blue exclusion (fig. 1). The NMDA-induced excitotoxicity in cultures treated chronically with 100 mM ethanol for 7 days was greater than that in control cultures (F = 10.32, P = .0014, ANOVA). The enhancement of NMDA-induced excitotoxicity after CEE was not caused by an inability to induce cell death in control cultures because maximal excitotoxicity, 96.6 ± 1.4% (mean ± S.E.M.) in control and 98.2 ± 0.9% in CEE cultures, was produced by 500 µM NMDA with no significant difference between control and CEE cultures (two-tailed t test, P > .1). The NMDA-induced excitotoxicity was mediated by NMDA receptors because it could be effectively blocked by coapplication during 5 min exposure to the competitive NMDA antagonist, APV (70 µM). The continued presence of ethanol reduced NMDA excitotoxicity to the same extent in both control and CEE cultures. The percent cell death induced by NMDA (50 µM) exposure in the absence of ethanol was 22.6 ± 2.6 (mean ± S.E.M.) in control cultures and 40.4 ± 4.7 in CEE, whereas excitotoxicity in presence of 100 mM ethanol was 4.8 ± 1.1 in control and 6.8 ± 1.9 in CEE cultures. Thus, enhanced excitotoxicity was only observed after ethanol withdrawal.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effect of CEE on NMDA-induced excitotoxicity in cultured hippocampal neurons. Excitotoxicity induced by the indicated concentration of NMDA (plus 3 µM glycine) in the absence and presence of 70 µM APV (NMDA+APV) after a chronic 7-day exposure to 100 mM ethanol as described under "Methods." Neuronal cell death was expressed as the percentage of total cell number (live + dead). Each data point for NMDA excitotoxicity in the absence of APV represents the mean ± S.E.M. from 17 to 39 measurements in control cultures and 18 to 39 measurements in CEE cultures derived from 15 separate experiments. Each data point for NMDA excitotoxicity in the presence of APV represents the mean ± S.E.M. from six measurements in control cultures and six measurements in CEE cultures derived from two separate experiments. ANOVA indicated a significant treatment effect of CEE (open squares vs. filled circles) on the percentage of cell death induced by NMDA (F = 10.32, P < .01). Asterisk (*) indicates points of significant difference revealed by post hoc analysis. Coapplication of APV during 5-min NMDA exposure blocked cell death in both control and CEE cultures (filled vs. open triangles, respectively).

The duration of ethanol exposure necessary for the enhancement of NMDA-induced excitotoxicity was examined in hippocampal cultures exposed to 100 mM ethanol for 2 days. NMDA-induced excitotoxicity was significantly greater in ethanol-treated cultures than controls after the 2-day CEE treatment (F = 15.65, P = .0001, ANOVA). Significant increases in excitotoxicity were observed in the 2-day CEE group in to 50 and 75 µM NMDA. A 1-day, 100 mM ethanol exposure did not significantly enhance NMDA-induced excitotoxicity (F = 3.74, P = .0549, ANOVA).

Recent evidence has shown that antagonists of NMDA receptors can prevent the enhancement of NMDA receptor function if given during the chronic ethanol treatment period (Hu and Ticku, 1995). To examine possible mechanisms for this enhancement and to determine whether NMDA antagonists would prevent the enhancement of NMDA receptor function, 70 µM APV was included in a 100 mM ethanol/7-day chronic exposure paradigm. No enhancement of NMDA-induced excitotoxicity was found (F = 0.52, P = .47, ANOVA) with concomitant ethanol/APV exposure (fig. 2A). However, when hippocampal cultures were treated chronically with 70 µM APV alone for 7 days, a significant increase in NMDA-induced excitotoxicity was observed (F = 8.16, P = .005, ANOVA) (fig. 2B). The lack of enhanced NMDA receptor function in the chronic ethanol + APV paradigm was not evident in positive control test wells treated with either chronic ethanol or APV alone for this group of neurons.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of chronic treatment of cultured hippocampal neurons with ethanol plus APV and APV alone on NMDA-induced excitotoxicity. (A) Hippocampal cultures were chronically exposed to 100 mM ethanol plus 70 µM APV (Et+APV) for 7 days then subjected to excitotoxicity induced by NMDA exposure after removal of antagonists, as described under "Methods." (B) NMDA-induced excitotoxicity after a chronic exposure to 70 µM APV for 7 days (chronic APV). Each data point represents the mean ± S.E.M. measurement of percent cell death in nonoverlapping fields (9-20, control; 8-20, chronic Et+APV; 21-25, control; 23-27, chronic APV) from four separate experiments. No significant differences were observed in NMDA-induced excitotoxicity with chronic Et+APV paradigm as indicated by ANOVA (F = 0.52, P > .05), whereas a significant difference was observed with the chronic APV paradigm (F = 8.16, P < .01, ANOVA).

Specificity of enhanced channel function after CEE. In contrast to the enhanced NMDA-induced excitotoxicity, no enhancement of KA-induced excitotoxicity was observed after a chronic exposure to 100 mM ethanol for 7 days (F = 0.23, P = .63, ANOVA). KA exposure produced a robust dose-dependent increase in neuronal cell death that was of similar magnitude in both ethanol-treated and control cultures (fig. 3A). In test sample wells serving as positive controls, a significant enhancement of NMDA-induced excitotoxicity was evident (F = 6.05, P = .018, ANOVA). The KA-induced excitotoxicity was sensitive to block by the NMDA antagonist, APV (70 µM), which reduced the percent cell death induced by 250 µM KA from 95.9 ± 1.0 to 49.7 ± 3.8 (mean ± S.E.M.) in control and 97.0 ± 0.9 to 49.4 ± 6.6 in CEE cultures. A combination of APV (70 µM) and the non-NMDA antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (6 µM), further reduced KA-induced excitotoxicity to 12.7 ± 2.9 (mean ± S.E.M.) and 7.9 ± 3.6 in control and CEE cultures, respectively. No differences were observed in the ability of antagonists to reduce KA-induced excitotoxicity in control and CEE cultures.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of chronic exposure to 100 mM ethanol for 7 days on excitotoxicity induced by kainic acid (A) and AMPA (B). (A) Cell death was induced by a 5-min exposure to the indicated concentration of kainic acid. Each data point represents the mean ± S.E.M. measurement of percent cell death in nonoverlapping fields (26-30, control; 27-30, CEE) from four separate experiments. ANOVA revealed no significant treatment effect of CEE (F = 0.18, P > .05). (B) A 5-min exposure to AMPA induced cell death in hippocampal cultures. Each data point represents the mean ± S.E.M. measurement of percent cell death in nonoverlapping fields (17-21, control; 17-19, CEE) from four separate experiments. No significant difference was observed as indicated by ANOVA (F = 1.19, P > .05).

Contribution of VSCCs to enhanced NMDA excitotoxicity after CEE. Chronic ethanol treatment has been reported to enhance the function of L-type VSCCs (Whittington et al., 1995; Whittington and Little, 1993). Such an enhancement could contribute to the excitotoxic damage produce by NMDA. To examine the role of VSCCs in cell death, control and CEE cultures were exposed to 50 mM KCl to activate VSCCs. KCl exposure produced no neuronal cell death in either control or CEE cultures (table 1). However, it is possible that a contribution of VSCCs is only evident in cooperation with NMDA receptor activation. Nifedipine, an antagonist of L-type VSCCs, was coapplied along with 50 µM NMDA to control and CEE cultures. Nifedipine (5 µM) inclusion resulted in a neuroprotective effect in control cultures, but not in CEE cultures (fig. 4). Neuronal cell death mediated by NMDA exposure was inhibited in the presence of nifedipine in control cultures, whereas nifedipine had no effect on NMDA-mediated cell death in CEE cultures. The percent cell death induced in control cultures in the absence and presence of nifedipine was 22.6 ± 2.6 (mean ± S.E.M.) and 14.3 ± 2.6, respectively. In CEE cultures, the percent cell death in the absence and presence of nifedipine was 40.4 ± 3.9 (mean ± S.E.M.) and 32.7 ± 4.6, respectively. Nifedipine or the vehicle, 0.5% DMSO, were not neurotoxic by themselves because no cell death was observed in the absence of NMDA. However, a small increase in excitotoxicity above control was observed with vehicle in the presence of NMDA in control neurons. Nifedipine did not produce a significant reduction in excitotoxicity in the CEE group when compared with either the NMDA or NMDA + DMSO group (fig. 4). The differences that were observed in excitotoxicity in the presence of nifedipine indicate that involvement of L-type VSCCs might be decreased after CEE because nifedipine reduced cell death in control but not CEE cultures. L-type channels did not contribute to CEE-enhanced excitotoxicity induced by NMDA because inhibition by nifedipine was not observed in CEE cultures.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   NMDA-induced neuronal cell death in the absence and presence of nifedipine (Nifed) after a chronic exposure to 100 mM ethanol for 7 days (CEE). All drug solutions were dissolved in CSS which did not induce cell death in and of itself. Nifedipine (nifedipine: 5 µM+0.05% DMSO as vehicle) or vehicle alone (0.05% DMSO) was coapplied with 50 µM NMDA for 5 min, after which neuronal cell death was measured 24 h later and expressed as the percentage of total cell number (live + dead) as described under "Methods." Each data point represents the mean ± S.E.M. of 17 to 21 determinations from three separate experiments. ANOVA indicated significant treatment (F = 27.31, P < .0001) and drug effects (F = 23.93, P < .0001). Scheffe post hoc comparisons indicated differences between NMDA and NMDA+Nifed (P = .032), NMDA+DMSO (P = .009) and NMDA+Nifed vs. NMDA+DMSO (P < .0001) in control neurons. No significant post hoc comparisons were found among the same groups in the CEE neurons. Significant increases in percent cell death in the CEE-treated neurons in the NMDA and NMDA+nifedipine drug conditions were also observed (Scheffe test, *P < .05).

NMDA-induced calcium transients after CEE. To determine whether enhanced NMDA receptor function is associated with the enhanced excitotoxicity observed after chronic antagonism, we examined NMDA-stimulated increases in intracellular calcium. The ability of ionotropic glutamate receptor activation to increase intracellular calcium was assessed after a chronic exposure to 100 mM ethanol for 7 days. A 3-s application of NMDA produced transient increases in the [Ca⁺⁺]_i, which returned to baseline after agonist removal in both control and CEE neurons (fig. 5, A and B, respectively). Peak NMDA-induced calcium responses occurred at NMDA concentrations of ~20 to 30 µM. When NMDA-induced calcium responses were compared in control versus CEE cultures, the NMDA-induced increases in [Ca⁺⁺]_i were found to be larger in CEE neurons than in control neurons (F = 7.96, P = .0051, ANOVA) at NMDA concentrations of 5 and 10 µM (fig. 5C). Conversely, calcium responses induced by KA (25 µM) were larger in control neurons than in CEE neurons treated with 100 mM ethanol for 7 days (table 2). However, the KA-induced calcium response was not consistently altered across several treatment paradigms. Considerable variability in the KA-induced calcium response was observed between paradigms (table 2). Because the experimental measurements within any given paradigm were made with sister cultures derived from a single culture preparation (five separate culture preparations for the 7-day CEE paradigm, table 2) the variability in the magnitude of kainate responses likely reflects interculture preparation variability.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   NMDA-induced increases in intracellular calcium after exposure to 100 mM ethanol for 7 days. Representative NMDA-induced calcium responses from single neurons in control (A) and CEE culture (B). Increases in [Ca++]i were induced in neurons exposed to various concentrations of NMDA for 3 s as indicated. (C) Comparison of NMDA-stimulated increases in [Ca++]i in control neurons and neurons treated chronically with 100 mM ethanol for 7 days (CEE). The increase in [Ca++]i ( [Ca++]i nM) reflects basal [Ca++]i values subtracted from peak agonist-induced calcium responses at each concentration. Values represent the mean ± S.E.M. of 26 to 31 determinations from 31 neurons for each data point in the control curve and 31 to 36 determinations from 36 neurons in the CEE curve. ANOVA revealed a significant treatment effect of CEE (F = 7.14, P < .01). Asterisk (*) denotes significance at individual concentrations as indicated by post hoc analysis.

View this table: [in this window] [in a new window]   TABLE 2 KA-induced calcium responses from control and treated cultures KA-induced calcium responses were not consistently altered across treatment paradigms (see "Methods"). Values represent the mean ± S.E.M. of increases in [Ca++]i ( [Ca++]i = peak agonist-stimulated [Ca++]i minus basal [Ca++]i) induced by 25 µM KA in control and treated neurons. Values in parentheses are number of neurons from two to five separate sister culture preparations. Statistical significance determined by two-tailed t test (*P < .05).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of chronic treatment of cultured hippocampal neurons with ethanol plus APV and APV alone on NMDA-induced increases in [Ca++]i. A, Hippocampal cultures were chronically exposed to 100 mM ethanol plus 70 µM APV (Et+APV) for 7 days. Following removal of antagonists NMDA-induced calcium responses ( [Ca++]i nM) were assessed in control and treated neurons as described in methods. B, NMDA-induced increases in [Ca++]i following chronic APV exposure. The treatment paradigm consisted of a chronic exposure to 70 µM APV for 7 days as described in methods. Calcium responses ( [Ca++]i nM) were induced by NMDA application in control and chronically treated neurons. Values represent the mean ± sem from: 12 determinations from 12 neurons for each data point in control curve (A, B) and 7 to 8 determinations from 8 neurons in the ethanol+APV curve (A), and 7 to 8 determinations from 9 neurons in the chronic APV curve (B). The data were derived from 2 separate experiments. A significant treatment effect was demonstrated by ANOVA (chronic ethanol+APV, F = 12.21, P < .001; chronic APV, F = 35.77, P < .001). Asterisk (*) denotes significance at individual concentrations as indicated by post hoc analysis.

Increased [Ca⁺⁺]_i induced by activation of VSCCs. Because all VSCCs can be activated by membrane depolarization, 50 mM KCl was used to elicit activation of VSCCs. KCl application produced increases in [Ca⁺⁺]_i of a magnitude similar to that of 10 to 20 µM NMDA (data not shown). When KCl-stimulated responses were examined in control versus CEE neurons, no differences were observed, which indicates that a 100 mM ethanol/7-day CEE does not enhance the VSCC-mediated calcium response (two-tailed t test, P = .54).

Differences in total cell number and basal intracellular calcium. The range of values for the mean total cell number was 98.0 ± 5.0 to 204.2 ± 7.6 in control cultures and 97.9 ± 4.6 to 179.1 ± 6.4 in CEE cultures. Significant differences in total cell number were found in two (2-day CEE and chronic ethanol + APV) of six treatment paradigms (two-tailed t test, P < .05). The largest difference was observed in the ethanol + APV paradigm, with a mean difference in total cell number of 38.1. However, no correlation between total cell number and percent cell death was evident in either paradigm (r² = 0.01, 2-day CEE; r² = 0.0009, chronic ethanol + APV). The differences in cell number most likely reflect variability in plating density rather than neurotoxicity during the chronic treatment.

	Discussion

Abstract Introduction Methods Results Discussion References

This study demonstrates that CEE sensitizes hippocampal neurons to excitotoxicity mediated by NMDA receptors. We have shown that the effect of CEE on NMDA-induced excitotoxicity does not involve AMPA/kainate or L-type VSCCs. The enhancement of excitotoxicity that was observed in hippocampal neurons is in agreement with other studies which demonstrate an enhancement of NMDA-induced excitotoxicity after CEE (Ahern et al., 1994; Blevins et al., 1995; Chandler et al., 1993; Iorio et al., 1993; Davidson et al., 1993). Our observations extend these earlier findings by demonstrating that no enhancement of excitotoxicity is observed when non-NMDA receptors are explicitly activated with selective agonists. The antagonist, APV, blocked NMDA-induced excitotoxicity and was equally effective in both the control and CEE cultures, which indicates that CEE does not alter the ability of the receptor to be blocked by this antagonist. Furthermore, the ability of ethanol to reduce cell death was not altered by CEE, which indicates that a loss of ethanol inhibition of NMDA receptors is not involved in the adaptation of NMDA receptors to CEE, as indicated previously by Chandler et al. (1993).

The enhancement of NMDA-induced excitotoxicity could be observed after 2 days of chronic exposure to 100 mM ethanol. This indicates that a relatively short time is necessary for adaptive mechanisms to occur leading to enhanced excitotoxicity after CEE. This finding could have implications for repetitive "binge" intoxication. It has been reported that short, repetitive ethanol administration will result in neuronal cell loss (Collins et al., 1996; Lundqvist et al., 1995).

Excessive stimulation of AMPA/KA receptors results in neurotoxicity (reviews, Coyle and Puttfarcken, 1993; Whetsell, 1996). Although these receptors are sensitive to inhibition by ethanol (Dildy-Mayfield and Harris, 1995; Lovinger et al., 1989; Martin et al., 1995) we did not observe evidence of an adaptive response to CEE with respect to excitotoxicity because there was no enhancement of AMPA- or KA-induced excitotoxicity after a 7-day chronic exposure to 100 mM ethanol. Furthermore, a large proportion of the neurotoxicity induced by non-NMDA agonists (i.e., AMPA, KA) appeared to involve activation of NMDA receptors, because APV could effectively reduce the percent cell death. The ability of APV to reduce KA-induced excitotoxicity likely reflects secondary synaptic glutamate release, because receptor activation can stimulate neuronal firing which ultimately results in transmitter release. Although AMPA and KA exposure produced excitotoxicity which appeared to be partly mediated by NMDA receptors, CEE did not result in an increased AMPA- or KA-induced excitotoxicity from enhanced NMDA receptor expression. It is possible that this reflects a less than full expression of NMDA receptor function, because glycine was not included during excitotoxic AMPA or KA exposure. It is also possible that the population of NMDA receptors activated by the secondary synaptic glutamate release is different than that which would be activated by direct NMDA application (e.g., synaptic vs. synaptic and somal receptors).

Antagonists to VSCCs decrease the severity of ethanol withdrawal hyperexcitability, which suggests that VSCCs are altered after chronic ethanol treatment (Huang and McArdle, 1993; Ripley et al., 1996; Whittington et al., 1995; Whittington and Little, 1993). However, we did not observe an alteration of KCl-induced increases in [Ca⁺⁺]_i after CEE. This agrees with other studies which have also reported no perturbation of depolarization-induced increases in [Ca⁺⁺]_i after CEE (Hu and Ticku, 1995; Iorio et al., 1992, 1993).

The role of VSCCs in neuronal cell death after CEE is unknown. We observed that KCl-induced depolarization did not result in neuronal cell death in either control or CEE cultures. The absence of cell death resulting from prolonged depolarization has been reported by other investigators (Churn et al., 1995; Tymianski et al., 1993). The failure of KCl to induce cell death was not the result of an inability to increase [Ca⁺⁺]_i, because a 50 mM KCl exposure produced increases in [Ca⁺⁺]_i similar in magnitude to that of low NMDA concentrations. Although during the course of the 5-min KCl exposure which was used to induce excitotoxicity, other factors such as channel inactivation could influence the total [Ca⁺⁺]_i. The lack of neuronal damage associated with KCl exposure is consistent with the idea that the route of calcium entry in hippocampal neurons is an important factor in determining neuronal cell death (Tymianski et al., 1993). Furthermore, this relationship is not altered after CEE.

Although activation of VSCCs does not induce cell death it is possible that VSCCs could play an associative role in NMDA-mediated excitotoxicity. When nifedipine was applied along with NMDA to control neurons a decrease in excitotoxicity was observed. However, no effect of nifedipine on NMDA-induced excitotoxicity was observed in CEE cultures. The lack of an effect of nifedipine on NMDA receptor-mediated excitotoxicity after CEE is consistent with what has been shown in cerebellar granule cells; however, nifedipine did not decrease excitotoxicity in untreated granule cells (Iorio et al., 1993). Thus, in hippocampal neurons a portion of the NMDA-induced excitotoxicity is mediated by activation of L-type VSCCs in control cultures. The decrease in the involvement of these channels in excitotoxicity after CEE to hippocampal neurons may result from a decrease in the numbers of channels or alterations in channel pharmacological properties or function. The contribution of L-type VSCCs to NMDA-induced excitotoxicity after CEE could be masked by the enhanced NMDA receptor-mediated excitotoxicity.

The ability of concomitant ethanol/APV exposure to prevent an enhancement of NMDA-induced cell death does suggest that the enhanced excitotoxicity after CEE requires active NMDA receptors. Furthermore, the lack of enhanced excitotoxicity observed after chronic ethanol + APV exposure is associated with the failure of this paradigm to produce a functional enhancement of the NMDA-induced calcium response. This finding suggests that functional NMDA receptors are necessary for the enhancement of receptor function resulting from CEE. Overall, these findings indicate that the enhanced excitotoxicity in hippocampal neurons that resulted after CEE selectively depends on alterations in functional NMDA receptors and not non-NMDA glutamate receptors or calcium channels.

The mechanisms that underlie enhancement of NMDA-induced excitotoxicity after CEE may involve alterations in the function of NMDA receptors. Enhancement of NMDA-induced increases in [Ca⁺⁺]_i was observed in all treatment paradigms except chronic ethanol + APV, whereas KA-induced increases in [Ca⁺⁺]_i were not altered consistently. The enhancement of NMDA-induced [Ca⁺⁺]_i agrees with evidence from other studies which have reported similar results (Ahern et al., 1994; Blevins et al., 1995; Hu and Ticku, 1995; Iorio et al., 1992, 1993). Evidence from receptor binding studies indicates that there are increases in receptor number with no change in affinity after CEE or chronic ethanol abuse (Freund and Anderson, 1996; Grant et al., 1990; Hoffman et al., 1995; Michaelis et al., 1990; Snell et al., 1993). However, the NMDA dose-response curve that we observed indicated no increase in the maximal NMDA-induced [Ca⁺⁺]_i increase, which would have been expected from a simple increase in receptor number. Comparison of the calcium dose-response curve after 7-day CEE with that from chronic APV exposure shows that enhancement of [Ca⁺⁺]_i in the 7-day CEE paradigm was present predominately at low NMDA concentrations (<20 µM), whereas in the chronic APV paradigm enhancement of [Ca⁺⁺]_i was present at all NMDA concentrations, which is consistent with a general increase in receptor number (Williams et al., 1992). These findings suggest a complex alteration of NMDA receptor function with CEE which may involve changes other than an increased density of receptors. It is possible that changes in NMDA receptor subunit composition could underlie these effects because receptor subunit composition can affect NMDA receptor properties such as agonist affinity, calcium permeability and increases in [Ca⁺⁺]_i (Grant et al., 1997; Koltchine et al., 1996; Kuner and Schoepfer, 1996; Monyer et al., 1994). Furthermore, several studies have reported that mRNA levels and protein expression of only certain NMDA subunits are elevated after chronic ethanol paradigms (Follesa and Ticku, 1995, 1996; Snell et al., 1996; Trevisan et al., 1994). However, evidence for changes in NMDA receptor subunit composition which could mediate the enhanced calcium response that we observed after CEE has yet to be demonstrated in cultured hippocampal neurons.

A correlation between the concentration of intracellular calcium and cell death was not observed under all of the conditions used in this study, which is consistent with evidence that the major determinants of cell death are the duration of agonist exposure, the route of calcium entry and the buffering capability of the neuron, and not the peak calcium concentration (Mattson et al., 1991; Tymianski et al., 1993). The alteration of the NMDA calcium response was correlated with the alteration of excitotoxicity in all but two paradigms. Enhancement of the NMDA-induced increase in [Ca⁺⁺]_i was observed in the 1-day CEE paradigm in which no enhancement of excitotoxicity was observed. In the chronic ethanol + APV paradigm a decrease in the NMDA calcium response was observed in treated neurons relative to control neurons; however, the excitotoxicity curve for both treated and control was not different. It is probable that in the former case a threshold level of intracellular calcium needed to enhance NMDA-induced excitotoxicity was not exceeded after the 1-day CEE paradigm. In the latter case, no change in excitotoxicity would necessarily be expected because peak intracellular calcium concentration is not tightly correlated with neuronal death.

Overall, the observations reported in this study indicate that in cultured hippocampal neurons the effect of CEE on agonist-induced increases in [Ca⁺⁺]_i and excitotoxicity are specific to NMDA receptors. Furthermore, the effect of CEE likely involves alterations in NMDA receptor properties. The enhanced excitotoxicity observed after CEE is associated with an enhanced NMDA receptor calcium response and little if any contribution from other ion channels.