Journal of Neurochemistry, 2001, 78, 396±405

Chronic ethanol exposure delays the `developmental switch'

of the NMDA receptor 2A and 2B subunits in cultured

cerebellar granule neurons

Lawrence D. Snell,1 Sanjiv V. Bhave,1 Boris Tabakoff and Paula L. Hoffman

Department of Pharmacology, University of Colorado Health Sciences Center, Denver, USA

Abstract

Chronic ethanol treatment of cultured neurons from various

brain areas has been found to increase NMDA receptor

function and to alter the levels of some NMDA receptor

subunit proteins. Because the cultured neurons are exposed

to ethanol during a period when the NMDA receptor is

undergoing developmental changes in subunit expression, we

wished to determine whether ethanol treatment alters this

developmental pattern. We found that 3 days of treatment of

cerebellar granule neurons with ethanol, which was previously

reported to increase NMDA receptor function, resulted in a

delay in the `developmental switch' of the NR2A and NR2B

subunits, i.e. the developmental decrease in NR2B and

increase in NR2A protein expression. As a result, the level

of NR2B was higher, and that of NR2A was lower, in the

ethanol-treated cells than in control cells. Cross-linking

experiments showed that the changes in total receptor subunit

proteins levels were re¯ected in cell-surface expressed

proteins, indicating changes in the amount of functional

receptors. These results were con®rmed by a higher potency

of glycine at the NMDA receptor in the ethanol-treated cells,

as determined by NMDA/glycine-induced increases in intra-

cellular Ca21. The results suggest that the mechanism by

which ethanol alters NMDA receptor expression in cultured

neurons, where receptors are undergoing development,

differs from the mechanism of ethanol's effect on NMDA

receptors in adult brain. Changes in the proportion of NR2A

and NR2B subunits may contribute to effects of ethanol on

neuronal development.

Keywords: cerebellar granule neurons, ethanol, NMDA

receptor development, NR2A and NR2B subunits.

J. Neurochem. (2001) 78, 396±405.

The NMDA subtype of glutamate receptor is composed of

multiple subunits. The mammalian NR1 subunit is widely

expressed throughout the brain, but does not form functional

homomeric receptors (Soloviev and Barnard 1997). A

response to NMDA that is similar to the response of native

receptors is observed when the NR1 subunit, which exists as

at least eight different splice variants (Nakanishi et al.

1992), is expressed in combination with one or more of the

family of NR2 subunits (NR2A±D) (Ishii et al. 1993; Mori

and Mishina 1995). The NR2 subunits, which are expressed

more discretely in brain than NR1, also confer pharmaco-

logical speci®city on responses of the heteromeric NMDA

receptors (Scheetz and Constantine-Paton 1994).

During CNS development, alterations in NMDA receptor

subunit expression take place. In particular, the NR2B

subunit is more highly expressed in the embryonic and

neonatal brain, and expression of the NR2A subunit

increases as the brain matures (Watanabe 1996). NMDA

receptors in primary neuronal cultures undergo develop-

mental changes in subunit expression that generally parallel

the changes that occur during CNS development in vivo

(e.g. Monyer et al. 1994; Bhave et al. 1999). This

`developmental switch' in subunit expression leads to

alterations in the functional and pharmacological properties

of the receptor (Williams et al. 1993; Feldmeyer and

Cull-Candy 1996; Kew et al. 1998).

Ethanol, acutely, is a potent inhibitor of NMDA receptor

function in various neuronal preparations (Tabakoff and

396 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 396±405

Resubmitted manuscript received March 29, 2001; accepted April 25,

2001.

Address correspondence and reprint requests to Dr P. L. Hoffman,

Department of Pharmacology, University of Colorado Health Sciences

Center, 4200 E 9th Ave, Box C236, Denver, CO 80262, USA.

E-mail: Paula.Hoffman@UCHSC.edu1L. D. Snell and S. V. Bhave contributed equally to this work.

Abbreviations used: BCA, bicinchoninic acid; BME, basal essential

medium; BS3, bis(sulfosuccinimidyl)suberate; [Ca21]i, intracellular

calcium concentration; PAGE, polyacrylamide gel electrophoresis;

PBS, phosphate-buffered saline; RPA, RNase protection assay; SDS,

sodium dodecyl sulfate.

Hoffman 1996). Chronic ethanol exposure of adult animals

has been reported to result in an increase in NMDA receptor

number, determined by ligand binding studies, and increased

expression of NMDA receptor subunits, including NR1,

NR2A and NR2B, in various brain regions (Grant et al.

1990; Follesa and Ticku 1995; Snell et al. 1996).

Chronic ethanol exposure of primary neuronal cultures

has also been reported to produce an increase in NMDA

receptor number, as measured by ligand binding studies, and

an increase in receptor function (Iorio et al. 1992, 1993;

Chandler et al. 1993; Ahern et al. 1994; Hoffman et al.

1995; Hu and Ticku 1995; Smothers et al. 1997). This

treatment also produces changes in expression of NMDA

receptor subunits (Hoffman et al. 1996; Follesa and Ticku

1996; Hu et al. 1996), and the implication of these studies

has been that the changes in NMDA receptor subunit expres-

sion mediate the observed increases in receptor function.

It has generally been accepted that the cultured neurons

represent a model in which the mechanism of ethanol's

effects on NMDA receptors in the brain can be investigated.

However, in contrast to adult brain, the cultured neurons are

exposed to ethanol during a period of development of

NMDA receptors. Thus, the molecular mechanism of

ethanol's effect on NMDA receptors may be quite different

in the two systems. In our previous studies of cerebellar

granule neurons, the cells were exposed to ethanol during

the period of the developmental switch between NR2B and

NR2A expression (Iorio et al. 1992, 1993; Hoffman et al.

1995), but the effect of ethanol was determined at only one

time point, following this switch. In this study, we evaluated

the time course of the developmental changes in the

expression of NMDA receptor subunits in cultured cere-

bellar granule neurons in the presence and absence of

ethanol. The results suggest that ethanol delays the

developmental change in NMDA receptor subunit expres-

sion in these neurons, and that this delay affects cell-surface

expressed receptors and therefore the pharmacological

properties of the NMDA receptors.

Materials and methods

Materials

Basal essential medium (BME), fetal bovine serum and the TriZol

reagent were obtained from Gibco-BRL (Grand Island, NY, USA).

The RPA II kit and the Maxiscript T7/T3 kit were obtained from

Ambion, Inc. (Austin, TX, USA). Fura-2 acetoxymethyl ester was

obtained from Molecular Probes (Eugene, OR, USA). Polyclonal

antibodies for the NR2A and NR2B subunits, and a `pan-antibody'

that detects NR2A, NR2B and NR2C, were kindly provided by Dr

MD Browning (Department of Pharmacology, University of

Colorado Health Sciences Center, Denver, CO, USA), and

monoclonal antibody for NR1 (that recognizes all splice variants)

was purchased from Pharmingen (San Diego, CA, USA). Horse-

radish peroxidase-coupled secondary antibodies were obtained

from Bio-Rad (Richmond, CA, USA). Renaissancee chemi-

luminescence reagents and Re¯ectione ®lm were obtained from

DuPont-New England Nuclear (Boston, MA, USA). All other

commercial reagents were from Sigma Chemical Corp. (St. Louis,

MO, USA).

Cell culture

Primary cultures of cerebellar granule neurons were prepared from

7-day-old Sprague±Dawley rat pups and grown in culture as

described previously (Iorio et al. 1992). Cerebellar granule neurons

(2 � 107 cells/100 mm dish) plated on tissue culture dishes coated

with poly l-lysine (10 mg/mL) were used to extract total proteins or

total RNA. For intracellular calcium ion ([Ca21]i) measurements,

cells (2 � 106 cells/well) were plated on glass coverslips coated

with polyethyleneimine. For chronic ethanol exposure, 100 mm

ethanol was included in the medium for 1 day (days 4±5 in culture)

or 3 days (days 4±7 in culture), as described previously (Iorio et al.

1992). This latter period of ethanol exposure was chosen to mimic

previous studies in which chronic ethanol treatment of cultured

cerebellar granule neurons was found to result in an `up-regulation'

of NMDA receptor function (Iorio et al. 1992). The ethanol

exposure procedure includes daily supplementation of the medium

with ethanol, in order to maintain a constant ethanol concentration,

as measured by gas chromatography (Tabakoff et al. 1976; Iorio

et al. 1992). All determinations were carried out with cells

maintained in culture for the number of days indicated in the

Results section.

RNase protection assays

Total RNA from cerebellar granule neurons was obtained with the

TriZol reagent. Antisense probes for NR1, NR2A and NR2B were

obtained by RT-PCR from rat brain mRNA as described previously

(Hoffman et al. 1996), and corresponded to nucleotide sequence

73±348 for NR1, 3979±4396 for NR2A and 3007±3368 for NR2B.

The DNA products were cloned into the Bluescript II SK(±) vector,

which was then linearized and used as a template in an [a-32P]UTP-

labeled cRNA probe synthesis reaction (Maxiscript T7/T3 kit).

Solution hybridization/RNase protection assays were carried out

according to the RPA II kit protocol. Protected fragments were

separated on a 5% denaturing polyacrylamide gel. A b-actin probe,

obtained from Ambion, was labeled to low speci®c activity and

used to correct for loading artifacts in the assay. Autoradiograms

were analyzed using the Bio-Rad GS-250 Molecular Imager and

phosphoranalyst image analysis software. The results are

expressed as the ratio of the volume (area � Phosphor counts) of

the NR1, NR2A or NR2B band to the b-actin band.

Western blot analysis

Cultures were washed twice with ice-cold phosphate-buffered

saline (PBS) and harvested in a buffer containing 2 mm EDTA,

2.3% SDS, 10% glycerol and 62.5 mm Tris (pH 6.9). To measure

the NR1, NR2A and NR2B subunits, the proteins, solubilized in the

sodium dodecyl sulfate (SDS)-containing sample buffer, were

subjected to polyacrylamide gel electrophoresis (PAGE) on 7.5%

polyacrylamide gels according to the procedures described by Snell

et al. (1996). The protein amount in each sample was estimated by

the bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA),

and 20 mg of solubilized proteins was loaded onto each lane of

the gels. Following electrophoretic separation, the proteins were

transferred to nitrocellulose membranes (0.22 mm, Schleicher and

Ethanol and NMDA receptor development 397

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 396±405

Schuell, Keene, NH, USA). After blocking with 5% non-fat dry

milk in Tris-buffered saline containing Tween-20, blots were

probed with the subunit-speci®c antibodies to the NR1, NR2A or

NR2B proteins (Hoffman et al. 1996; Snell et al. 1996). Blots were

then incubated with horseradish peroxidase-conjugated goat IgG

(goat antimouse for anti-NR1 or goat antirabbit for NR2A and

NR2B). Immunoreactive bands were visualized using a chemi-

luminescence method. The NR1 antibody recognized a major band

of < 100 kDa, and a second band of < 110 kDa. Both bands were

included in the quantitation. The NR2A and NR2B antibodies each

recognized a major band of < 165 kDa. The minor band detected

by the NR2B antibody (see Fig. 1) was not observed in cells

transfected with NR2B (Snell et al. 1996), and was therefore not

included in the quantitation. Quantitation of protein bands was

performed by image analysis as described previously (Snell et al.

1996).

Cross-linking of surface-expressed NMDA receptors

Cultures were washed with PBS and incubated with 1 mg/mL

bis(sulfosuccinimidyl)suberate (BS3) in PBS for 30 min at 378C,

as described previously (Hall and Soderling 1997). Following

incubation, the cells were washed in PBS containing ethanolamine

(50 mm) to quench unreacted BS3, and were extracted in hot SDS

(1%). The cell lysate was stored at 2708C until use in immunoblot

analysis as described above.

Measurement of intracellular Ca21 concentration ([Ca21]i)

The [Ca21]i was determined by measuring Fura-2 ¯uorescence, as

described previously (Iorio et al. 1992; Snell et al. 1994). In brief,

cerebellar granule neurons were loaded with 5 mm Fura-2-

acetoxymethylester for 60 min at 378C. The glass coverslips were

then rinsed with Mg21-free cell buffer (145 mm NaCl, 5 mm KCl,

10 mm CaCl2, pH 7.4) and transferred to a cuvette containing

2 mL of cell buffer maintained at 378C with constant stirring.

Fluorescence was measured using an SLM-Aminco spectro¯uori-

meter (excitation at 340 and 380 nm; emission at 505 nm). The

data were stored on a NEC 286 computer using the software from

SLM-Aminco (Urbana, IL, USA). Determinations of Rmax and Rmin

were carried out as described previously (Snell et al. 1994).

General procedure

To generate glycine concentration±response curves, glass cover-

slips with adherent cells were placed in a cuvette ®tted with a

superfusion system attached to a peristaltic pump. The coverslip

was perfused with cell buffer for 2 min at a ¯ow rate of 4 mL/min

and the basal levels of [Ca21]i were recorded for 30 s. NMDA

(100 mm) was added and the NMDA-induced change in the levels

of [Ca21]i was recorded for 30 s. Glycine was then added to

produce the increasing ®nal concentrations indicated in the Results

(Fig. 5), i.e. a cumulative glycine concentration±response curve

was generated. After each addition of glycine, the change in

[Ca21]i was recorded for 30 s.

For each stimulation, the effect of NMDA alone or NMDA plus

added glycine on [Ca21]i was calculated by subtracting the value

obtained before the addition of NMDA (i.e. baseline) from the peak

value obtained after the addition of NMDA or NMDA plus each

concentration of added glycine. Results are expressed as the change

in the ratio of emission at 340 and 380 nm excitation (D340/380

ratio), a measure of intracellular free Ca21 levels (Grynkiewicz

et al. 1985).

Statistical analysis

Levels of mRNA were normalized to levels of b-actin in the RPA.

The mean of the ratios (i.e. NMDA receptor subunit mRNA/actin

mRNA) for data obtained on day 4 in vitro for each subunit was

calculated and values obtained on other days were then normalized

to this mean, taken as 100%. Levels of NMDA receptor subunit

proteins on days 1, 5 and 7 in vitro were also normalized to the

levels of the appropriate protein on day 4 in vitro. This normal-

ization was performed separately on each blot or gel, and values are

expressed as percent of day-4 values in order to compare among

experiments. The data obtained for mRNA and protein levels over

days in culture were subjected to one-way anova (data for protein

and mRNA levels on day 4 in vitro were omitted from the analysis)

followed by post hoc Student-Newman-Keuls tests, using the

Sigmastat 1.0 statistical program. For the ethanol treatment

experiments, data obtained from the ethanol-treated cells (mRNA

or protein levels) were expressed as percent of values obtained on

the same day in control cells, taken as 100%. For each day in vitro

(day 5 or day 7), one-sample t-tests were used to ascertain

differences from control (100%).

Data obtained from measurements of intracellular Ca21 levels

were also analyzed using anova followed by post hoc comparisons.

A baseline endogenous concentration of glycine was present in all

experiments, as illustrated previously (Bhave et al. 1999) by the

presence of a response to the addition of NMDA alone. Iterative

curve ®tting was therefore used to determine best ®t lines for

glycine concentration±response data, using a modi®cation of a two-

equivalent binding site model for monophasic ®ts as described by

Fig. 1 NMDA receptor subunit mRNA and protein levels in cere-

bellar granule neurons treated chronically with ethanol and control

neurons. Cerebellar granule neurons obtained from 7-day-old

Sprague2Dawley rats were maintained in culture for the number of

days indicated. On days 1 and 4 in vitro, RNA and protein were

extracted from control cells. On day 4 in vitro, ethanol (100 mM) was

added to some cells, and on day 5 or day 7 in culture, as indicated,

RNA and protein were extracted from the control and ethanol-treated

cells. mRNA levels for the NMDA receptor subunits were determined

with the RNase protection assay, and NMDA receptor subunit

proteins were analyzed by immunoblotting, as described in the text.

398 L. D. Snell et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 396±405

Kew et al. (1998):

Response � Responsemax

1 1KD

�A 1 g�� �2

Responsemax is the maximum [Ca21]i ratio (in the presence of

100 mm NMDA and 10 mm or greater added glycine concentra-

tion), KD is the dissociation constant for glycine, [A] is the

concentration of added glycine, and g represents the endogenous

glycine concentration. This model is derived from electrophysio-

logical studies of the activation kinetics of NMDA channels,

indicating that the binding of NMDA and glycine are independent

and that two molecules of glycine, binding with equal af®nity (i.e.

nH � 1), are required for full activation. The calculated endo-

genous glycine concentration (0.11 ^ 0.03 mm in control cultures

and 0.03 ^ 0.02 mm in ethanol-treated cultures) was added to each

exogenous glycine concentration to give the corrected glycine

concentration. The data were replotted using the corrected glycine

concentrations. The response to NMDA, added alone, was plotted

as a function of the calculated endogenous glycine value. The con-

centration±response curves were then ®tted with the two-equivalent

binding site equation excluding the term for the endogenous

glycine (g).

Values are presented as mean ^ SEM. A value of p , 0.05 was

taken to indicate statistical signi®cance.

Results

Development of NMDA receptors in cerebellar granule

neurons

Protein and mRNA levels for the NMDA receptor subunits

showed generally similar developmental patterns over the

®rst 7 days in culture (Figs 1, 2a and 3a). NR1 mRNA and

protein levels increased to a maximum level at day 5 and

remained constant until day 7. NR2A mRNA levels also

increased over time in culture, reaching a maximum at day 5

and remaining constant to day 7, whereas NR2A protein

levels continued to increase slightly between days 5 and 7.

The NR2B mRNA and protein levels increased to a

maximum level at days 4 and 5, and then decreased between

days 5 and 7. Neither we nor others (Vallano et al. 1996)

detected NR2C mRNA in cerebellar granule neurons at this

Fig. 2 Developmental pattern of NMDA

receptor subunit mRNA levels (a) and effect

of ethanol treatment on mRNA levels (b).

Cerebellar granule neurons obtained from

7-day-old Sprague2Dawley rats were main-

tained in culture for the number of days

indicated. (a) On the indicated day in vitro

(DIV), cells were harvested, RNA was

extracted and mRNA levels for the three

NMDA receptor subunits were quantitated

by RNase protection assay, as described in

the text. Data are normalized to the level of

b-actin mRNA, and are expressed as per-

cent of the mRNA level on day 4 in vitro.

ANOVA revealed signi®cant changes over

time for all subunits [NR1, F � 4.6

(d.f. � 2,13), p , 0.031; NR2A, F � 12.0

(d.f. � 2,13), p , 0.001; NR2B, F � 11.0

(d.f. � 2,13), p , 0.002]. Data represent

mean ^ SEM (n � 3±7). p , 0.05, post

hoc comparisons. (b) Ethanol (100 mM) was

added to the cells for 24 h (days 4±5

in vitro) or 72 h (days 4±7 in vitro), as

described in the text. Following ethanol

treatment, cells were harvested, RNA was

extracted and mRNA levels for the NMDA

receptor subunits were quantitated by

RNase protection assay. Values obtained

from ethanol-treated cells are expressed as

percent of values obtained from control

cells on the same day in vitro (DIV). Data

represent mean ^ SEM (n � 5). *p , 0.05,

compared with control cells (one-sample

t-test).

Ethanol and NMDA receptor development 399

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 396±405

time in culture, and we also did not detect NR2C protein or

mRNA in control or ethanol-treated cells (data not shown).

Effect of ethanol treatment on development of NMDA

receptors

Ethanol treatment for 24 h (days 4±5 in culture) resulted in

an increase in mRNA levels on day 5, compared with

control cells, for all of the NMDA receptor subunits,

although the increase in NR2A mRNA did not reach

statistical signi®cance ( p � 0.1) (Figs 1 and 2b). When

cells were treated with ethanol for 3 days (days 4±7 in

culture), although the concentration of ethanol in the culture

medium remained relatively constant over this time, as

assessed by gas chromatographic analysis (Tabakoff et al.

1976), mRNA levels for the NR1 and NR2B subunits

returned to control levels by day 7 in culture (Figs 1 and

2b). The mRNA for NR2A was signi®cantly lower than

control at this time.

Although the levels of the NMDA receptor subunit

proteins appeared to re¯ect the levels of mRNA in control

cells during development, the response of the proteins to

Fig. 3 Developmental pattern of NMDA

receptor subunit protein levels (a) and effect

of ethanol treatment on protein levels (b).

(a) Cerebellar granule neurons were pre-

pared as described in the text and legend

to Fig. 1. On the indicated day in vitro

(DIV), cells were harvested and proteins

extracted as described in the text. NMDA

receptor subunit proteins were quantitated

by immunobloting. Data are expressed as a

percent of the levels of each subunit protein

determined on day 4 in vitro (taken as

100%). ANOVA revealed signi®cant changes

over time for all subunit proteins [NR1,

F � 4.5 (d.f. � 2,9), p , 0.04; NR2A, F �10.2 (d.f. � 2,9), p , 0.005; NR2B, F �14.9 (d.f. � 2,9), p , 0.001]. Data represent

mean ^ SEM (n � 4±5). *p , 0.01±0.05,

post hoc comparisons. (b) Effect of ethanol

treatment on NMDA receptor subunit pro-

teins. Ethanol (100 mM) was added to the

cells on day 4 in vitro and cells were har-

vested, and protein extracted, either on

day 5 or day 7 in vitro. NMDA receptor sub-

unit proteins were quantitated by immuno-

bloting. Values from ethanol-treated

neurons are expressed as a percentage of

the values obtained from control cells on

the same day in vitro (DIV). Data represent

mean ^ SEM (n � 5±13). *p , 0.05 com-

pared with control (one-sample t-test).

Fig. 4 Effect of ethanol treatment on the surface expression of

NR2A and NR2B proteins in cerebellar granule neurons. Cerebellar

granule neurons were prepared and maintained in the absence or

presence of 100 mM ethanol on days 4±7 in culture, as described in

the text. Cells were treated with BS3 for 30 min and subjected to

immunoblotting (Materials and methods). Representative immuno-

blots are shown.

400 L. D. Snell et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 396±405

ethanol exposure differed from that of mRNA in most cases

(Figs 1, 2b and 3b). Ethanol treatment for 24 h or 3 days had

no effect on NR1 protein levels. NR2A and NR2B protein

levels were also not affected by 24 h of ethanol exposure,

but NR2A protein levels were reduced in the ethanol-treated

cultures, compared with control cultures, following 3 days

of ethanol exposure. In contrast, following 3 days of ethanol

exposure, NR2B protein levels were higher in the ethanol-

treated neurons than in control neurons. Because a goal of

this study was to determine developmental changes in

NMDA receptor subunit expression that accompany pre-

viously observed alterations in NMDA receptor function in

ethanol-treated cells, the investigation was not extended to

determine whether there were further changes in NR2A and

NR2B proteins.

Effect of ethanol treatment on cell-surface expression of

NMDA receptor subunits

To determine the effect of 3 days of ethanol exposure (days

4±7 in culture) on cell surface expression of NMDA

receptor subunits, we treated the cells with the membrane-

impermeable cross-linking reagent BS3, which effectively

removes all surface receptors (Hall and Soderling 1997).

Immunoblots after BS3 treatment show the remaining

intracellular receptor subunit proteins migrating at the

expected position, as the cell surface receptors are cross-

linked, and the resulting aggregates either do not enter the

gels, or can be detected as high molecular mass species (Hall

and Soderling 1997). Preliminary studies showed that

20 min exposure of cells to BS3 resulted in a maximum

decrease in NMDA receptor subunit immunoreactivity at the

expected position in the gels, with no further decrease after

30 min of BS3 exposure. Therefore, control and ethanol-

treated cultures were treated with BS3 for 30 min. This

treatment did not decrease the levels of immunoreactive

b-actin, which is completely intracellular (data not shown).

Figure 4 shows the levels of remaining (intracellular)

immunoreactive NR2A and NR2B in control and ethanol-

treated cells following BS3 exposure. Treatment of control

cells with BS3 reduced the amount of NR2A at the expected

position in the gel by 79%, and reduced the amount of

NR2B by 86% (Table 1). A similar percentage reduction in

levels of NR2A and NR2B was observed in the ethanol-

treated cells (Table 1). These data indicate that < 80% of

NR2A and 85% of NR2B are normally expressed at the cell

surface of both control and ethanol-treated cells. However,

because the total amount of NR2B was higher in ethanol-

treated cells (Table 1, Fig. 3b), the absolute amount of

membrane-expressed NR2B was also higher in the ethanol-

treated cells (11.5 ^ 0.49 versus 8.5 ^ 0.35 OD units,

Table 1). Similarly, because the total amount of NR2A was

lower in ethanol-treated than control cells, the absolute

amount of membrane-expressed NR2A was also lower in the

ethanol-treated cells (10.12 ^ 0.39 versus 7.34 ^ 0.52 OD

units, Table 1). Bis(sulfosuccinimidyl)suberate treatment

resulted in similar decreases in NR1 levels at the expected

position in the gel in control (40 ^ 16%) and ethanol-

treated cells (35 ^ 10%; n � 3 experiments). These results

indicate that < 60±65% of NR1 is expressed at the surface

of the cerebellar granule neurons under both conditions, and

that there is no signi®cant change in the amount of cell

surface-expressed NR1 in ethanol-treated cells.

Effect of ethanol treatment on NMDA/glycine-stimulated

increases in intracellular Ca21 ([Ca21]i)

Figure 5 shows that the response to NMDA (100 mm) plus

glycine was higher in neurons treated with ethanol for 3

days in vitro than in control neurons. We also found that

Table 1 Distribution of NR2A and NR2B proteins in membrane and intracellular fractions of cerebellar granule neurons.

NR2A

Control Ethanol treated

NR2B

Control Ethanol treated

Total immunoreactivity 12.39 �^ 2.28 9.03 �^ 1.21* 9.88 �^ 1.14 13.30 �^ 1.10*

Immunoreactivity remaining

after BS3 treatment

2.26 �^ 0.39 1.68 �^ 0.52 1.37 �^ 0.35 1.80 �^ 0.40

Immunoreactivity in membrane 10.12 �^ 0.39 7.34 �^ 0.52 8.50 �^ 0.35 11.50 �^ 0.49

% in membrane 79.30 �^ 5.08 83.80 �^ 3.14 85.89 �^ 3.98 87.17 �^ 2.17

% intracellular 20.69 �^ 5.08 16.17 �^ 3.14 14.10 �^ 3.98 12.82 �^ 2.17

Cerebellar granule neurons were prepared as described in the text and maintained in the absence (control) or presence (ethanol-treated) of

100 mM ethanol on days 4±7 in culture. Cells were treated with BS3 for 30 min and subjected to immunoblotting for NMDA receptor subunits as

described in the text. Total immunoreactivity (i.e. immunoreactivity in extracts of cells not treated with BS3), and immunoreactivity remaining after

BS3 treatment (i.e. intracellular immunoreactivity) represent measured values (OD units), and statistical analyses were carried out only on these

values. All other values are calculated from these measures (e.g. immunoreactivity in membrane � total immunoreactivity minus immunoreactivity

remaining after BS3 exposure). Data represent mean ^ SEM (n � 3 experiments). *p , 0.05, compared to control (ANOVA and post hoc

comparisons).

Ethanol and NMDA receptor development 401

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 396±405

chronic ethanol exposure of cerebellar granule neurons

resulted in an increase in glycine potency (glycine EC50:

control, 0.31 ^ 0.09 mm; ethanol-treated, 0.07 ^ 0.02 mm;

p , 0.04, Student's t-test).

Discussion

There have been a number of demonstrations that chronic

ethanol ingestion by animals increases the expression of

NMDA receptor subunits in brain (Trevisan et al. 1994;

Follesa and Ticku 1995; Snell et al. 1996), and increases

NMDA receptors per se, as measured using ligand binding

procedures (Grant et al. 1990; Gulya et al. 1991; Sanna et al.

1993; Snell et al. 1993). Furthermore, chronic ethanol

exposure has been found to increase the function of the

NMDA receptor (i.e. maximal response to NMDA), as well

as to alter the levels of NMDA receptor subunit proteins, in

various neuronal culture preparations, including cerebellar

granule neurons (Iorio et al. 1992; Ahern et al. 1994; Follesa

and Ticku 1996; Hoffman et al. 1996; Hu et al. 1996;

Chandler et al. 1997; Smothers et al. 1997). The implication

of these studies has been that, with respect to the effects of

ethanol exposure on NMDA receptors, the neuronal cultures

represent models for the adult brain, and that the changes in

NMDA receptor subunit proteins underlie altered NMDA

receptor function. The results of this study expand on

previous work, in particular by assessing cell-surface

expression of the NMDA receptor subunit proteins, and

challenge these assumptions, as we have shown that, at least

for primary cultures of cerebellar granule neurons, the effect

of chronic ethanol exposure is to alter the normal

developmental pattern of the NMDA receptor. In the adult

brain, although the functional outcome of chronic ethanol

exposure, i.e. `upregulation' of the NMDA receptor, may

appear similar to the effect in cultured cells, a different

mechanism must underlie ethanol's effect. In addition, as

discussed below, our results suggest that changes in the

levels of NMDA receptor subunit proteins may not, in

themselves, be suf®cient to account for all of the changes

in NMDA receptor function that have been observed in

cultured cells exposed chronically to ethanol.

The developmental changes in NMDA receptor subunit

expression noted in the control cultured cerebellar granule

neurons in this study are similar to those reported previously

(Bhave et al. 1999), and, in general, to development of

NR2A and NR2B in vivo and in vitro in the cerebellum

(Watanabe et al. 1992; Monyer et al. 1994; Wang et al.

1995; Vallano et al. 1996; Wenzel et al. 1997). The effect of

ethanol on NR2A and NR2B protein levels in cerebellar

granule neurons appeared to re¯ect a delay in the

`developmental switch' in these neurons, i.e. the decrease

in the NR2B subunit was less pronounced in the presence of

ethanol, as was the increase in the NR2A subunit. As a

result, on day 7 in culture, NR2A protein was lower in

ethanol-treated than control cells, as we reported previously

(Hoffman et al. 1996), whereas NR2B was higher in the

ethanol-treated than control cells. In contrast to NR2A and

NR2B, there was no effect of ethanol treatment on the level

of NR1 protein in this study. We previously observed a

small (20%) increase in NR1 protein in cerebellar granule

neurons exposed to ethanol for 4 days in vitro (Hoffman

et al. 1996). The difference in effects on NR1 between this

and previous studies may re¯ect the longer ethanol exposure

in the earlier study, and/or the dif®culty in quantitating

small changes in protein levels.

It is of interest that, in this study, differential effects of

ethanol exposure on NMDA receptor subunit protein and

Fig. 5 NMDA/glycine-stimulated increase in intracellular Ca21 in

control and ethanol (EtOH)-treated cerebellar granule neurons. Cells

were treated with 100 mM ethanol for 72 h, starting on day 4 in vitro,

as described in the text. Changes in intracellular Ca21 levels were

measured using Fura-2 ¯uorescence following treatment with

100 mM NMDA and the indicated concentrations of glycine (inset

shows a representative tracing). The data represent cumulative

concentration±response curves for glycine, and are expressed as

the change in the ratio of emission at 340 and 380 nm excitation.

Iterative curve ®tting was used to determine best-®t lines for the

glycine concentration±response data and to calculate EC50 values

and the endogenous glycine concentrations. Data have been

replotted using the corrected glycine concentrations. The data for

the lowest glycine concentrations in each case represent the

response to 100 mM NMDA, added alone, plotted as a function of

the calculated endogenous glycine concentrations (see Materials

and methods). ANOVA revealed a signi®cant effect of ethanol treat-

ment [F � 9.7 (d.f. � 1,77), p , 0.03]. Data represent mean ^ SEM

of 6±7 observations at each glycine concentration.

402 L. D. Snell et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 396±405

mRNA levels were observed. For example, the increases in

mRNA observed after 24 h of ethanol exposure were not

re¯ected by changes in protein levels. However, 3 days of

ethanol treatment did result in decreases in both NR2A

mRNA and protein, suggesting that the decrease in NR2A

expression could be due to a change in transcription. In

contrast, there was no ethanol-induced increase in NR2B

mRNA to accompany the increase in NR2B protein after

3 days of ethanol exposure. These data suggest that ethanol

may affect NR2B protein levels independent of mRNA

levels in developing cerebellar granule neurons. There is

precedent for such an effect, as ethanol treatment has

previously been noted to decrease the rate of degradation of

cytochrome P450IIE1, resulting in an increase in the level of

this protein without a change in mRNA levels (McGehee

et al. 1994). The differential responses of the NR2A and

NR2B subunits suggest that ethanol exposure can affect the

regulation of expression of various NMDA receptor subunits

by different mechanisms. The effect of ethanol treatment

may re¯ect the in¯uence of synaptic activity on develop-

ment of the NMDA receptor, as cortical neurons chronically

exposed to glutamate receptor antagonists also showed a

delay in the developmental increase in NR2A mRNA

(Hoffmann et al. 2000). Interestingly, like ethanol exposure,

glutamate receptor antagonist treatment did not affect the

developmental change in NR2B mRNA in the cortical

neurons (Hoffmann et al. 2000).

The cross-linking experiments showed that the increase in

NR2B protein and the decrease in NR2A protein in the

ethanol-treated cells were re¯ected in the cell-surface

expression of these two proteins. That is, the results

indicated that there was an increase in the absolute amount

of NR2B, and a decrease in the absolute amount of NR2A,

at the cell surface, although the proportions (percentages)

of these proteins at the cell surface versus those at the

intracellular compartment were not altered by the ethanol

treatment. The altered amounts of cell-surface expressed

proteins after chronic ethanol exposure are compatible

with the conclusion that there are more functional

NR2B-containing receptors, compared with those containing

NR2A, or a greater proportion of NR2B relative to NR2A in

heterotrimeric receptors, inserted into the cell membrane.

Either of these changes should affect the pharmacological

properties of the receptor. In particular, the potency of

glycine as a co-agonist at the NMDA receptor has been

shown to be as much as 10-fold higher for expressed

receptors containing NR1/NR2B subunits, compared with

NR1/NR2A subunits (Planells et al. 1993; Laurie and

Seeburg 1994; Priestley et al. 1995). Similarly, Kew et al.

(1998) showed that NMDA receptors in cortical neurons of

young rats, which express a relatively greater amount of

NR2B, had higher af®nity for glycine than receptors in

neurons of older rats. Our ®nding of higher potency of

glycine in the ethanol-exposed (3 days) cerebellar granule

neurons, which display relatively higher levels of NR2B

than control cells, is consistent with the earlier observations,

as well as with the ®nding of Blevins et al. (1995) of

increased sensitivity to glycine and ifenprodil (an NR2B-

selective antagonist) in cortical neurons exposed to ethanol

for 4 days. No measures of NMDA receptor subunit levels

were included in the study by Blevins et al. (1995),

however. Our results are also consistent with our previous

®nding that NR2B levels and glycine potency are higher in

cerebellar granule cells grown for 4 days in culture, than in

those grown for 7 days in culture (Bhave et al. 1999). As

would be predicted, because the ethanol treatment appeared

to partially delay the developmental decrease of NR2B

protein and the increase of NR2A protein, the potency of

glycine on day 7, in cells grown in the presence of ethanol

from day 4 to day 7 (EC50: 120 nm), is approximately

midway between the glycine potencies found previously

when NMDA receptor function was measured on day 4

(EC50: 43 nm) and day 7 (EC50: 288 nm) in vitro in control

cells (Bhave et al. 1999). It is noteworthy, however, that

Blevins et al. (1997) also reported increased sensitivity of

NMDA receptors to ifenprodil following 24-h ethanol

exposure of HEK293 cells transfected with NR1, NR2A

and NR2B subunits, without a detectable change in subunit

protein levels.

In addition to the change in glycine potency, ethanol

exposure, both in this and in our earlier studies, also

increased the maximal response of the NMDA receptor to

NMDA and glycine (Fig. 4 and Iorio et al. 1992). However,

in contrast to these results, the maximal NMDA/glycine

response was not different between control neurons grown

for 4 or 7 days in culture (Bhave et al. 1999), even though

the developmental difference in NMDA receptor subunit

levels is greater than the change produced by chronic ethanol

exposure, and the accompanying alteration in glycine

potency suggests that the developmental change in subunit

levels is re¯ected in alterations in functional receptors.

One characteristic of NMDA receptors that we have not

yet examined in the ethanol-treated neurons is the localiz-

ation of the receptors. It has been reported that, during

development, there is a differential targeting of the NMDA

receptor subunits to synaptic versus extrasynaptic receptors

in cerebellar granule neurons (Rumbaugh and Vicini 1999).

As the NR2A subunit increases, it is ®rst inserted into

synaptic receptor populations, although this change is

delayed in the extrasynaptic population. Similarly, the

decrease in NR2B occurs later in extrasynaptic, compared

with synaptic, receptors. Chronic ethanol treatment may

therefore have its greatest effect on synaptic NMDA

receptors, because it causes a delay in the developmental

changes. Rao and Craig (1997) reported that chronic

treatment of hippocampal neurons with an NMDA receptor

antagonist increased the number of immunocytochemically

stained clusters of NMDA receptors, and resulted in a

Ethanol and NMDA receptor development 403

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 396±405

greater proportion of the receptors being localized to

synaptic regions of the neurons. This treatment also resulted

in increased NMDA receptor responses (Obrietan and Van

den Pol 1995). Because the strength of synaptic transmission

depends on the number of receptors clustered at post-

synaptic sites, it is possible that changes in the synaptic

localization of receptors may contribute to the increased

maximal response to NMDA observed after chronic ethanol

exposure. If so, the measurement of total NMDA receptor

subunit protein levels may be insuf®cient to predict all

aspects of the functional response to NMDA in cerebellar

granule neurons or in other ethanol-treated neurons (e.g.

Follesa and Ticku 1996; Smothers et al. 1997).

The consequences of the delay in the developmental

switch of NR2A and NR2B caused by chronic ethanol

treatment, in addition to alterations in the pharmacological

properties of the NMDA receptor, may include effects on

neuronal development. A temporal correlation has been

reported between migration of cerebellar granule neurons,

NMDA receptor activity and expression of the NR2B

subunit (Feldmeyer and Cull-Candy 1996). Therefore, if the

observed changes in the expression of NR2B caused by

ethanol exposure also occur during in vivo development,

neuronal migration could be affected. In addition, there is

evidence that NMDA receptors containing NR2B show a

slower decay of NMDA-induced excitatory post-synaptic

currents than those containing NR2A (Flint et al. 1997).

In vivo, the period of slow kinetics of NMDA receptor

function coincides with the limited period during early post-

natal development when activity-dependent changes in

synaptic plasticity can take place (Feldmeyer and Cull-

Candy 1996). Alterations in the switch between NR2A and

NR2B expression in developing neurons, produced by

ethanol, may therefore have a substantial impact on the

synaptic plasticity which is necessary for the formation of

neural interconnections.

Acknowledgements

This work was supported in part by National Institute on

Alcohol Abuse and Alcoholism (NIAAA) (AA 9005) and the

Banbury Fund. The authors thank Ms Karin Nunley for expert

technical assistance.

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