Chronic Ethanol Exposure Attenuates the Anti-Apoptotic Effectof NMDA in Cerebellar Granule Neurons

Sanjiv V. Bhave, Lawrence D. Snell, Boris Tabakoff, and Paula L. Hoffman

Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado, U.S.A.

Abstract: Ethanol, added to primary cultures of cerebel-lar granule neurons simultaneously with NMDA, was pre-viously shown to inhibit the anti-apoptotic effect ofNMDA. The in vitro anti-apoptotic effect of NMDA isbelieved to mimic in vivo protection against apoptosisafforded by innervation of developing cerebellar granuleneurons by glutamatergic mossy fibers. Therefore, theresults suggested that the presence of ethanol in thebrain at a critical period of development would promoteapoptosis. In the present studies, we examined the effectof chronic ethanol exposure on the anti-apoptotic actionof NMDA in cerebellar granule neurons. The neuronswere treated with ethanol in vitro for 1–3 days in theabsence of NMDA. Even after ethanol was removed fromthe culture medium, as ascertained by gas chromatogra-phy, the protective effect of added NMDA was signifi-cantly attenuated. The decreased anti-apoptotic effect ofNMDA was associated with a change in the properties ofthe NMDA receptor, as indicated by a decrease in ligandbinding, decreased expression of NMDA receptor sub-unit proteins, and decreased functional responses includ-ing stimulation of increases in intracellular Ca21 and in-duction of brain-derived neurotrophic factor expression.The latter effect may directly underlie the attenuated pro-tective effect of NMDA in these neurons. The resultssuggest that ethanol exposure during development canhave long-lasting effects on neuronal survival. Thechange in the NMDA receptor caused by chronic ethanoltreatment may contribute to the loss of cerebellar granuleneurons that is observed in animals and humans exposedto ethanol during gestation. Key Words: Apoptosis—Cerebellar granule neurons—N-Methyl-D-aspartatereceptor—Ethanol.J. Neurochem. 75, 1035–1044 (2000).

Ethanol has repeatedly been shown to acutely inhibitthe function of theN-methyl-D-aspartate (NMDA) recep-tor in various neuronal preparations (see Hoffman andTabakoff, 1996). Following chronic exposure of adultanimals or cultured neurons to ethanol and ethanol with-drawal, “up-regulation” of neuronal NMDA receptornumber and function as well as increases in expression ofNMDA receptor subunits have been reported (Grantet al., 1990; Iorio et al., 1992; Chandler et al., 1993;Ahern et al., 1994; Follesa and Ticku, 1995, 1996; Hoff-

man et al., 1995; Hu and Ticku, 1995; Snell et al., 1996a;Smothers et al., 1997). This increased receptor functionhas been suggested to represent an adaptive response tocontinued exposure of the receptor to ethanol inhibition(Hoffman and Tabakoff, 1996) and in vivo to contributeto signs of ethanol withdrawal (Grant et al., 1990; Gulyaet al., 1991; Snell et al., 1996b).

In contrast, when animals are exposed chronically toethanol during gestation, NMDA receptor function gen-erally appears to be decreased in the offspring. Forexample, prenatal exposure of rats to ethanol resulted ina reduction of NMDA-mediated depolarization, mea-sured in a hippocampal slice preparation (Morrisett et al.,1989), as well as reduced ligand binding to the hip-pocampal NMDA receptor (Savage et al., 1991; Diaz-Granados et al., 1997). A similar reduction in NMDAreceptor binding was observed in hippocampus of guineapigs that had been exposed to ethanol during gestation(Valles et al., 1995). In addition, NMDA receptor-medi-ated stimulation of increases in intracellular calcium wasreduced in neurons obtained from rats treated prenatallywith ethanol, as was expression of the NMDA receptorsubunits NR2A and NR2B (but not NR1) (Lee et al.,1994; Spuhler-Phillips et al., 1997; Hughes et al., 1998).These findings suggest that in contrast to the adaptivechanges in the adult brain and some cultured neurons,chronic ethanol exposure during gestation may interferewith the development of the NMDA receptor, leading toa decrease in receptor number and/or function.

We have previously used primary cultures of cerebel-lar granule neurons as a model to investigate the inter-actions of ethanol with the NMDA receptor (Hoffmanet al., 1989, 1995, 1996; Iorio et al., 1992). These neu-rons can be maintained in culture for relatively longperiods if they are grown in the presence of a depolar-

Received February 18, 2000; revised manuscript received May 3,2000; accepted May 4, 2000.

Address correspondence and reprint requests to Dr. P. L. Hoffman atDepartment of Pharmacology, University of Colorado Health SciencesCenter, 4200 E. 9 Ave., Box C236, Denver, CO 80262, U.S.A. E-mail:Paula.Hoffman@UCHSC.edu

Abbreviations used:BDNF, brain-derived neurotrophic factor; PBS,phosphate-buffered saline; SDS, sodium dodecyl sulfate.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 2000 International Society for Neurochemistry

izing concentration (25 mM) of KCl (Balazs et al.,1988). Under these conditions, ethanol acutely inhibitsNMDA receptor function, and the NMDA receptor isup-regulated following chronic ethanol exposure andwithdrawal, as in the adult brain (Hoffman et al., 1989,1996; Iorio et al., 1992). In addition, these neurons, likeadult neurons, are susceptible to glutamate/NMDA-in-duced excitotoxicity, which is increased after chronicethanol exposure (Iorio et al., 1993). When the cerebellargranule neurons are cultured in the presence of a phys-iological concentration of KCl (5 mM), however, theyare considered to be “immature” neurons, which undergoapoptotic death after 4–5 days in culture (Bala´zs et al.,1988; Yan et al., 1994). This death can bepreventedbytreatment of the cells with NMDA, and we have shownthat ethanol, added simultaneously with NMDA, attenu-ates this protection through inhibition of NMDA receptorfunction, leading to enhanced apoptosis (Bhave andHoffman, 1997). The purpose of the present study was todetermine how chronic ethanol treatment and withdrawalof “immature” cerebellar granule neurons affect NMDAreceptor function. Our results demonstrate that this treat-ment can lead to a long-term disruption of NMDA re-ceptor function, producing increased apoptosis. Thesefindings may provide a mechanism underlying neuronalloss that can arise from chronic alcohol intake duringpregnancy.

MATERIALS AND METHODS

MaterialsGlycine and NMDA were obtained from Research Bio-

chemicals (Natick, MA, U.S.A.). Basal essential medium andheat-inactivated fetal bovine serum were obtained from Gibco-BRL (Grand Island, NY, U.S.A.). Fura-2-acetoxymethyl esterwas obtained from Molecular Probes (Eugene, OR, U.S.A.).The ApopTag kit was obtained from Intergen Co. (Purchase,NY, U.S.A.). The brain-derived neurotrophic factor (BDNF)Emax immunoassay kit was obtained from Promega Corp.(Madison, WI, U.S.A.). Anti-NR1 monoclonal antibody waspurchased from Pharmingen (San Diego, CA, U.S.A.), andanti-NR2A and NR2B polyclonal antibodies were kindly pro-vided by Dr. Michael Browning (University of Colorado HealthSciences Center, Denver, CO, U.S.A.). Enhanced chemilumi-nescence reagents and [3H]MK-801 were obtained from Du-Pont-NEN (Boston, MA, U.S.A.). Anti-b-actin monoclonalantibody and all other reagents were obtained from SigmaChemical Corp. (St. Louis, MO, U.S.A.).

Cell culturePrimary cultures of cerebellar granule neurons were pre-

pared as described previously (Bhave and Hoffman, 1997;Bhave et al., 1999a,b). In brief, cerebella were dissected from7-day-old Sprague–Dawley rat pups. Cells were dissociated bytrypsinization and resuspended in basal essential medium con-taining 10% heat-inactivated fetal bovine serum, 5 mM KCl, 2mM glutamine, and 100mg/ml gentamicin (GibcoBRL). Forassessing apoptosis, neurons were plated on glass coverslips (23 106 cells/well) or on eight-chambered microscope slides(Falcon Culture Slide; 0.53 106 cells/well) coated with poly-ethylenimine (100mg/ml). For intracellular calcium ([Ca21]i)measurements, cells (23 106 cells/well) were plated on glass

coverslips coated with polyethylenimine. Cerebellar granuleneurons plated in tissue culture dishes coated with poly-L-lysine (10mg/ml) were used for the extraction of total proteinfor analyzing BDNF levels (53 106 cells/well in a six-wellmultiwell dish) and expression of NMDA receptor subunitproteins (2 3 107 cells/100-mm dish). Neurons (23 106

cells/well) were plated in 24-well tissue culture dishes coatedwith polyethylenimine for the analysis of [3H]MK-801 binding.

In most experiments, cerebellar granule neurons were treatedchronically with 100 mM ethanol for 3 days, as previouslydescribed (Iorio et al., 1992; Hoffman et al., 1996). In brief,ethanol (6.2ml of 95% ethanol/ml of medium) was added to themedium 24 h after the cells were plated (day 1 in vitro). Themedium was supplemented daily with ethanol to maintain theproper concentration [assessed by gas chromatography (Taba-koff et al., 1976)]. The cultures treated with ethanol weremaintained within a larger dish containing 100 mM ethanol toreduce the loss of ethanol due to evaporation from the medium.Following the chronic ethanol exposure, on day 4 in vitro inmost experiments, cells were washed repeatedly to removeethanol prior to any further treatment. Control cells weretreated identically. This washing procedure reduced the numberof cells by;7% in both control and ethanol-treated cultures, asassessed by measuring fluorescein fluorescence (Iorio et al.,1993). The ethanol concentration in the medium followingwashing was ascertained by gas chromatography (Tabakoffet al., 1976). In some experiments, cells were exposed to 25,50, or 100 mM ethanol for 72 h (days 1–4 in vitro) beforeethanol was removed and NMDA was added on day 4 in vitro.To evaluate the time course of the effect of ethanol, cells wereexposed to 100 mM ethanol for 24, 48, or 72 h (i.e., days 3–4,2–4, or 1–4 in vitro) before washout and the addition ofNMDA on day 4 in vitro. To assess whether the effect ofexposure to ethanol was reversible, neurons were grown in thepresence or absence of 100 mM ethanol for 72 h (from day 1 to4 in vitro). Ethanol was washed out, and some cells weretreated with NMDA for 24 h. In these cells, apoptosis wasassessed on day 5 in vitro. Other cells were returned to condi-tioned medium for 24 h before the addition of NMDA (on day5 in vitro). Apoptosis was assessed in these cells 24 h afteraddition of NMDA, on day 6 in vitro. The conditioned mediumused for these experiments was obtained from control cells onday 4 in vitro and was stored frozen at270°C until used.Conditioned medium was used to avoid the possibility of celldeath due to the presence of glutamate in fresh medium(Schramm et al., 1990; Marini and Paul, 1992), although thiseffect has been reported only with cells maintained in mediumcontaining 25 mM KCl.

Measurement of apoptosisExcept where indicated, in experiments designed to assess

the protective effect of NMDA, NMDA dissolved in condi-tioned medium containing 5 mM KCl was added (10ml/ml) tothe culture medium on day 4 in vitro, and apoptosis wasdetermined 24 h later (day 5 in vitro) (Bhave et al., 1999a).Vehicle (conditioned medium) was added to control cultures asappropriate.

To assess apoptosis, the neurons were fixed and apoptoticcell death was determined with the ApopTag kit, according tothe manufacturer’s instructions (Bhave and Hoffman, 1997).This method provides for in situ fluorescent labeling of the39-OH ends of fragmented DNA. Total cell number is assessedby staining the fixed cells with propidium iodide. Fluorescencewas detected with an epifluorescence microscope (Nikon;1003 objective). The total (propidium iodide-labeled) and apo-

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1036 S. V. BHAVE ET AL.

ptotic (fluorescein-labeled) cells were manually counted inthree randomly chosen fields on each coverslip by an investi-gator who was unaware of the treatments.

Measurements of [Ca21]i[Ca21]i was determined by measuring fura-2 fluorescence,

as previously described (Snell et al., 1994). In brief, cerebellargranule neurons were loaded with 5mM fura-2-acetoxymethylester for 60 min at 37°C. The glass coverslips were then rinsedwith Mg21-free cell buffer (145 mM NaCl, 5 mM KCl, 10 mMglucose, 10 mM HEPES, 1 mM Na2HPO4, and 1 mM CaCl2,pH 7.4) and transferred to a cuvette containing 2 ml of cellbuffer maintained at 37°C with constant stirring. Fluorescencewas measured using an SLM-Aminco spectrofluorometer (ex-citation at 340 and 380 nm; emission at 505 nm). The data werestored on a computer using the software from SLM-Aminco(Urbana, IL, U.S.A.). Determinations ofRmax and Rmin werecarried out as previously described (Snell et al., 1994).

Glass coverslips with adherent neurons were placed in acuvette fitted with a superfusion system attached to a peristalticpump. The coverslip was perfused with cell buffer for 2 min ata flow rate of 4 ml/min, and the basal levels of [Ca21]i wererecorded for 30 s. Glycine (10mM) was added, and the changein the level of [Ca21]i was recorded for 30 s. Increasingconcentrations of NMDA (0.03–30mM NMDA) were thenadded in a stepwise manner, and changes in the level of [Ca21]iafter each addition were recorded. Results are expressed as theratio of emission at 340-nm and 380-nm excitation (340/380ratio), a measure of intracellular free Ca21 levels (Grynkiewiczet al., 1985).

Whole-cell [3H]MK-801 bindingThe number of NMDA receptors was estimated by quanti-

fying the number of specific (1)-[3H]MK-801 binding sites, aspreviously described (Chuang et al., 1992; Hoffman et al.,1995). Ethanol-exposed cells and corresponding control cellswere rinsed three times with 0.5 ml of ice-cold phosphate-buffered saline (PBS). Prewarmed PBS (0.5 ml, 37°C) contain-ing 100mM L-glutamate, 100mM glycine, and 30mM MgSO4

was added to each well, followed by either 4 or 40 nM (1)-[3H]MK-801 (22.5 Ci/mmol). All assays were allowed to reachequilibrium for 15 min at 37°C. The binding was terminated bywashing the cells three times with 0.75 ml of ice-cold PBS.Finally, 0.5 ml of hot 1% sodium dodecyl sulfate (SDS) wasadded to each well to solubilize the cells. Following shaking for60 min at room temperature, aliquots from each well wereremoved for protein determination (bicinchoninic acid; Pierce,Rockford, IL, U.S.A.). The remaining content of each well wastransferred to scintillation vials, scintillant was added (UltimaGold; Packard, Meridian, CT, U.S.A.), and radioactivity wasdetermined in a liquid scintillation spectrometer. Specific bind-ing was calculated as total binding minus nonspecific bindingdetermined in the presence of unlabeled (1)MK-801 (20mM).

Specific (1)-[3H]MK-801 binding in each well was calcu-lated as picomoles per milligram of protein and was averagedfor similarly treated wells. The total protein in cells maintainedin the absence (control) or presence of ethanol was similar(35.5 6 1.9 and 32.76 1.7 mg/well in control and ethanol-treated cells, respectively). (2)-MK-801 or ketamine wasadded to some wells at their approximate IC50 concentrations(0.2 and 10mM, respectively) to assess specificity of (1)-[3H]MK-801 binding to the NMDA receptor. As expected,these ligands displaced;50% of (1)-[3H]MK-801 binding(data not shown).

Western blot analysisCultures were washed with ice-cold PBS, extracted in hot

SDS (1%), and then frozen at280°C (Hoffman et al., 1996;Bhave et al., 1999a). For immunoblotting, the samples werethawed and protein concentrations were determined (bicincho-ninic acid method). Each sample was then solubilized by ad-dition of dithiothreitol and urea (final concentrations, 150 mMand 2.4M, respectively) followed by boiling for 3 min. Solu-bilized samples (20mg of total protein/lane) were subjected toSDS polyacrylamide gel electrophoresis (7.5% for NR1,NR2A, and NR2B and 10% forb-actin), and proteins weretransferred to nitrocellulose membranes (0.22mm; Schleicherand Schuell, Keene, NH, U.S.A.) according to proceduresdescribed previously (Snell et al., 1996a). After blocking, blotswere probed with subunit-specific antibodies to NR1, NR2A,NR2B (Snell et al., 1996a), or b-actin (1:5,000). Blots werethen incubated with horseradish peroxidase-conjugated goatIgGs (goat anti-mouse for NR1 andb-actin and goat anti-rabbitfor NR2A and NR2B). Immunoreactive bands were visualizedusing the enhanced chemiluminescence method. The NR1 an-tibody recognized a major band of;110 kDa and a secondband of;100 kDa. Both bands were included in the quantita-tion, as these bands had previously been reported when theantibody was used to detect NR1 protein in membranes pre-pared from HEK293 cells transfected with NR1 cDNA (Siegelet al., 1994). The NR2A and NR2B antibodies each recognizeda single band of;165 kDa. Theb-actin antibody recognized asingle band of;42 kDa. Bands were quantitated by imageanalysis using a Bio-Rad GS-250 molecular imager and Phos-phorAnalyst image analysis software. The results are calculatedas volume (area3 phosphor counts) of the appropriate bands,and levels of NMDA receptor subunits are expressed as per-centage of control (Snell et al., 1996a).

Analysis of BDNF levelsThe level of BDNF protein in the cerebellar granule neurons

was determined using the BDNFEmax immunoassay kit in anantibody sandwich format. Cerebellar granule neurons werewashed and extracted in a lysis buffer (20 mM Tris, 137 mMNaCl, 1% Nonidet P-40, 10% glycerol, 1 mM phenylmethyl-sulfonyl fluoride, 10mg/ml aprotinin, 1mg/ml leupeptin, and0.5 mM sodium vanadate), and determination of intracellularBDNF levels was carried out after acid treatment according tothe manufacturer’s instructions.

Statistical analysisAll values are presented as means6 SEM. As most data are

expressed as ratios or percentages, statistical significance wasdetermined by the Mann–WhitneyU test or by the Kruskal–Wallis nonparametric ANOVA or two-way ANOVA on datasubjected to the arcsin square root transformation, followed bypost hoc multiple comparisons, using the SigmaStat program(Jandel Scientific Software, San Rafael, CA, U.S.A.). EC50

values were calculated using the NFIT curve-fitting programand were compared using the confidence limits of the regres-sion lines (Sokal and Rohlf, 1981). A value ofp , 0.05 wasconsidered significant.

RESULTS

Effect of chronic ethanol exposure and withdrawalon anti-apoptotic effect of NMDA

As previously reported (Yan et al., 1994), cerebellargranule neurons maintained in medium containing 5 mMKCl undergo apoptosis over time in culture. In our ex-

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periments, the percentage of apoptotic cells was as fol-lows: day 3, 26%; day 4, 32%; day 5, 38%; day 6, 54%.As shown in our previous work (Bhave and Hoffman,1997; Bhave et al., 1999b), addition of 100mM NMDAto the cerebellar granule neurons on day 4 in vitroreduced apoptosis (measured on day 5 in vitro) by;60%. However, when the cells were treated with 100mM ethanol from day 1 to day 4 in vitro, the anti-apoptotic effect of NMDA was significantly attenuated;that is, NMDA produced only;30% protection (Fig. 1).This decrease in the anti-apoptotic effect of NMDA wassimilar to what we had previously observed when etha-nol was added to the cells simultaneously with NMDAfor 24 h (Bhave and Hoffman, 1997; Bhave et al.,1999b). However, in the present experiments, ethanolwas removed from the cells prior to the addition of

NMDA. At the end of the ethanol treatment period, theethanol concentration in the medium was 1036 2 mM (n5 3), but after the cells were washed, and before theaddition of NMDA, the ethanol concentration in themedium was below the level of detection (,5 mM).Therefore, the presence of ethanol was not necessary toobserve a reduction in the protective effect of NMDA.

Chronic ethanol treatment alone did not significantlyincrease apoptosis (Fig. 1) and also did not significantlyalter the number of viable cerebellar granule neurons, asdetermined with fluorescein diacetate (Iorio et al., 1993).Fluorescein fluorescence in the ethanol-exposed cellswas 966 3% of that in control cells (n5 12).

The effect of chronic ethanol treatment on the anti-apoptotic action of NMDA was concentration and timedependent. Figure 2A shows that 3 days of exposure to

FIG. 1. Effect of chronic ethanol exposure on the anti-apoptotic effect of NMDA incerebellar granule neurons. Cerebellar granule neurons were maintained in mediumcontaining 5 mM KCl plus or minus 100 mM ethanol for days 1–4 in vitro. On day 4in vitro, cells were washed to remove ethanol and then treated with 100 mM NMDAfor 24 h, as described in Materials and Methods. Apoptosis was assessed with theApopTag kit on day 5 in vitro. A: Top panels show apoptotic cells (fluorescein labeled)and the bottom panels show total number of cells (propidium iodide labeled) in arepresentative field in a typical experiment. B: Results are expressed as number(percent) of apoptotic (fluorescein-positive) cells per total cell number (propidiumiodide-labeled cells). Values represent the means 6 SEM of 17–23 observations infour separate experiments. Kruskal–Wallis ANOVA revealed a significant effect oftreatment (H 5 56.85, df 5 3, p , 0.001). *p , 0.05 compared with control withoutNMDA; **p , 0.05 compared with chronic ethanol without NMDA; 1p , 0.05compared with control with NMDA (post hoc comparisons).

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25 mM ethanol, and withdrawal, produced a significantreduction in the anti-apoptotic effect of NMDA, and thiseffect was further reduced by treatment with 50 or 100mM ethanol. As shown in Fig. 2B, the protective effectof NMDA was reduced by;55% following 24 h ofexposure to 100 mM ethanol and washout, whereas thelonger periods of ethanol exposure reduced the responseto NMDA by ;75%.

We also investigated the reversibility of the effect ofchronic ethanol exposure. As described in Materials andMethods, some cells in these experiments were main-tained for 24 h in conditioned medium following ethanoltreatment and washout, before NMDA was added on day5 in vitro. The results in Fig. 3 show that the anti-apoptotic effect of NMDA in control cells appeared to beless when NMDA was added on day 5 in vitro as com-pared with day 4 in vitro, although this difference wasnot statistically significant. This apparent decreased ef-fectiveness of NMDA is presumably due to the fact thatover this period, apoptotic processes in a greater numberof cells have reached a point where the cells can nolonger be rescued by NMDA. Nevertheless, the protec-tive effect of NMDA in the cells treated chronically with

ethanol was reduced to about the same extent, comparedwith control cells, even after the cells had been ethanol-free for 24 h.

Effect of chronic ethanol treatment on NMDAreceptor characteristics

In experiments to assess the properties and function ofthe NMDA receptor, cerebellar granule neurons wereexposed to ethanol as described above, and ethanol waswashed out (withdrawal) prior to treatment of the cellswith NMDA or other manipulations. The functionalproperties of the receptor were first assessed by measur-ing NMDA-induced increases in intracellular calcium([Ca21]i) with fura-2 fluorescence, as previously de-scribed (Snell et al., 1994). NMDA produced a concen-tration-dependent increase in [Ca21]i (EC50 0.58mM) incontrol neurons. The response to all concentrations ofNMDA was significantly reduced in the cells exposedchronically to 100 mM ethanol (Fig. 4). As previouslyreported (Iorio et al., 1992), chronic exposure to 25 or 50mM ethanol also reduced the response to NMDA (datanot shown). In addition, NMDA potency was signifi-cantly decreased in the ethanol-exposed cells (EC50 1.59mM; p , 0.05, confidence limits) (Fig. 4).

Another measure of NMDA receptor function is theability of NMDA to induce expression of BDNF. Thiseffect is particularly important in the context of thepresent work, as we have previously shown that BDNFmediates the anti-apoptotic effect of NMDA in theseneurons (Bhave et al., 1999b). As shown in Fig. 5A,

FIG. 3. Reversal of the effect of chronic ethanol exposure on theanti-apoptotic action of NMDA. Cerebellar granule neurons weremaintained in medium containing 5 mM KCl in the presence orabsence of 100 mM ethanol for days 1–4 in vitro (DIV). Afterethanol was washed out, some cells were treated with NMDA(100 mM ) for 24 h, as described in Materials and Methods, andapoptosis was assessed using the ApopTag kit on day 5 in vitro.After removal of ethanol, other cells were maintained for 24 h inconditioned medium in the absence of ethanol before addition ofNMDA on day 5 in vitro. In these cells, as well as in a parallelgroup of control (non-ethanol-treated) cells, apoptosis was as-sessed on day 6 in vitro. Results are expressed as the percentdecrease in apoptosis produced by NMDA. Kruskal–WallisANOVA revealed a significant effect of treatment (H 5 37.8, df5 3, p , 0.001). *p , 0.05 compared with respective control(post hoc comparisons).

FIG. 2. Concentration- and time-dependent effects of ethanolexposure on the anti-apoptotic action of NMDA. A: Cerebellargranule neurons were maintained in medium containing 5 mMKCl in the presence or absence of the indicated concentrationsof ethanol for days 1–4 in vitro and were then treated with NMDA(100 mM ) for 24 h, as described in Materials and Methods. Cellswere washed to remove ethanol prior to addition of NMDA.Apoptosis was assessed with the ApopTag kit on day 5 in vitro.Results are expressed as the percent decrease in apoptosisproduced by NMDA. Values represent the means 6 SEM of15–17 observations in two separate experiments. Kruskal–WallisANOVA revealed a significant effect of treatment (H 5 41.5, df5 3, p , 0.001). *p , 0.05 compared with all other groups; **p, 0.05 compared with 50 and 100 mM ethanol (post hoc com-parisons). B: Control cerebellar granule neurons were main-tained in medium containing 5 mM KCl for days 1–4 in vitro andthen treated with NMDA (100 mM ) for 24 h, as described inMaterials and Methods. Other groups of cerebellar granule neu-rons were exposed to ethanol (100 mM ) for 24, 48, or 72 h (days3–4, 2–4, or 1–4 in vitro, respectively) before washout of ethanoland addition of NMDA for 24 h. Apoptosis was assessed with theApopTag kit on day 5 in vitro. Results are expressed as thepercent decrease in apoptosis produced by NMDA. Values rep-resent the means 6 SEM of nine observations. Kruskal–WallisANOVA revealed a significant effect of time of ethanol exposure(H 5 25.35, df 5 3, p , 0.001). *p , 0.05 compared with all othergroups; **p , 0.05 compared with 2 and 3 days of ethanolexposure (post hoc comparisons).

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chronic ethanol treatment decreased NMDA-induced ex-pression of BDNF in a concentration-dependent manner.This effect appeared to be maximal after 24 h of ethanol(100 mM) treatment (Fig. 5B). Chronic ethanol treatmentalone (100 mM ethanol on days 1–4 in vitro) had noeffect on the baseline levels of BDNF (control, 8.46 1.0pg; chronic ethanol, 8.96 1.1 pg; n5 6).

To determine whether the decreased responses toNMDA reflected a decreased number of NMDA recep-tors, [3H]MK-801 binding to intact cells was measuredas previously described (Hoffman et al., 1995). Cerebel-lar granule neurons exposed to chronic treatment with100 mM ethanol, and withdrawal, showed significantlyreduced binding at both concentrations of ligand used (aconcentration close to theKD value and a concentrationthat produces maximal binding) (Hoffman et al., 1995)(Fig. 6). These results are consistent with a decreasednumber of NMDA receptors in the ethanol-exposed cells.

Changes in the level of NMDA receptor subunit pro-teins could contribute both to the decreased NMDAreceptor binding and to the decreased functional re-sponse to NMDA. As shown in Fig. 7, NR1, NR2A, andNR2B subunits are expressed on day 4 in vitro in cere-bellar granule neurons grown in medium containing 5mM KCl. Neither we nor others (Vallano et al., 1996)have detected expression of NR2C in the cerebellar gran-ule neurons under these conditions. When the cells weregrown in the presence of 100 mM ethanol for 3 days(days 1–4 in vitro), levels of NR1 were unchanged, butboth NR2A and NR2B levels were significantly reduced

by 30–40% (Fig. 7). Levels ofb-actin were not affectedby the chronic ethanol exposure (data not shown).

DISCUSSION

The ability of NMDA to protect cultured cerebellargranule cells against apoptosis is believed to reflect thesituation that occurs in vivo during development, whenthe cerebellar granule neurons are innervated by theglutamatergic mossy fibers (Altman, 1982; Bala´zs et al.,1988). We previously showed that ethanol inhibited theprotective effect of NMDA when ethanol and NMDAwere added simultaneously to the cultured neurons(Bhave and Hoffman, 1997). Those results suggestedthat the presence of ethanol in the brain at a criticaldevelopmental period would increase apoptosis of thecerebellar granule neurons, perhaps contributing to theloss of these neurons that is a characteristic of the fetalalcohol syndrome (Miller, 1992). Similarly, it has beenreported that treatment of postnatal rats with NMDAreceptor antagonists or ethanol at critical developmentalperiods caused widespread apoptosis in the brain (Ikono-

FIG. 5. Effect of chronic ethanol exposure on NMDA-inducedBDNF expression in cerebellar granule neurons. A: Cerebellargranule cells were prepared and maintained in medium contain-ing 5 mM KCl, in the absence or presence of the indicatedconcentrations of ethanol, on days 1–4 in vitro, as described inMaterials and Methods. On day 4 in vitro, cells were washed toremove ethanol and treated with conditioned medium or NMDA(100 mM ). Twenty-four hours later, on day 5 in vitro, cells wereextracted for analysis of BDNF levels. Results are expressed asNMDA-induced BDNF formation in cells exposed to ethanol as apercent of that seen in control cells (100%). Basal values ofBDNF were not affected by ethanol treatment and have beensubtracted. Values represent the means 6 SEM of six observa-tions in two separate experiments. Kruskal–Wallis ANOVA re-vealed a significant effect of ethanol treatment (H 5 27.92, df5 3, p , 0.001). *p , 0.05 compared with all other groups (posthoc comparisons). B: Cerebellar granule neurons were main-tained in medium containing 5 mM KCl in the presence orabsence of 100 mM ethanol for 1, 2, or 3 days in vitro (days 3–4,2–4, or 1–4, respectively). On day 4 in vitro, cells were washedto remove ethanol and were treated with NMDA (100 mM ) for24 h, as described in Materials and Methods. On day 5 in vitro,cells were extracted for analysis of BDNF levels. Results areexpressed as NMDA-induced BDNF formation in cells exposedto ethanol as a percent of that seen in control cells (100%). BasalBDNF levels were not affected by ethanol treatment and havebeen subtracted. Values represent the means 6 SEM of sixobservations in two separate experiments. Kruskal–WallisANOVA revealed a significant effect of time of ethanol exposure(H 5 16.0, df 5 3, p , 0.001). *p , 0.05 compared with all othergroups (post hoc comparisons).

FIG. 4. Effect of chronic ethanol exposure on NMDA-inducedincrease in [Ca21]i in cerebellar granule neurons. Cerebellargranule neurons were cultured in the absence or presence of 100mM ethanol on days 1–4 in vitro, and NMDA-induced changes in[Ca21]i were determined with fura-2 fluorescence followingwashout of ethanol on day 4 in vitro. Neurons were exposed toincreasing concentrations of NMDA (0.03–30 mM ) in the pres-ence of 10 mM glycine, and the change in [Ca21]i was monitored.Data are expressed as the 340/380 ratio, as described in Mate-rials and Methods. Values represent means 6 SEM of six ob-servations. Two-way ANOVA revealed a significant effect ofethanol (F 5 34.28, df 5 1, p , 0.001), a significant effect ofNMDA concentration (F 5 194, df 5 7, p , 0.001), and asignificant treatment–concentration interaction (F 5 9.62, df 5 7,p , 0.001).

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midou et al., 1999, 2000). The present results indicate,however, that when the cerebellar granule cells are ex-posed to ethanol for prolonged periods ($24 h) in theabsence of added NMDA, the protective effect ofNMDA is reduced even if ethanol is no longer presentwhen NMDA is added to the cells. The pro-apoptoticeffect of ethanol persisted for up to 24 h after ethanolwithdrawal. These findings suggest that ethanol exposureof the developing brain may enhance apoptosis even ifethanol is no longer present at the critical period ofcerebellar granule neuron innervation. It is also impor-tant to note that a significant reduction in the protectiveeffect of NMDA was observed after chronic exposure ofthe cells to 25 mM ethanol, a concentration close to thelegal limit of intoxication.

Our results demonstrate that, at least in part, the re-duced protective effect of NMDA is due to decreasedfunction of the NMDA receptor associated with the eth-anol exposure. Both the initial response to NMDA (in-crease in intracellular Ca21) and the ability of NMDA toinduce BDNF expression were significantly lowered inthe ethanol-exposed neurons. The decreased effect ofNMDA on BDNF expression may be a direct result ofthe deficit in the Ca21 response to NMDA. It has beendemonstrated that Ca21 influx through NMDA receptorscan increase expression of BDNF mRNA and release ofBDNF protein from hippocampal and cortical neurons(Zafra et al., 1991; Ghosh et al., 1994). The increasedBDNF expression results from phosphorylation of calci-um/cyclic AMP response element binding protein or aclosely related protein (Tao et al., 1998). A calcium/cyclic AMP response element-like sequence is present inthe BDNF promoter (Tao et al., 1998), suggesting thatBDNF expression in cerebellar granule neurons may beregulated by Ca21 in a similar fashion.

We previously showed that BDNF mediates the anti-apoptotic effect of NMDA in cerebellar granule neurons(Bhave et al., 1999b). Therefore, the decreased NMDA-induced BDNF expression is likely to directly underliethe attenuated protective effect of NMDA in the ethanol-treated cells. This hypothesis is in line with earlier workof Balazs and colleagues (Hack et al., 1993), who impli-cated Ca21/calmodulin kinase in the protective effect ofNMDA. Stimulation of this kinase by NMDA, whichwould be expected to be reduced in the ethanol-exposedcells, has been reported to activate the BDNF promoter(Shieh et al., 1998). However, the difference in timecourse for the two phenomena (i.e., the protective effectof NMDA continued to decrease between 24 and 48 h ofethanol exposure, whereas the effect of ethanol exposureon NMDA-induced BDNF expression was maximal after24 h) suggests the possibility that signal transductionpathways downstream of the NMDA receptor, which areinvolved in the protective effects of NMDA-inducedBDNF expression (i.e., phosphatidylinositol 39-kinaseand AKT) (Bhave et al., 1999b) may also be affected bychronic ethanol exposure.

Furthermore, our preliminary studies have indicatedthat depolarization (25 mM KCl)-induced increases inintracellular Ca21, measured in the presence of MK-801,are reduced in cells treated for days 1–3 in vitro with 100

FIG. 6. Effect of chronic ethanol exposure on [3H]MK-801 bind-ing in cerebellar granule neurons. Cerebellar granule neuronswere obtained and cultured in the presence or absence of 100mM ethanol on days 1–4 in vitro as described in Materials andMethods. On day 4 in vitro, cells were washed to remove etha-nol, and whole-cell [3H]MK-801 binding was assessed using theindicated concentrations of ligand. Specific [3H]MK-801 bindingwas calculated as pmol/mg of protein. Values represent themeans 6 SEM in three separate experiments. *p , 0.05 com-pared with control (Student’s t test).

FIG. 7. Effect of chronic ethanol exposure on the expression ofNMDA receptor subunits in cerebellar granule neurons. Cerebel-lar granule neurons were obtained and cultured in the absenceor presence of 100 mM ethanol on days 1–4 in vitro as describedin Materials and Methods. Total protein was extracted from thecells on day 4 in vitro, and expression of NMDA receptor subunitproteins was assessed with subunit-specific antibodies (Snellet al., 1996a). Immunoblots for NR1, NR2A, and NR2B from oneset of cultures are shown. The mean 6 SEM values from thequantitative densitometry of immunoreactive bands from four tofive sets of cultures are illustrated in the graph. The densitometryvalues obtained from cells maintained in the presence of ethanolare expressed as a percent of those obtained from control cells,set at 100%. *p , 0.05 compared with appropriate control(one-sample t test).

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mM ethanol compared with control cells. It appears thatdiffering pathways mediate the anti-apoptotic effects ofdepolarization and NMDA on cerebellar granule neu-rons, although both depend on the initial Ca21 influx (seeBhave et al., 1999b). These initial data therefore suggestthat chronic ethanol exposure may alter the protectiveeffect not only of NMDA but also of other stimuli suchas depolarization. Although the reduced effect of NMDAappears to depend, at least in part, on changes in NMDAreceptor properties, elucidation of the mechanism pro-ducing altered responses to other agents would requirefurther investigation.

It is noteworthy that we did not observe changes in thebasal level of BDNF in the ethanol-treated neurons. Thebasal level of endogenous BDNF is likely regulated by anumber of factors. It has been reported that chronicethanol exposure reduced the level of unidentified neu-rotrophic factors produced by cortical astrocytes (Kimand Druse, 1996) and that chronic exposure of rats toethanol throughout gestation reduced plasma levels ofinsulin-like growth factor-1 in the offspring (Breeseet al., 1993). However, we found that chronic ethanolexposure of the cerebellar granule neurons interferedmore selectively with BDNF expression induced byNMDA.

Our work also suggests a possible mechanism for thedecrease in NMDA receptor function in the ethanol-exposed neurons. Although it is theoretically possiblethat chronic ethanol treatment could select out a popula-tion of neurons with low levels of NMDA receptors orNMDA receptor function, the fact that cell number andtotal protein levels were not substantially altered by theethanol treatment reduces the likelihood of this explana-tion. It seems more likely that ethanol treatment alters theproperties of the NMDA receptor, similar to previousreports (e.g., Savage et al., 1991; Follesa and Ticku,1995; Hoffman et al., 1995; Hu and Ticku, 1995; Snellet al., 1996a; Diaz-Granados et al., 1997; Hughes et al.,1998). These receptors exist as a heteromeric complex ofNR1 and one or more NR2 subunits (NR2A–D) (Ishiiet al., 1993; Mori and Mishina, 1995). We have previ-ously found that ethanol exposure delays the develop-mental changes in NMDA receptor subunit expression incerebellar granule neurons cultured in medium contain-ing 25 mM KCl (Snell et al., 1996c). The NMDA recep-tor also undergoes developmental changes in cells grownin 5 mM KCl, as in the present study. Studies of NMDAreceptor subunit mRNA levels lead to the conclusion thatboth NR2A and NR2B mRNA are increasing during thetime that the neurons were exposed to ethanol in thepresent study (days 1–4 in vitro) (Vallano et al., 1996).Assuming that developmental changes in mRNA levelsare reflected in protein levels, ethanol treatment maydelay these changes, leading to lower levels of NR2Aand NR2B proteins. Interestingly, NR1 expression, inboth the present and the previous study (Snell et al.,1996c), was not affected by ethanol treatment, eventhough NR1 mRNA levels also increase in control cellsduring the period of ethanol exposure (Vallano et al.,

1996). Similarly, Hughes et al. (1998) found no effect ofprenatal ethanol treatment on the level of NR1 in fore-brain or hippocampus of the offspring, although NR2Aand NR2B were lowered.

The decreased levels of NR2A and NR2B could leadto a decrease in receptor number, which would accountfor the decreased MK-801 binding and decreasedNMDA-induced stimulation of increases in intracellularCa21 and BDNF expression found in the ethanol-ex-posed neurons. Although these changes might also alterreceptor subunit composition, receptors containingNR2A or NR2B appear to have similar affinities forNMDA (Laurie and Seeburg, 1994; Lynch et al., 1995).Therefore, it is difficult to ascribe the observed change inpotency of NMDA to stimulate increases in intracellularCa21 to the altered levels of receptor subunit proteins. AsNR2B in particular but also NR2A (Kornau et al., 1995;Niethammer et al., 1996; Wyszynski et al., 1997; Strackand Colbran, 1998; Yamada et al., 1999) have beenimplicated in determining the synaptic localization of theNMDA receptor, it is possible that the decrease in thesesubunit proteins produced by chronic ethanol exposurecould interfere with the proper localization of receptorwithin the neurons, leading to altered functional proper-ties. Another possibility is that the ethanol treatmentresults in altered posttranslational modification, for ex-ample, phosphorylation of NMDA receptor subunits,which has been reported to affect agonist affinity (Du-rand et al., 1993).

The decrease in NMDA receptor expression and func-tion following chronic ethanol treatment of the “imma-ture” cerebellar granule neurons is compatible with pre-vious studies of the effect of prenatal ethanol exposureon the NMDA receptor, as mentioned in the introductorysection, that is, decreased electrophysiological and bio-chemical responses to NMDA as well as decreasedNMDA receptor binding and decreased expression ofNMDA receptor subunit proteins. There have also beenprevious reports that chronic in vitro ethanol treatmentreduces NMDA receptor responses in developing cere-bellar Purkinje or granule neurons, as well as cerebellarmacroneurons, obtained from embryonic rats and main-tained in culture (Zou et al., 1995; Gruol and Parsons,1996; Gruol et al., 1998). However, in these studies, theneurons were purposely maintained in ethanol at the timethat the NMDA responses were measured. Because eth-anol acutely inhibits the response to NMDA (Hoffmanand Tabakoff, 1996), the influence of the chronic ethanoltreatment in these studies cannot be accurately deter-mined.

On the other hand, we have previously shown thatchronic ethanol exposure of cerebellar granule neuronsobtained from postnatal rats and grown in 25 mM KCl(“mature” neurons) produces an up-regulation of NMDAreceptor function, when this function is measured in theabsence of ethanol. This change resulted in increasedsusceptibility of the neurons to glutamate-induced exci-totoxicity (necrosis) (Iorio et al., 1992, 1993). Similarresults were reported for cultured cortical (Chandler

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et al., 1993; Ahern et al., 1994) and hippocampal(Smothers et al., 1997) neurons. Chronic ethanol treat-ment also results in NMDA receptor up-regulation in theadult brain (Grant et al., 1990; Gulya et al., 1991; Snellet al., 1996a,b). The results suggest that the response ofthe NMDA receptor to chronic ethanol exposure dependsto a significant degree on the stage of receptor and/orneuronal development during which the neurons are ex-posed to ethanol. Nevertheless, with respect to NMDAreceptor function, chronic ethanol exposure results inenhanced toxicity in both mature and immature neurons,although the type of toxicity, that is, apoptosis versusnecrosis, differs.

Overall, the present studies provide further evidencefor a mechanism by which ethanol exposure during de-velopment can lead to an inappropriate loss of cerebellargranule neurons. It has been demonstrated that the cere-bellum is one of the brain areas that is most susceptibleto neuronal loss as a result of fetal alcohol exposure(Miller, 1992). Our work demonstrates that changes inthe NMDA receptor following ethanol exposure arelong-lived and may result in increased loss of cerebellargranule cells through apoptosis even after ethanol hasbeen eliminated from the extracellular milieu.

Acknowledgment: The authors thank Karin Nunley andJeremy Gerspacher for technical assistance. This work wassupported in part by the Banbury Fund and NIAAA (AA9005,AA3527).

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