chronic ethanol exposure changes dopamine d2 …if-pan.krakow.pl/pjp/pdf/2010/4_649.pdf · receptor...

Chronic ethanol exposure changes dopamine D2

receptor splicing during retinoic acid-induced

differentiation of human SH-SY5Y cells

Catrin Wernicke1, Julian Hellmann2, Ulrich Finckh3,

Hans Rommelspacher1,2

�Charité, Universitätsmedizin Berlin, Campus Charité Mitte, Klinik für Psychiatrie und Psychotherapie,

Dorotheenstr. 94, 10117 Berlin, Germany

�Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Klinik für Psychiatrie und Psychotherapie,

Sektion Klinische Neurobiologie, Eschenallee 3, 14050 Berlin, Germany

�Institut für Humangenetik, Universitätsklinikum Eppendorf, Hamburg, Germany

Correspondence: Catrin Wernicke, e-mail: [email protected]

Abstract:

There is evidence for ethanol-induced impairment of the dopaminergic system in the brain during development. The dopamine D2

receptor (DRD2) and the dopamine transporter (DAT) are decisively involved in dopaminergic signaling. Two splice variants of

DRD2 are known, with the short one (DRD2s) representing the autoreceptor and the long one (DRD2l) the postsynaptic receptor. We

searched for a model to investigate the impact of chronic ethanol exposure and withdrawal on the expression of these proteins during

neuronal differentiation. RA-induced differentiation of human neuroblastoma SH-SY5Y cells seems to represent such a model. Our

real-time RT-PCR, Western blot, and immunocytochemistry analyses of undifferentiated and RA-differentiated cells have demon-

strated the enhanced expression of both splice variants of DRD2, with the short one being stronger enhanced than the long one under

RA-treatment, and the DRD2 distribution on cell bodies and neurites under both conditions. In contrast, DAT was down-regulated by

RA. The DAT is functional both in undifferentiated and RA-differentiated cells as demonstrated by [�H]dopamine uptake. Chronic

ethanol exposure during differentiation for up to 4 weeks resulted in a delayed up-regulation of DRD2s. Ethanol withdrawal caused

an increased expression of DRD2l and a normalization of DRD2s. Thus the DRD2s/DRD2l ratio was still disturbed. The dopamine

level was increased by RA-differentiation compared to controls and was diminished under RA/ethanol treatment and ethanol with-

drawal compared to RA-only treated cells. In conclusion, chronic ethanol exposure impairs differentiation-dependent adaptation of

dopaminergic proteins, specifically of DRD2s. RA-differentiating SH-SY5Y cells are suited to study the impact of chronic ethanol

exposure and withdrawal on expression of dopaminergic proteins during neuronal differentiation.

Key words:

chronic ethanol, alcohol withdrawal, dopamine D2 receptor, splice variants, development, differentiation, DAT, human

neuroblastoma cells

Abbreviations: BCA – bicinchoninic acid, DAPI – 4’,6-diamidino-

2-phenylindole, DAT – dopamine transporter, DBH – dopa-

mine-�-hydroxylase, DMSO – dimethylsulfoxide DRD2 – do-

pamine D2 receptor, FBS – fetal bovine serum, FITC – fluores-

ceine isothiocyanate, HEK cells – human embryonic kidney

cells, hPBGD – human porphobilinogen deaminase housekeeping

gene, l – long, nCAM – neural-cell-adhesion-molecule, NET –

norepinephrine transporter, PMSF – phenylmethylsulfonylfluorid,

RA – retinoic acid, RT – reverse transcriptase, s – short, TEA –

triethanolamine, TRITC – tetramethylrhodamine isothiocyanate

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Introduction

The mesolimbic dopaminergic system exerts key

functions in rewarding properties of ethanol and other

drugs [16, 25, 62, 64]. It was demonstrated that the

DRD2 is important for the reinforcing and motivating

effects of ethanol as DRD2 knock-out mice demon-

strated reduced ethanol drinking [37] and ethanol-

conditioned place preference [10]. In humans, the lev-

els of DRD2 in the nucleus accumbens and the amyg-

dala were lower in alcoholics compared to healthy

subjects [20, 41, 58]. Furthermore, healthy relatives

of subjects with alcohol dependence demonstrated

higher levels of DRD2 in the respective brain regions

compared to healthy persons without family history of

alcohol dependence [61]. Whether the reduced DRD2

level in alcoholics is a cause or consequence of alco-

hol consumption was not clear for a long time. It was

repeatedly demonstrated that DRD2 expression influ-

ences ethanol consumption: Animal studies revealed

that over-expression of DRD2 in the nucleus accum-

bens reduces ethanol self-administration [53–55], and

that reduced DRD2 expression enhances ethanol con-

sumption [4, 35]. Moreover, association studies re-

vealed putative low expressing alleles to be associated

with alcohol dependence [26]. There is also evidence

that ethanol consumption influences DRD2 expres-

sion: One-week ethanol treatment of male Wistar rats

reduced D2-like receptor density by 43% in the stria-

tum [60]. Furthermore, Sari et al. [43] demonstrated

increased DRD2 binding density in the anterior re-

gions of the nucleus accumbens shell and core but not

in the striatum in alcohol-preferring rats which had

access to ethanol for 14 days compared to those which

had access to water only. Also Kim et al. [24] reported

on an increase of DRD2 mRNA in the caudate puta-

men and nucleus accumbens of rats after chronic in-

take of ethanol. In rhesus monkeys moderate-level al-

cohol exposure in early gestation and during whole

gestation reduced the striatal DRD2 binding/dopa-

mine synthesis ratio in adulthood, whereas middle-

to-late gestation ethanol exposure increased this ratio

[45]. In mice, prenatal ethanol exposure did not alter

DRD2 binding but increased responses to dopaminer-

gic drugs [7]. Thus, the consequence of ethanol con-

sumption on DRD2 density seems to depend on

amount, duration, and time point of ethanol consump-

tion. There is a strong evidence that ethanol will have

the greatest influence in periods of brain develop-

ment, like the fetal and adolescence periods.

There is another aspect, which has to be consid-

ered; two isoforms of DRD2 exist derived from alter-

native splicing of exon 6, which results in a 29 amino

acids deletion in the third intracellular loop of the

short isoform (DRD2s) compared to the long one

(DRD2l) [15]. The long isoform is preferentially

involved in postsynaptic response, whereas the short

isoform predominantly exerts presynaptic autoinhibition

of dopamine synthesis and release [23, 33, 59]. Further-

more, it was demonstrated that the splice variants couple

to different G-proteins [34]. A reduced DAT activity re-

sults in a higher concentration of dopamine in the synap-

tic cleft and vice versa. Thus, the balance between the

actions of both splice variants of DRD2 and DAT is

critically involved in dopaminergic neurotransmission

and long-term adaptation of dopaminergic pathways.

We were searching for a suited cell model for

studying the impact of long-term exposition of etha-

nol on the regulation of both splice variants of DRD2.

As primary mesencephalic embryonic cells derived

from animals have a limited survival period [17],

a permanent cell line would have the advantage of an

unlimited survival. The human neuroblastoma SH-

SY5Y cell line seemed to represent a suited model for

several reasons [5]. It consists of a mixture of neuron-

like and epithelium-like cell populations. Retinoic

acid (RA) treatment induces differentiation into the

neuron-like phenotype with long neurite-like pro-

cesses and a network of cell-cell connections whereas

epithelium-like cells are suppressed [14, 21, 22]. It

was demonstrated, that SH-SY5Y cells express tyro-

sine hydroxylase [6], and DRD2 [14]. Furthermore,

the expression of DAT [38] but also of norepinephrine

transporter (NET) [56, 66] and dopamine-hydroxylase

(DBH) [6] was reported. In view of the expression of

proteins characteristic for dopaminergic neurones, we

investigated whether SH-SY5Y cells express both iso-

forms of DRD2 and whether the expression of DRD2

and DAT are influenced by RA-induced differentia-

tion and chronic ethanol. Furthermore, the levels of

dopamine were determined.

Materials and Methods

Chemicals

Cell culture: Minimal essential medium with Earl’s salt

(MEM-Earle) supplemented with 2.2 g/l NaHCO3, fetal

650 ��

bovine serum (FBS) and phosphate buffered saline

(PBS) were purchased from Biochrom AG Seromed,

Berlin, Germany. Penicillin (100 I.E./ml), streptomy-

cin (100 µg/ml), retinoic acid (RA) and dimethylsul-

foxide (DMSO) were from Sigma, Taufkirchen, Ger-

many.

RT-PCR: high pure RNA isolation kit, 1st strand

cDNA synthesis kit for RT-PCR, LightCycler Fast-

Start DNA Master hybridization probes kit and Light

Cycler-h-PBGD housekeeping gene set were all from

Roche Applied Science, Mannheim, Germany. Prim-

ers and FRET-probes were designed and synthesized

by Tib Molbiol Berlin, Germany.

Protein isolation, Western blot and immunocyto-

chemistry: PMSF (Roche, Mannheim Germany), bicin-

choninic acid (BCA) protein assay reagent A (Pierce,

Thermo Fisher Scientific Inc., Waltham, USA), copper

(II) sulfate solution, reagent B (Sigma, Taufkirchen,

Germany), protein molecular weight marker IV of

Roche (Mannheim, Germany), Ponceau-S red, bovine

serum albumin (BSA, Sigma, Taufkirchen), antifade

solution containing 4’,6-diamidino-2-phenylindole (DAPI

II, Vysis, Abbott Laboratories, Wiesbaden, Germany),

and LumiLightPLUS kit from Roche.

Antibodies: mouse monoclonal anti-DRD2 anti-

body (1:400) and the polyclonal goat anti-DAT anti-

body (H-80; 1:400, both from Santa Cruz Biotechnol-

ogy, Santa Cruz, USA), and the peroxidase conju-

gated secondary antibodies anti-mouse IgG-antibody

and anti-goat IgG-antibody (both from Sigma,

Taufkirchen, Germany; 1:1000). Polyclonal rabbit-

anti-neurofilament antibody (Chemicon, Temecula,

USA), mouse monoclonal anti-human-neural-cell-

adhesion-molecule (nCAM) antibody (Chemicon, Te-

mecula, USA); secondary antibodies used in immuno-

cytochemistry were all from Sigma, Taufkirchen,

Germany: anti-mouse-IgG-antibody conjugated with

fluoresceine isothiocyanate (FITC) and anti-rabbit-

IgG-antibody conjugated with tetramethylrhodamine

isothiocyanate (TRITC), anti-rabbit-IgG-FITC anti-

body and anti-mouse-IgG-TRITC antibody.

GBR 12909 (Tocris, Bristol, UK), CellTiter 96

AQueous One Solution Cell Proliferation Assay of

Promega (Madison, USA)

Cell culture

The SH-SY5Y neuroblastoma cell line was obtained

from GewebeResourcenZentrum Braunschweig, Ger-

many. Cells were seeded with an initial density of 1 ×

105 cells/cm2 in cell flasks (Nunc, Wiesbaden, Ger-

many) and grown in MEM-Earle supplemented with

2.2 g/l NaHCO3, 10% (v/v) FBS and penicillin

(100 I.E./ml)/streptomycin (100 µg/ml). Cells were

maintained at 37°C in a saturated humidity atmos-

phere containing 5% CO2. Initial experiments were

conducted to find the optimal conditions for studying

the expression of both splice variants of DRD2. Con-

trols were cells grown for 7 days. For differentiation

experiments, RA was dissolved in DMSO to a con-

centration of 15 mg/ml (stock solution). The final

concentration of RA in the cell medium was

10 µmol/l. RA was added to the medium one day after

seeding the cells. Six days later, the cells were har-

vested. For immunocytochemistry, cells were grown

in chamber slides (PermanoxTM, Nunc, Wiesbaden,

Germany). Chamber slides were pre-coated with

poly-L-lysine under sterile conditions by adding

200 µl of a 1% (w/v) poly-L-lysine solution to the

chambers and subsequent incubation at 37°C for

30 min. The excess solution was removed followed

by washing the slides twice with sterile PBS. Thereaf-

ter, the chamber slides were air dried under sterile

conditions. Cells were seeded at a density of 105

cells/cm2 and cultivated under the same two conditions

as described above for the cell culture. Before fixation,

the medium was aspirated, cells were washed once

with PBS, and the chambers were removed. To fix the

cells on the slides, slides were incubated for 5 min in

ice cold methanol. Subsequently, the slides were air

dried and frozen at –20°C until further use.

Ethanol exposure

Cells were incubated in small (21 cm2) or medium

(75 cm2) cell flasks with RA for 3 days after seeding,

before adding ethanol to the medium at a final con-

centration of 100 mmol/l. RA was present during the

whole experiment. The medium was changed twice a

week. Control cells were cultured simultaneously

with RA, but without ethanol. The values determined

at each time-point are the results of four to six inde-

pendent experiments. In addition, two independent

experiments were conducted to investigate with-

drawal from ethanol for 3 h and 24 h, respectively.

RNA isolation

RNA was isolated using the high pure RNA isolation

kit which includes a DNase treatment.

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Adaptation of DRD2 splice variants to chronic ethanol��

Real-time RT-PCR

Reverse transcription (RT) of 0.5–1 µg RNA of each

isolate was performed with random primers using the

1st strand cDNA synthesis kit for RT-PCR.

Real-time RT-PCR was performed with the fluores-

cence resonance energy transfer (FRET)-probe format

in a LightCycler system (Roche Applied Science,

Mannheim, Germany) using the LightCycler FastStart

DNA Master hybridization probes kit and the appro-

priate primers and probes designed and synthesized

by Tib Molbiol. Primers (DRD2 and DAT) and probes

(DAT) were designed spanning exon boundaries to

exclude possible DNA fragments. Furthermore, two

different reverse primers for DRD2 were used in

separate capillaries to distinguish between the long

and the short isoform. Table 1 shows the applied

primers and probes. Fifty ng of cDNA were added to

each reaction tube. Cycling conditions were: denatu-

ration at 94°C for 5 min, 40 times cycling of denatura-

tion at 94°C for 5 s, primer and probe annealing at

58°C (DRD2) and 63°C (DAT) for 7 s, and elongation

at 72°C for 10 s. The amount of LC-Red specific fluo-

rescence was measured in each cycle after probe an-

nealing and analyzed with the LightCycler software.

The relative amount of target was calculated as

amount of target of each probe compared to the

amount of the human porphobilinogen deaminase

housekeeping gene (hPBGD, from the LightCy-

cler–h-PBGD housekeeping gene set) as reference

gene of the same probe. In ethanol experiments, no

standards of the housekeeping gene were included,

because of the limited space in the LightCycler. The

quantification was performed by the 2-��Ct method

[29]. Each probe was analyzed in two independent

PCR runs. The paired two-tailed t-test of the Graph-

652 ��

Tab. 1. Sequences of primers and probes

Function Sequence Genome bank reference numberNM_000795 (DRD2)NM_001044 (DAT)

Position of oligonucleotides

hPBGD housekeeping gene Premix of primers and probes provided by Roche, Mannheim, Germany

Forward primer for DRD2l and

DRD2s

5’-ggactcaataacgcag/accagaa

exon 4 exon 5

682–704

Reverse primer for DRD2s 5’-cgggcagcctc/ctttagt

exon 7 exon 5

986–882

Reverse primer for DRD2l 5’-ggtgagtacagttgcc/ctttagt

exon 6 exon 5

902–884

FRET-probes for DRD2 5’-caatgaagggcacgtagaaggagac-FL

exon 5

5’-LC Red640-atggaggagtagaccacgaaggccg-PH

exon 5

775–751

749–726

Forward primer for DAT 5’-cgtct/gtttggattgacg

exon 6 exon 7

1051–1068

Reverse primer for DAT 5’-aaggagaagacgacgaagc

exon 8

1228–1210

FRET-probes for DAT 5’-gctacaaccaagttcaccaacaactgctacag-FL

exon 7

5’-LC Red640-acgcgattgtcaccacctccatc-PH

exon 8

1129–1159

1162–1184

Shown are the sequences of primers and probes used in the real-time RT-PCR analysis, and their position in the sequence of the respective ge-nome bank reference number

Pad Prism program (GraphPad Prism software, San

Diego, CA, USA) was used to analyze the data.

Protein isolation and Western blot analysis

Washed cell pellets were re-suspended in 10 fold vol-

ume lysis buffer containing 20 mmol/l Tris HCl, pH

8.0, 1 mmol/l EDTA, 2 mmol/l �-mercaptoethanol, 1%

(w/v) sodium cholate, and 0.1 mmol/l PMSF, ho-

mogenized by using a B-12 sonifyer (Branson Sonic

Power Company, Danbury, CT, USA), and incubated

for 1 h at 4°C. Probes were centrifuged for 10 min at

2,000 × g and 4°C. The supernatant was used for fur-

ther analysis. An aliquot was removed and the protein

concentration determined using the bicinchoninic acid

(BCA) protein assay reagent A and copper(II) sulfate

solution, reagent B.

The protein was precipitated by methanol/chloro-

form (3:1, v/v). Each probe was diluted in Laemmli

buffer and the proteins were denatured for 10 min at

95°C. Forty µg per lane were loaded on a 10% (v/v)

acrylamide gel with a 5% (v/v) collecting gel. The

protein molecular weight marker IV of Roche was

used as a reference.

After running the gel for 6–8 h, the protein was

blotted onto polyvinylidene fluoride microporous

membrane (Millipore, Schwalbach, Germany) over-

night at 150 mV/cm2. Ponceau-S red staining of the

blot was performed to assess even blotting and equal

amounts of protein per lane and to identify molecular

weight marker. Immunostaining was performed fol-

lowing a standard protocol [1] using the mouse mono-

clonal anti-DRD2 antibody (1:400) and the polyclonal

goat anti-DAT antibody (H-80; 1:400), and the per-

oxidase conjugated secondary antibodies anti-mouse

IgG-antibody and anti-goat IgG-antibody (1:1000).

The LumiLightPLUS kit from Roche was used to pro-

duce a chemiluminescence signal, which was detected

after exposition to a Kodak film. The developed film

was scanned and molecular weight determination and

semi-quantification of bands were conducted using

the Aida software version 4.3 of raytest (Strauben-

hardt, Germany). For relative quantification, the blot

was stained with Coomassie blue and the staining was

recorded and quantified using the LAS 3000 imager

and the Aida software 4.3 of raytest (Straubenhardt).

Previous experiments revealed that the Coomassie

staining of the Western blots is as good as �-actine

staining for relative quantification (data not shown).

The investigation was conducted in two to three inde-

pendent experiments. The two-tailed t-test of Graph-

Pad Prism software was used to analyze the differ-

ences.

Immunocytochemistry

The following antibodies were used for immunostain-

ing: primary antibodies (1:200): mouse monoclonal anti-

DRD2 antibody, polyclonal rabbit-anti-neurofilament an-

tibody, polyclonal rabbit-anti-DAT antibody, mouse

monoclonal anti-human-neural-cell-adhesion-molecule

(nCAM) antibody; secondary antibodies (1:400): anti-

mouse-IgG antibody conjugated with fluoresceine

isothiocyanate (FITC) and anti-rabbit-IgG antibody

conjugated with tetramethylrhodamine isothiocyanate

(TRITC; for DRD2 and neurofilament), anti-rabbit-

IgG-FITC antibody and anti-mouse-IgG-TRITC anti-

body (for DAT and nCAM). DRD2 and neurofilament

were stained simultaneously with FITC and TRITC,

revealing green fluorescence for DRD2 and red fluo-

rescence for neurofilament. The same procedure was

applied for DAT and nCAM. All antibodies were di-

luted in PBS containing 1.5% (w/v) bovine serum al-

bumin (BSA). For immunostaining, slides were rehy-

drated for 5 min in PBS and blocking of non-specific

binding sites were performed with 10% (w/v) BSA in

PBS for 20 min. The primary antibody was incubated

for 1 h at 37°C. After three washing steps for 5 min in

PBS, the secondary antibody was incubated for 45

min at 37°C, followed again by three washing steps.

Slides were cover-slipped with antifade solution con-

taining 4’,6-diamidino-2-phenylindole (DAPI II). Im-

ages were obtained using an epifluorescence micro-

scope (Axiophot, Zeiss, Oberkochen, Germany)

equipped with a cooled charge-coupled device camera

(CCD- 1300DS) and analyzed using FISHview soft-

ware expo 2.0 (Applied Spectral Imaging, Migdal

Ha’emek, Israel). The investigation was done at least

three times for each condition and antibody. As nega-

tive control additionally slides were incubated with

secondary antibody only.

[3H]dopamine uptake assay

Uptake experiments were performed following the

protocol of Storch et al. [52] with minor modifica-

tions. Cells were seeded in 24 well plates at a density

of 2.5 × 105 cells/well. For differentiation, cells were

treated with RA for one week. Undifferentiated cells

were seeded for 3 days before conducting uptake ex-

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periments. Uptake was stopped after 10 min incuba-

tion by adding ice cold uptake buffer. Nonspecific up-

take was determined in simultaneously conducted as-

says at 0°C as well as in assays in the presence of the

DAT-specific inhibitor GBR 12909 (3 µmol/l). The

cells had been pre-incubated with the inhibitor for

10 min. An aliquot of each probe was removed for

protein quantification using the BCA protein assay

reagent A and copper(II) sulfate solution, reagent B.

Vmax and Km were calculated by nonlinear regression

using the GraphPad Prism program. The net uptake

was calculated as difference between the data re-

vealed at 37°C and 0°C (net-1), and at 37°C with

GBR12909 (net-2), respectively. This procedure was

chosen to investigate whether the DAT alone, or (ad-

ditionally) other transporters were responsible for the

uptake.

Cell viability assay

Cells were seeded at a density of 105 cells/well in 96

well plates and grown either in medium or medium

with 10 µmol/l RA for 3 days. Hundred mmol/l

ethanol was added to half of the wells. Cell viability

was determined after 3, 10, 17, 24, and 31 days in cul-

ture (representing 0 days, 1, 2, 3, and 4 weeks of etha-

nol exposure) using the CellTiter 96 AQueous One

Solution Cell Proliferation Assay of Promega.

Measurement of dopamine levels in cells

Cells were seeded in flasks at a density of 1.2 × 105

cells per cm2 and grown in medium with 10 µmol/l

RA. After 3 days medium was renewed and 100 mmol/l

ethanol was added to parts of the flasks. After incuba-

tion for further 7 days, cells were harvested. For with-

drawal experiments, parts of the RA/ethanol treated

cells were incubated 24 h with RA-containing me-

dium before harvesting one day later. Controls were

untreated cells which were seeded at the same density

as mentioned above, but were harvested after 3 days

of seeding. The differences in treatment periods were

chosen, as we wanted to start with the same cell den-

sity. However, untreated cells have a high mitosis

rate, whereas RA-treatment leads to a strong mitosis

inhibition. Thus, 3 day cultivation led to around 50%

confluence in the case of untreated cells. For each

condition three independent experiments were con-

ducted. Dopamine levels were assayed in cell ho-

mogenates using high-performance liquid chromatog-

raphy (HPLC) with coulochemical detection as

described previously [30]. Cell lysates of undifferenti-

ated, RA-differentiated and RA/ethanol treated cells

were transferred to a centrifugal filter device and then

centrifuged (13,000 × g, 10 min, +4°C) using a Bio-

fuge (Heraeus, Hanau, Germany). The resulting elu-

ent was immediately applied to the HPLC apparatus.

Dopamine was separated by using a Nova-Pak C-18

column, 3.9 × 150 mm, a HPLC pump (Knauer, Ber-

lin, Germany), an autosampler (Waters 717 plus, Mil-

ford, MA, USA) and a coulochemical detector (ESA,

Chelmsford, MA, USA). The mobile phase consisted

of 1.5% triethanolamine (TEA), 3.41 mol/l 1-octane

sulfonic acid, 53.7 µmol/l EDTA and 2% methanol

(v/v, isocratic conditions). The pH was adjusted to 3.9

with acetic acid. The flow rate was 0.4 ml/min, and

the temperature was kept at 20°C. The electrode was

set at 350 mV. The detection limit for dopamine was

approximately 15 fmol. An aliquot of each probe was

removed for protein quantification using the BCA

protein assay reagent A and copper(II) sulfate solu-

tion, reagent B.

Results

Dopaminergic status of RA-differentiated cells

DRD2: Figure 1a shows a representative Western blot

for DRD2. The analysis revealed specific bands at ap-

proximately 52 kDa, 65 kDa, 87 kDa and 102 kDa.

The enhancement of the 52 kDa band seen by RA

treatment (Fig. 1b) was significant (p = 0.0187, t =

7.209, df = 2). The DRD2l and DRD2s specific anti-

bodies provided by Calbiochem were not sufficiently

specific. They also marked bands of the molecular

weight marker (data not shown), but did not mark

specific bands in the protein lysate. That is why we

used the monoclonal antibody from Santa Cruz Bio-

technology, which revealed clear bands in the lysate,

but did not discriminate DRD2l and DRD2s isoforms.

Immunocytochemistry revealed the distribution of the

DRD2 (green fluorescence) on cell bodies and pro-

cesses [Fig. 2a (undifferentiated), Fig. 2d (RA-

differentiated)]. Immunostaining of neurofilament

(red fluorescence) was visible in the undifferentiated

(Fig. 2b) and differentiated cells (Fig. 2e). Also small

654 ��

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a cb

fed

Fig. 2. Immunostaining of DRD2 (green fluorescence, Fig. 2a, d) andneurofilament (red fluorescence, Fig. 2b, e) in undifferentiated andRA-differentiated SH-SY5Y cells. Undifferentiated cells: a–c, RA-treated cells: d–f. In undifferentiated cells the cell bodies and shortcell process are stained by DRD2 antibody (Fig. 2a). The immu-nostaining with neurofilament antibody was scarcely visible (Fig. 2b).In RA-treated culture cell bodies and long cell processes are stainedwith both DRD2 and neurofilament (Fig. 2d–f). The arrows indicatebranches, and the arrow head growth cones. In the merged figures(2c, f) nuclei are stained with DAPI (blue). All images were taken witha 750-fold magnification. The investigation was done in three inde-pendent experiments with four independent slides in each experi-ment

a b c

fed

* * *

Fig. 3. Immunostaining of DAT (green fluorescence) and nCAM (redfluorescence) in SH-SY5Y cells treated as described in Figure 1. Un-differentiated cells: a–c, RA-treated cells: d–f. In undifferentiated cells(Fig. 3a) some areas of the soma are strongly stained (arrow) and insome areas DAT signals are missing (arrow head). The staining ofnCAM (Fig. 3b) is scarcely visible compared to differentiated cells(Figs. 3e, h). Some (asterisk) but not all (double arrow) cell processesshow DAT immunostaining in undifferentiated cells (Figs. 3a–c). InRA-differentiated cells long neurites and somata are stained withboth DAT and nCAM (Fig. 3d–f). The arrows indicate branches. In themerged figures (3c, f) nuclei are stained with DAPI (blue). All imageswere taken with a 750-fold magnification. The investigation was donein three independent experiments with four independent slides ineach experiment

102 kDa

87 kDa

65 kDa

52 kDa

co RA

co RA

75 kDa

0.1

0.05

0

102 87 65 52

DRD2 expression

controlRA

peaks [kDa]

De

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sre

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tota

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tota

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control RA

treatment

DAT expression1.0E + 00

5.0E - 01

0.0E + 00

*

*

a b

c d

Fig. 1. Western blot analysis for DRD2 and DAT in undifferentiated and RA-differentiated SH-SY5Y cells. SH-SY5Y cells were grown in culturefor 3 days. Thereafter, the medium was replaced by fresh medium (controls) and fresh medium containing retinoic acid (10 µmol/l), re-spectively. The cells were cultivated for further 7 days and finally collected. Western blot analysis was conducted with DRD2 (1a) and DAT (1c)specific antibodies. In Figure 1a specific bands for DRD2 are visible at approximately 102 kDa, 87 kDa, 65 kDa and 52 kDa in all cells. The latterband is enhanced in RA-treated cells. Figure 1b shows the mean and standard deviation of the corresponding relative quantification of DRD2expression recorded by the Aida software (raytest). In Figure 1c a specific band for DAT is visible at approximately 75 kDa in all cells, witha lower expression in the RA- treated cells. Figure 1d shows the mean and standard deviation of the corresponding relative quantification ofDAT expression. Figures 1b and 1d represent the results of three independent experiments. * p < 0.05

cell processes were visible in undifferentiated cells.

They became much longer and more prominent fol-

lowing differentiation, showing ramifications, and

bundles of neurite-like processes, all of them specifi-

cally stained by DRD2 and neurofilament antibodies

(Fig. 2d–f). Furthermore, growth cones were visible

and clearly stained by both antibodies (Fig 2d–f, ar-

row heads).

The findings of the real-time RT-PCR studies are

presented in Figures 4a and 4b. Both splice variants

were detectable under both conditions with the long

isoform more prominent compared to the short one

(76-fold more prominent in controls, 41-fold in RA-

treated cells; reciprocal values of Fig. 4a). The

amount of mRNA was increased 1.3-fold in the case

of DRD2l and 2.4-fold (p = 0.0218, t = 6.663, df = 2)

in the case of DRD2s after one week of RA treatment

compared to undifferentiated cells (Fig. 4b). The dif-

ference of the DRD2s/DRD2l-ratio (Fig. 4a) between

controls and RA-treated cells was significant (t-test:

p = 0.0365, t = 5.089, df = 2).

DAT and dopamine uptake: The Western blot

showed a specific, single band at approximately 75 kDa

for DAT (Fig. 1c). The intensity of the band was sig-

nificantly reduced in the RA (p = 0.0186, t = 3.829, df

= 4) treated cells (Fig. 1d), amounting to 71% of con-

trol cells. Immunocytochemistry [Fig. 3a (undifferen-

tiated), Fig. 3d (RA-differentiated)] revealed the pres-

656 ��

a

b

c

Fig. 4. Changes of the amount of the short and the long splice variantof DRD2 under different conditions. Cells were treated as describedin Figure 1. Figure 4a: DRD2s/DRD2l ratio of mRNA in undifferentiated,and RA-treated SH-SY5Y cells. Figure 4b: Percentage of the relativeamount (normalized to the housekeeping gene hPBGD) of mRNA ofthe long and short isoform of DRD2 after RA-treatment compared toundifferentiated cells (100%). Figure 4c: Percentage of the relativeamount (normalized to the housekeeping gene hPBGD) of mRNA ofDAT after RA-treatment compared to undifferentiated cells (100%).The figures represent the mean and standard deviation of two inde-pendent cell culture experiments. Furthermore, each probe was ana-lyzed by two independent real-time RT-PCR runs. The paired two-tailed t-test was used to analyze the data. * p < 0.05

[3H]dopamine uptake in

undifferentiated SH-SY5Y cells

0 25 50 75 100 1250.0 × 10–00

2.5 × 10–07

5.0 × 10–07

37°C-0°C

37°C-GBR

dopamine concentration [µmol/L]

do

pam

ine

up

take

[µm

ol/µ

gp

rote

in/1

0m

in]

a

[3H]dopamine uptake in retinoic acid

differentiated SH-SY5Y cells

0 25 50 75 100 125

37°C-0°C

37°C-GBR

dopamine concentration [µmol/L]

0.0 × 10–00

2.5 × 10–07

5.0 × 10–07

do

pam

ine

up

take

[µm

ol/µ

gp

rote

in/1

0m

in]

b

Fig. 5. Dopamine uptake in undifferentiated and RA-differentiatedSH-SY5Y cells. The graphs show the specific net uptake of [�H]dopa-mine as the difference between 37�C condition and 0�C (net-1), and37�C and 37�C in combination with 3 µmol/l of the specific inhibitor ofDAT (GBR 12909)(net-2), respectively, in undifferentiated (upperplot) and RA-differentiated cells (lower plot). The mean values arepresented as the mean ± SEM of two independent experiments. Be-tween undifferentiated and RA-differentiated cells the net-1 uptake(37�C–0�C) was significantly different (p < 0.05), whereas the differ-ence in the net-2 uptake (37�C-GBR 12909) was at the trend levelonly (p < 0.1)

ence of the DAT on cell bodies and neurite-like pro-

cesses (green fluorescence). In undifferentiated cells

there were strong signals in a distinct area of the

soma. Some, but not all neurite-like processes were

marked (Fig. 3a). In RA-differentiated cells nearly all

neurite-like processes were marked until the distal

end, and the cell body signals were less pronounced

than in undifferentiated cells, suggesting trafficking

of the DAT protein along the neurite-like processes

(Fig. 3d). Figures 3b and 3e demonstrate labeling with

the neuronal marker nCAM. Staining was weaker in

undifferentiated cells compared to the differentiated

cells. The results of real-time RT-PCR are presented

in Figure 4c. DAT-mRNA was decreased to 35% in

RA-differentiated cells (p = 0.0120, t = 9.054, df = 2).

Although the changes detected were qualitatively

equal to the Western blot, they differed quantitatively.

This discrepancy in the extent of changes may be due

to methodological problems of the real-time RT-PCR,

specifically with the primers and probes used for

DAT. In control experiments with human embryonic

kidney (HEK) 293 cells over-expressing human DAT,

we found that the efficacy of DAT-PCR decreased

dramatically, if the amount of the target-transcript was

below a certain threshold (data not shown). This prob-

lem could not be solved by changing the PCR

parameters. That is why we did not use this method to

quantify DAT transcription in chronic ethanol experi-

ments in which the amount of DAT was apparently

below that of RA-only controls. To investigate

whether the undifferentiated and RA-differentiated

cells express functional transporter protein, [3H]dopa-

mine uptake was measured. In undifferentiated cells

(Fig. 5a), the experiments revealed a Vmax of 2.6 ×

10–13 (± 7.2 × 10–14) mol/µg protein and 10 min

(net-1, 37°C–0°C), and 1.8 × 10–13 (± 5.8 × 10–14)

mol/µg protein and 10 min (net-2, 37°C-GBR). In dif-

ferentiated cells (Fig. 5b), the Vmax, for both net-1 and

net-2, was 1.2 × 10–13 (± 2.6 × 10–14, ± 2.5 × 10–14)

mol/µg protein and 10 min. The Km was 1.5 × 10–5

mol/L under all conditions. There was a trend towards

a difference of the net-2 uptake between undifferenti-

ated and differentiated cells (paired t-test: p = 0.0628,

t = 2.210, df = 7); the Vmax of both conditions was not

significantly different (t-test). The net-1 uptake was

significantly different between undifferentiated and

differentiated cells (paired t-test: p = 0.0317, t = 2.676,

df = 7), although, the Vmax was also not significantly

different (t-test).

Dopamine concentration: The dopamine concen-

tration (Fig. 6) was 0.30 ± 0.032 pg dopamine/ng pro-

tein in untreated controls and was significantly enhanced

to 1.38 ± 0.375 pg dopamine/ng protein cells treated for

one week with RA (p = 0.015, t = 4.058, df = 4).

Chronic ethanol exposure

Cell viability: Cell viability was assessed in undiffer-

entiated and RA-differentiated cells with and without

ethanol exposure after different incubation periods in

culture up to 31 days in culture. Chronic exposure

with 100 mM ethanol for 28 days did not impair cell

viability in undifferentiated cells as can be conducted

from the ratios of nearly 1 during the whole time-

course (Fig. 7). RA-induced differentiation led to a re-

duced ratio in the cell viability assay compared to un-

�� 657


cell viability

0

0.2

0.4

0.6

0.8

1

1.2

1.4

day 3

(day 0)

day 10

(day 7)

day 17

(day 14)

day 24

(day 21)

day 31

(day 28)

time of RA (upper lane) and ethanol (lower lane) treatment

RA/co

RA+EtOH/co

EtOH/co

ratio

Fig. 7. Cell viability of SH-SY5Y cells under different conditions. Timecourse of the relative cell viability of SH-SY5Y cells treated either withRA (10 µmol/l), ethanol 100 mmol/l), and in combination with ethanolcompared to undifferentiated control cells

Fig. 6. Dopamine contents in SH-SY5Y cells. Cells were grown for 10days with 10 µmol/l retinoic acid (RA; 10µmol/l), and for three days withRA and for further 7 days with RA and 100 mmol/l ethanol (RA/EtOHand RA/EtOH-withdrawal), respectively. Ethanol-withdrawal lasted24 h. Controls were grown in medium for three days only to preventovergrowing. * p < 0.050, (*) p < 0.100

differentiated cells (ratio ~0.6, p = 0.0003, t = 11.44,

df = 4). This is the result of a reduced cell division of

RA-treated cells. The outcome of cells treated with etha-

nol in combination with RA was also significantly dif-

ferent from undifferentiated cells (ratio ~0.6, p = 0.0021,

t = 7.031, df = 4) but not from RA-treated cells.

DRD2 transcription: Chronic RA treatment led to

an increase of mRNA of both splice variants (Fig.

8a-b) seen from day 10 of RA treatment on until day

31 of RA treatment with the short splice variant being

enhanced more than the long one (Fig. 8c). Long-term

RA-treatment resulted in a continuously increasing

658 ��

Tab. 2. Two-way ANOVA of transcription changes with respect totreatment and time.

p-value df F

D2l/hPBGD

co vs. ETOH treatmenttime

0.0003

< 0.000115

15.576.6

co vs. w3 treatmenttime

< 0.0001

< 0.0001

13

375.445.2


< 0.0001

0.0269

13

265.73.7

ETOH vs. w3 treatmenttime

< 0.0001

< 0.0001

13

313.252.1


< 0.0001

0.0301

13

215.73.6

D2s/hPBGD


< 0.0001

< 0.0001

15

69.3166.4


0.0933< 0.0001

13

3.177.6


< 0.0001

< 0.0001

13

31.542.7


< 0.0001

< 0.0001

13

74.764.0


< 0.0001

< 0.0001

13

123.330.7

D2s/D2l


< 0.0001

< 0.0001

15

107.439.0


< 0.0001

0.0148

13

66.54.4


< 0.0001

0.0018

13

22.67.0


0.0010

0.0004

13

14.39.2


0.0167

< 0.000113

6.716.8

Shown are the results of the two-way ANOVA analysis of the data rep-resented in Figure 8 with respect to the different cell culture condi-tions (treatment) and time of cell cultivation (time). Abbreviations: co– RA-only treated cells; ETOH – cells treated with RA and ethanol; w3– cells treated with RA and ethanol and withdrawal of ethanol for 3 h;w24 – cells treated with RA and ethanol and withdrawal of ethanol for24 h; D2l/hPBGD – ratio of cDNA of the long splice variant of DRD2with respect to the housekeeping gene; D2s/hPBGD – ratio of cDNAof the short splice variant of DRD2 with respect to the housekeepinggene; D2s/D2l – ratio of cDNA of the short splice variant of DRD2 withrespect to the long splice variant; df – degree of freedom

DRD2S/DRD2L

0.00E+00

2.00E-02

4.00E-02

6.00E-02

8.00E-02

day 3

(3h)

day 4

(day 1)

day 10

(day 7)

day 17

(day 14)

day 24

(day 21)

day 31

(day 28)

time of RA (upper lane) and ethanol (lower lane)

treatment

rati

o

co

EtOH

3 h_w

24 h_w

DRD2S/hPBGD

0.00E+00

2.00E-02

4.00E-02

6.00E-02

8.00E-02

1.00E-01

day 3

(3h)

day 4

(day 1)

day 10

(day 7)

day 17

(day 14)

day 24

(day 21)

day 31

(day 28)


treatment

rati

o

co

EtOH

3 h_w

24 h_w

DRD2L/hPBGD

0.00

0.50

1.00

1.50

2.00

2.50

3.00

day 3

(3h)

day 4

(day 1)

day 10

(day 7)

day 17

(day 14)

day 24

(day 21)

day 31

(day 28)


treatment

ratio

co

EtOH

3 h_w

24 h_w

a

b

c

Fig. 8. mRNA of DRD2s and DRD2l under different conditions. Timecourse of the relative amount of mRNA of DRD2l (a) and DRD2s (b)compared to hPBGD housekeeping gene and of the ratio of DRD2sto DRD2l (c) during RA (10 µmol/l) treatment (black columns) andduring combined treatment with RA and ethanol (100 µmol/l; light col-umns) for up to 4 weeks and subsequent ethanol withdrawal for 3 h(diagonal stripped columns) and 24 h (stripped columns). The valuesdetermined at each time-point are the results of four to six independ-ent experiments. In addition, two independent experiments wereconducted to investigate withdrawal from ethanol for 3 h and 24 h, re-spectively. Shown are the mean and standard deviation. Statisticaldata are presented in Table 2

DRD2s/DRD2l-ratio (see Fig. 8c). After 31 days of

RA-treatment the amount of DRD2l was 2.1-fold, and

of DRD2s 4.2-fold increased compared to day 3 (p <

0.0001, t = 9.993, df = 8; p < 0.0001, t = 15.31, df = 8).

This resulted in a significant difference of the

DRD2s/DRD2l-ratio between the first (3 days) and

last (31 days) time point (p = 0.0001, t = 9.097, df =

8). Ethanol treatment enhanced the increase of DRD2l

(Fig. 8a), and significantly diminished the increase of

DRD2s (Fig. 8b). This led to a significantly decreased

ratio of DRD2s/DRD2l compared to RA-only treated

cells (Fig. 8c). The statistical data for the ethanol

treatment are represented in Table 2. Withdrawal from

ethanol induced an enhanced expression of DRD2l

compared to RA-treated cells after 3 h and 24 h. Fur-

thermore, withdrawal increased the expression of

DRD2s leading to normalized amounts after 3 h of

withdrawal compared to RA-only treated cells, and to

an enhanced amount after 24 h of withdrawal compared

to RA-only treated cells (Fig. 8b). Because the expres-

sion of DRD2l (Fig. 8a) was enhanced more than the ex-

pression of DRD2s (Fig. 8b) during withdrawal, the

DRD2s/DRD2l ratio was still reduced after withdrawal

from ethanol for 3 h and 24 h (Fig. 8c).

Dopamine concentration: The dopamine concen-

tration (Fig. 6) was 0.30 ± 0.032 pg dopamine/ng pro-

tein in untreated controls and was significantly en-

hanced to 1.38 ± 0.375 pg dopamine/ng protein cells

treated for one week with RA (p = 0.015, t = 4.058, df

= 4). Combined RA/ethanol treatment diminished the

up-regulation seen under RA-treatment alone to 0.61

± 0.186 pg dopamine/ng protein; the difference to

RA-treated cells and controls was at the trend level (p =

0.061, t = 2.588, df = 4 and p = 0.078, t = 2.358, df = 4,

respectively). Ethanol withdrawal for 24 h resulted in

a further reduction of the dopamine concentration to

0.41 ± 0.156 pg dopamine/ng protein compared to RA-

treated cells (p = 0.028, t = 3.367, df = 4).

Discussion

Dopaminergic status of RA-differentiated

SH-SY5Y cells

We were searching for a suited dopaminergic model

to investigate the influence of ethanol on DRD2 splic-

ing. There are conflicting results whether SH-SY5Y

cells represent a more dopaminergic or noradrenergic

cell type, as the expression of DRD2 [14], DAT [38],

DBH [6], and NET [56] were reported. We detected

specific bands for DRD2 with molecular weight of

65, 87 and about 102 kDa and in the RA treated cells

an additional band at approximately 52 kDa. These

findings are in agreement with published data for

DRD2 [13, 32]. Although the deduced molecular

weight of DRD2 is 48 and 51 kDa for the short and

the long isoform respectively, a higher molecular

weight may represent glycosylated proteins [9, 32].

Immunocytochemistry revealed the distribution of

DRD2 in the areas of the cell bodies and neurites. On

the protein level it was not possible to distinguish be-

tween DRD2l and DRD2s because suited antibodies

were not available. Using real-time RT-PCR, we dem-

onstrated that both splice variants of DRD2 are ex-

pressed and that the expression of the short isoform is

more enhanced than the expression of the long iso-

form during RA-differentiation. This is consistent

with the observation of a functional RA response ele-

ment (RARE) in the promoter of the DRD2 gene [42].

In our experiments the ratio of DRD2s to DRD2l

changed from 1:77 in controls to 1:41 in RA treated

cells, indicating a nearly twofold increase in alterna-

tive splicing of the short isoform. Recently, Oomizu et

al. [36] (suggested that the splicing factor-2/alterna-

tive splicing factor (SF-2/ASF), which belongs to the

serine and arginine (SR) repeats containing splicing

factors, represents the responsible factor for alterna-

tive splicing of DRD2. After 6 h of RA treatment

Truckenmiller et al. [57] found an up-regulation of

gene expression of splicing factor arginine/serine-rich

4 (SFRS4), another member of SR proteins, and of

several small nuclear ribonucleic proteins, which may

also contribute to splice site selection [2].

Using [3H]dopamine uptake in undifferentiated and

RA-differentiated cells we demonstrate the function-

ality of DAT. In accordance with our Western blot

analysis, the Vmax (net-2) was slightly but not signifi-

cantly decreased in RA-differentiated cells compared

to undifferentiated ones. The difference in the paired

t-test seen between net-1 and net-2 in undifferentiated

cells indicate that in addition to DAT further specific

transport mechanisms (e.g., NET) must be present un-

der this condition. In RA-differentiated cells, there

was no difference between net-1 and net-2 uptake,

emphasizing the dopaminergic status of RA-

differentiated SH-SY5Y cells. Contrary to us, Pres-

graves et al. [38] found a slight but significant en-

�� 659


hancement of DAT after 3 days of RA treatment of

SH-SY5Y cells in Western blot analysis. We do not

have an explanation for this discrepancy. It is known

that direct and indirect dopaminergic activation leads

to down-regulation of DAT action in vivo [27]. There

is evidence that DAT is physically and functionally

coupled to G protein-coupled receptors. For instance,

Lee et al. [28] demonstrated a physical coupling be-

tween DRD2s and DAT in human embryonic kidney

(HEK) cells, artificial expressing both proteins. They

found an activation of DAT function by co-expressing

DRD2s. Bolan et al. [8] used the same cell model ex-

pressing both proteins and reported on an activation

of DAT function by short-time DRD2 agonist treat-

ment. In HEK cells co-expressing DAT and DRD3

Zapata et al. [65] demonstrated for the DAT-activity

an up-regulation after short-time (1 min) DRD3 acti-

vation and a down-regulation after prolonged (30

min) DRD3 activation. As no prolonged DRD2 ago-

nist treatment was reported, it is not clear whether

a longer DRD2 agonist treatment will also result in

a down-regulation of DAT-function, which would be

in accordance with in vivo findings [27]. RA treat-

ment increased the dopamine content of SH-SY5Y

cells, indicating a continuously enhanced dopaminergic

neurotransmission. Thus, the up-regulation of DRD2s

in SH-SY5Y cells during RA-induced differentiation,

as demonstrated in our study, may influence the

slightly decreased expression/function of DAT during

RA treatment. The expression of nCAM was weak in

undifferentiated cells and enhanced after RA and

RA/BDNF treatment, confirming a recent report [47].

As reported by others, RA differentiation stimulates

the expression of a large number of genes, including

tyrosine kinase B (TrkB), the receptor for BDNF [12,

22, 57]. As differentiation leads to a reduced rate of

proliferation, the cell viability assay revealed a re-

duced cell number in differentiated cells from day 3

of RA treatment on compared to undifferentiated cells

until the end of the experiment. This is in agreement

with our previous findings [21], where RA-treatment

alone was sufficient to maintain long-lasting differen-

tiation in SH-SY5Y cells.

Chronic ethanol exposure

Dopaminergic neurotransmission participates in the

development and maintenance of alcoholism [e.g. 44,

46, 51, 63]. Furthermore, prenatal exposure to ethanol

impairs the development of most CNS neurotransmit-

ter systems. Neurochemical, behavioral, and pharma-

cological studies suggest the developing dopaminer-

gic systems may be particularly targeted [11, 18, 40].

Dietary ethanol consumption for 3 weeks before, and

continuing throughout pregnancy decreased both

DAT and tyrosine hydroxylase (TH) mRNA expres-

sion in the VTA of adult male offspring. This is in

agreement with our findings that dopamine is reduced

in RA/ethanol treated cells compared to RA-treated

one. Morphological changes in dopaminergic neurons

have been reported from rats prenatally exposed to

ethanol, including smaller cell bodies, retarded den-

dritic growth and branching in the SNpc [49]. This is

in agreement with previous studies [21], where

RA/ethanol treatment, although inducing differentia-

tion of SH-SY5Y cells, as indicated by cells con-

nected with a network of neurite-like cell processes,

the neurites were shorter compared to RA-only

treated cells and the cell clusters with abundant and

branched neurites were missing [21]. No significant

cell loss was induced by chronic ethanol treatment of

differentiated cells, as shown by the cell viability as-

say. No dopaminergic cell loss has been observed in

children with fetal alcohol syndrome, and animals ex-

posed to ethanol during embryonic and fetal period

[45]. However, a reduced DRD2 density [31, 45] and

activity of midbrain dopaminergic neurons [48] has

been reported. In our cell model the expression of

DRD2l was unchanged, whereas DRD2s was dimin-

ished until the end of observation. The finding is con-

sistent with the report of Oomizu et al. [36], who also

found a decrease of DRD2s mRNA in the anterior pi-

tuitary of rats after chronic ethanol treatment. Further-

more, the dopamine concentration in cells was re-

duced in RA/ethanol treated cells compared to RA-

only treated cells after one week of exposure. As the

amount of DRD2l increased more than the amount of

DRD2s during withdrawal for 3 and 24 h, the

DRD2s/DRD2l ratio was still reduced compared to

RA-only treated cells indicating a long-lasting altera-

tion in dopaminergic function. This was also demon-

strated by a continued reduction of dopamine concen-

tration after 24 h withdrawal.

Apomorphine-induced hypoactivity is exerted via

presynaptic DA receptors localized in the nucleus ac-

cumbens [39]. In contrast, apomorphine hypersensi-

tivity is thought to be mediated by postsynaptic meso-

limbic DA receptors [18]. Mice were exposed to pre-

natal ethanol on gestation days 6–18. At 90 days of

age, male offspring was monitored in an open field

660 ��

following low apomorphine injections. Apomorphine

was less effective in reducing locomotor activity com-

pared to control mice. The results suggest disturbance

of presynaptic DRD2 receptors by prenatal ethanol

exposure and that the exposure results in long-lasting

perturbation of DA receptor sensitivity [3]. Rats re-

ceived liquid ethanol diet beginning on gestational

day 6 through 20 of pregnancy. Twenty eight-day-old

offspring received systemic apomorphine. The dose-

response curve of ethanol-treated rats was biphasic,

showing increased locomotor activity following low

doses of apomorphine and less activity at the highest

dose compared to control rats [18]. These findings

were not confirmed in a recent study with rats. No

prenatal treatment effects of ethanol on drug-induced

behaviors were observed for dopamine D2 receptor

agonists or antagonists [50]. These findings demon-

strate that ethanol exposition in utero affects dopa-

minergic neurons in offspring. The apomorphine

challenge experiments may indicate that presynaptic

DRD2, that means the short variant (see introduction)

is affected whereas the postsynaptically expressed

long variant seems less impaired.

Our results demonstrate that RA differentiated

SH-SY5Y cells are well-suited to study the impact of

chronic ethanol exposure on the development of

DRD2. Further work is required to characterize the

molecular mechanisms involved.

Acknowledgments:

The authors would like to thank Sabine Strauss, Regina Hill, and

Anke Sänger for excellent technical assistance. Special thanks to

Christiane Bommer (Institute for Medical Genetics, Charité) for

technical support in fluorescence microscopy. The work was partly

supported by Bundesministerium für Bildung und Forschung,

Germany, 01GZ0309.

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Received:

August 18, 2009; in revised form: December 8, 2009.

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