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a
-Synuclein expression in the brain and blood during abstinence from
chronic alcohol drinking in mice
Barbara Ziolkowska
a
, Agnieszka Gieryk
a
, Agnieszka Wawrzczak-Bargiela
a
, Tomasz Krowka
a
,
Dorota Kaminska
a
, Agnieszka Korkosz
b
, Przemyslaw Bienkowski
b
, Ryszard Przewlocki
a
,
*
a
Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, Sm˛etna 12, 31-343 Krakow, Poland
b
Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Sobieskiego 9, 02-957 Warsaw, Poland
article info
Article history:
Received 9 October 2007
Received in revised form 27 March 2008
Accepted 1 April 2008
Keywords:
a
-Synuclein
Alcohol drinking
Two-bottle choice
Abstinence
Amygdala
Craving
abstract
a
-Synuclein is a presynaptic protein proposed to serve as a negative regulator of dopaminergic neuro-
transmission. Recent research has implicated
a
-synuclein in chronic neuroadaptations produced by
psychostimulant and opiate use, as well as in genetically determined susceptibility to alcoholism in
humans. The aim of our study was to characterize the changes in
a
-synuclein expression after short-term
abstinence from chronic alcohol drinking in mice.
Male C57BL/6J mice were allowed to drink increasing concentrations of alcohol in the two-bottle choice
procedure. Then the mice were given constant access to an 8% alcohol solution and water for 32 days, and
were sacrificed 2 h, 24 h or 48 h after alcohol withdrawal. RT-PCR, in situ hybridization and Western
blotting techniques were used to measure
a
-synuclein mRNA and protein levels in the brain and blood.
a
-Synuclein protein levels were elevated by up to 80% in the amygdala of mice withdrawn from alcohol
for 24 h or 48 h. No changes in
a
-synuclein levels were found in the mesencephalon or striatum/
accumbens. The levels of
a
-synuclein mRNA remained unchanged in all brain regions examined (the
striatum, nucleus accumbens, amygdala, substantia nigra, ventral tegmental area).
a
-Synuclein mRNA
was up-regulated in the whole blood 48 h after alcohol withdrawal.
The accumulation of
a
-synuclein in the amygdala, observed in this study, seems to be a common feature
of alcohol and opiate abstinence. This finding suggests a role of
a
-synuclein in common neuroadaptations
produced by long-term alcohol and drug use. Although
a
-synuclein expression in the blood seems un-
related to that in the brain, it may serve as a peripheral biomarker of chronic alcohol consumption.
Ó2008 Elsevier Ltd. All rights reserved.
1. Introduction
a
-Synuclein is a protein abundantly expressed by neurons, and is
present almost exclusively in presynaptic terminals (Iwai et al.,
1995; Clayton and George, 1998, 1999). It is best known for its in-
volvement in neuropathology, especially Parkinson’s disease and
other synucleinopathies, whereas its physiological roles remain
unclear (Lotharius and Brundin, 2002; Marti et al., 2003; Vekrellis
et al., 2004). Its proposed physiological functions include the regu-
lation of neurotransmitter release and reuptake, presynaptic vesicle
recycling and the role of a molecular chaperone (reviewed by
Vekrellis et al., 2004). Reports focusing on brain dopaminergic
neurons, which express
a
-synuclein at high levels, have suggested
that the protein might negatively regulate the function of dopami-
nergic cells by several mechanisms.
a
-Synuclein was found to inhibit
activity or expression of enzymes involved in dopamine synthesis
(Perez et al., 2002; Baptista et al., 2003), affect function of the
dopamine transporter (Lee et al., 2001;Wersinger and Sidhu, 2003)
and inhibit dopamine release in response to repetitive stimulation
(Abeliovich et al., 2000; Yavich et al., 2004; Larsen et al., 2006). This
suggests participation of
a
-synuclein in the regulation of reward and
reinforcement mechanisms mediated by the mesocorticolimbic
dopaminergic system, a conclusion supported by the observation of
an altered rate of intracranial self-stimulation of medial forebrain
bundle in
a
-synuclein knockout mice (Oksman et al., 2006).
Several recent studies have also implicated
a
-synuclein in sub-
stance dependence and abuse, processes known to involve abnor-
malities in dopaminergic function. Elevated levels of the protein
were found in the brains of cocaine addicts (Mash et al., 2003; Qin
et al., 2005) and in rodents treated with psychostimulants (Brenz
Verca et al., 2003; Fornai et al., 2005). Moreover, we previously
reported a long-lasting accumulation of
a
-synuclein in the mouse
striatum and amygdala following withdrawal from chronic mor-
phine treatment (Ziolkowska et al., 2005). Taking into account the
inhibitory influence of
a
-synuclein on dopaminergic neurotrans-
mission, we proposed that the up-regulation of
a
-synuclein
*Corresponding author. Tel.: þ48 12 66 23 218; fax: þ48 12 637 45 00.
E-mail address: nfprzewl@cyf-kr.edu.pl (R. Przewlocki).
Contents lists available at ScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
Neuropharmacology 54 (2008) 1239–1246
Contents lists available at ScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
0028-3908/$ – see front matter Ó2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2008.04.001
produced both by psychostimulants and opiates may contribute to
reduction of dopaminergic system activity, which is characteristic
of withdrawal states from various drugs of abuse (Ziolkowska et al.,
2005).
Interestingly, a growing body of evidence suggests that
a
-
synuclein might also play a role in alcohol addiction. In rats, the
a
-synuclein gene is localized within the quantitative trait locus for
alcohol consumption, and its basal expression in the hippocampus
and striatum is higher in an inbred alcohol-preferring strain than in
a nonpreferring strain, apparently due to a single nucleotide dif-
ference in the gene sequence (Liang et al., 2003).
a
-Synuclein gene
polymorphisms, which correlate with alcohol dependence and
craving phenotypes, were also identified in human alcohol addicts
(Bonsch et al., 2005b; Foroud et al., 2007). On the other hand, the
level of
a
-synuclein protein and mRNA in the blood was higher in
alcoholic patients during early abstinence than in healthy control
subjects (Bonsch et al., 2004, 2005a). Moreover, the blood level of
a
-synuclein expression of those patients correlated with alcohol
craving (Bonsch et al., 2004, 2005a). However, these human studies
did not address the question if high blood levels of
a
-synuclein
expression in alcoholics were constitutive or, alternatively, reflec-
ted an increase in the expression resulting from chronic alcohol
consumption.
Thus, the available data indicate that individual variations in
a
-synuclein gene sequence may contribute to the genetically
determined susceptibility to alcoholism. They also suggest that al-
cohol drinking may regulate
a
-synuclein expression in the blood,
which is related to craving in alcohol-dependent subjects. Whereas
a direct causative relationship between peripheral
a
-synuclein
expression and craving seems doubtful, the effect of alcohol
drinking on
a
-synuclein levels in the brain has not yet been
assessed. Moreover, it is not known if any relationship exists be-
tween expression of
a
-synuclein in the brain and blood. To further
our understanding of the role of
a
-synuclein in alcohol addiction, it
would also be interesting to compare the putative effects of alcohol
on
a
-synuclein expression in the brain to those of opiates and
psychostimulants (Brenz Verca et al., 2003; Mash et al., 2003;
Fornai et al., 2005; Qin et al., 2005; Ziolkowska et al., 2005).
In order to address the above issues, we employed a mouse
model of chronic voluntary alcohol drinking, in which we assessed
the levels of
a
-synuclein mRNA and protein both in the blood and in
brain regions encompassing the dopaminergic pathway, involved
in the regulation of motivation and reward. In contrast to human
studies, the scope of which is limited for obvious reasons, the use of
this simple animal model permits a more thorough evaluation of
a
-synuclein expression changes produced by long-term alcohol
exposure. Such an approach seems to be an essential step towards
understanding the putative functional role of
a
-synuclein in alco-
holism and other addictive disorders.
2. Methods
2.1. Animals
The experiments were performed on male C57BL/6J mice (bred in the Medical
Research Center, Warsaw, Poland) weighing 25–30 g at the start of behavioral pro-
cedures. The mice were kept under standard conditions, on a 12/12 h light/dark
cycle, with free access to rodent chow (Labofeed H, Kcynia, Poland) and tap water.
Treatment of the mice in the present study was in full accordance with the ethical
standards laid down in the respective European (Directive no. 86/609/EEC) and
Polish regulations. The experimental protocol was approved by the ethics com-
mittee on animal studies.
2.2. Alcohol drinking in the two-bottle choice procedure
C57BL/6J mice present high two-bottle choice ethanol preference across a wide
range of ethanol concentrations and consume pharmacologically relevant amounts
of ethanol (He et al., 1997; Peirce et al., 1998). In our preliminary experiment
(Bienkowski et al., unpublished), C57BL/6J mice (n¼6) were given free choice
between 8% (v/v) ethanol and water. The animals were killed by decapitation 2 h
into the dark phase, and samples of trunk blood were collected. Blood ethanol levels
exceeded 50 mg% confirming that the mice consumed pharmacologically relevant
amounts of ethanol.
The two-bottle choice model of oral alcohol self-administration was also used in
the present study (Korkosz et al., 2004). The animals were housed singly in standard
Plexiglas boxes equipped with two graduated drinking tubes. They were allowed 7
days for acclimatization, during which both drinking tubes were filled with tap
water, and baseline water drinking was assessed. On the following days, one of the
tubes was filled with water and the other one with an ethanol solution. The alcohol
concentration was increased gradually from 2% (for 2 days) through 4% (4 days) to
reach 8%. Then the mice were exposed to the 8% ethanol solution for 32 days, and
had free choice between alcohol solution and water throughout the experiment; the
control group received water in both tubes. The ethanol solutions were replaced
daily and the drinking tubes were rotated every day to prevent position preference.
Fig. 1 shows the time course of alcohol solutions vs. water consumption during the
experiment. In mice exposed to 8% ethanol, the average (S.D.) daily consumption of
alcohol was 11.43 1.75 g/kg. The preference for 8% ethanol, calculated according to
the formula: [ethanol intake/total fluid intake] 100%, equalled 91.03 9.69%.
The bottles containing alcohol were withdrawn immediately after the end of the
dark period. During abstinence, the two bottles were filled with water. The mice
were randomly assigned to different experimental groups and sacrificed by de-
capitation 2 h, 24 h or 48 h after alcohol withdrawal. These withdrawal periods were
dictated by our previous observation that the changes in
a
-synuclein levels resulting
from chronic morphine exposure developed only af ter the drug withdrawal
(Ziolkowska et al., 2005). The experimental groups did not differ in terms of ethanol
consumption and preference (all pvalues >0.05). In the preliminary experiment, no
handling-induced convulsive activity, spontaneous signs of withdrawal hyperex-
citability (Chan et al., 1991) or other signs of alcohol withdrawal (e.g. body tremor,
piloerection) were observed after 2 h, 24h or 48 h of abstinence.
2.3. RNA isolation from blood and reverse transcription-real-time PCR
Blood was collected after decapitation into ethylenediaminetetraacetic acid
(EDTA)-containing tubes, frozen and stored at 70
C until RNA isolation. Total RNA
was extracted from the whole blood by the method of Chomczynski and Sacchi
(1987), with further modifications, using the TRIzol reagent (Invitrogen, Carlsbad,
CA, USA). RNA was then purified using the RNeasy Mini Kit columns (Qiagen, Hilden,
Germany) according to the manufacturer’s instructions. Quality of the total RNA was
assessed based on the intensity of 28S and 18S rRNA bands after denaturing agarose
gel electrophoresis followed by ethidium bromide staining, and by the spectro-
photometric ratio A260/A280 (1.9–2.1). RNA concentration was measured using the
fluorescent reagent RiboGreen (Molecular Probes, Eugene, Oregon, USA).
Reverse transcription was performed using Omniscript reverse transcriptase
(Qiagen) and oligo(dT
16
) primer at 37
C for 60 min. Quantitative real-time RT-PCR
reactions were performed, according to the manufacturer’s protocol, using the
a
-synuclein TaqMan Gene Expression Assay (Mm00447333_m1) from Applied
Biosystems (Foster, CA, USA). The reactions were run on the iCycler device (BioRad,
Hercules, CA, USA) with the 3.0a software version. Amplification efficiency for the
assay was determined by running a standard dilution curve. Expression of hypo-
xanthine guanine phosphoribosyl transferase 1 (Hprt1) transcript was quantified to
control for variation in cDNA amounts. The threshold cycle values were calculated
automatically by iCycler IQ 3.0a software with default parameters. Abundance of
RNA was calculated as 2
(threshold cycle)
.
2.4. In situ hybridization and image analysis
After sacrifice, the brains were removed from the skulls, frozen on dry ice and
stored at 70
C. Then they were cut into 12-
m
m thick coronal sections on a cryostat
0 2 4 68 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
0.00
0.05
0.10
0.15
0.20
water
alcohol
2% 8%
4%
da
y
daily intake [ml/g]
Fig. 1. Daily intake of alcohol solution and water by C57BL/6J mice during the course of
the experiment (mean S.E.M., n¼24). The intake is expressed in milliliters of liquid
per gram of body weight. Alcohol concentrations on consecutive days of the experi-
ment are indicated above the upper curve.
B. Ziolkowska et al. / Neuropharmacology 54 (2008) 1239–12461240
microtome (CM 3050 S, Leica Microsystems, Nussloch, Germany), the sections were
thaw-mounted on gelatin-chrom-alum-coated slides and processed for in situ hy-
bridization according to the method of Young et al. (1986). Briefly, the sections were
fixed with 4% paraformaldehyde, washed with PBS and acetylated by incubation
with 0.25% acetic anhydrite (in 0.1 M triethanolamine and 0.9% sodium chloride).
The sections were then dehydrated using increasing concentrations of ethanol (70–
100%), treated with chloroform for 5 min, and rehydrated with decreasing concen-
trations of ethanol.
The sections were hybridized for approximately 15 h at 37
C with an oligonu-
cleotide probe complementary to nucleotides 329–377 of the mouse
a
-synuclein
cDNA (Hong et al., 1998)(5
0
-TGTCTTCTGAGCGACTGCTGTCACACCAGTCACCACTG
CTCCTCCAACA-3
0
). The hybridization buffer contained 50% formamide, 4SSC (i.e.
0.6 M sodium chloride and 0.06 M sodium citrate), 1Denhardt’s solution, 0.25 mg/
ml yeast tRNA, 0.5 mg/ml salmon sperm DNA, 50 mg/ml dextran sulphate and
10 mM dithiothreitol. The probe was labeled with
35
S-dATP by the 3
0
-tailing reaction
using terminal transferase (Roche Diagnostics, Mannheim, Germany) and applied to
the slides in the concentration of approximately 16 000 dpm/
m
l.
After hybridization, the slices were washed three times for 20 min with 1SSC/
50% formamide at 40
C, and twice for 50 min with 1SSC at room temperature. The
slices were then dried and exposed to Fujifilm (Tokyo, Japan) phosphorimager im-
aging plates for 4 days. The hybridization signal was digitized using the Fujifilm BAS-
5000 phosphorimager and the Image Reader software.
The in situ hybridization signal was analyzed using the MCID Elite system
(Imaging Research, St. Catharines, Ontario, Canada). Mean signal density, expressed
in photostimulated luminescence units/mm
2
, was measured in selected brain re-
gions in the Fujifilm BAS-5000 images. The regions included: the substantia nigra
(pars compacta), ventral tegmental area (VTA), dorsal striatum, nucleus accumbens
core and shell, lateral and basolateral nuclei of amygdala (see Fig. 2 for the regions
definition in the autoradiograms). The two latter amygdaloid nuclei were chosen for
the analysis because of a particularly high
a
-synuclein expression; other amygdaloid
nuclei cannot be delineated in the autoradiograms because of a much lower and
homogenous
a
-synuclein mRNA signal, which does not exceed markedly that in the
surrounding tissue (Fig. 2). For each brain structure, data were collectedfrom at least
four sections per animal, bilaterally.Background signal was measured over the white
matter (corpus callosum) and was subtracted from the hybridization signal in the
regions of interest.
2.5. Brain dissection
After decapitation, brains were removed from the skulls and dissected rapidly.
We collected samples containing (1) the rostral part of caudate/putamen plus
nucleus accumbens (referred to as striatum/accumbens), (2) amygdala (the tissue
about 0.5 mm below rhinal fissure including the majority of the amygdalar nuclei
located between the piriform cortex on the one side, and the optic tract and
ventral part of lateral ventricles, substantia innominata and ventral border of the
caudate-putamen on the other), and (3) mesencephalon. Tissue samples were
frozen on dry ice immediately after dissection and stored at 70
C until protein
extraction.
2.6. Western blot
The dissected tissue samples were homogenized in hot 2% sodium dodecyl
sulphate (SDS), boiled for 8 min and cleared by centrifugation (20 800gfor 30 min).
Protein concentration in the supernatant was determined using the BCA Protein
Assay Kit (Sigma–Aldrich, St. Louis, MO, USA). Samples (containing 65
m
g of pro-
tein) were heated for 10 min at 95
C in loading buffer (50 mM Tris–HCl, 2% SDS, 2%
ß-mercaptoethanol, 8% glycerol, 0.1% bromophenol blue) and resolved by SDS-
PAGE on 12% polyacrylamide gels. The amount of protein per lane was optimized in
pilot studies, in which Western blots were performed using different amounts of
protein per sample (20–150
m
g) extracted from each experimental brain region. For
quantitative analysis of
a
-synuclein, the amount of total protein was chosen, for
which twofold differences in protein content were linearly reflected by the assay.
After the gel electrophoresis, proteins were electrophoretically transferred to
nitrocellulose membranes (Trans-Blot; BioRad). To control equal gel loading, gels
were stained with Coomassie Brilliant Blue R250 after protein transfer, and
intensity of staining was compared between lanes. The blots were blocked using 5%
albumin (Sigma–Aldrich) in Tris-buffered saline (TBS) for 1 h and incubated over-
night at 4
C with the sheep anti-
a
-synuclein polyclonal antibody (dilution 1:500;
Abcam, Cambridge, UK). The blots were then incubated with a peroxidase-
conjugated secondary antibody (donkey anti-Sheep IgG, Abcam) at a dilution of
1:3000 for 1 h at room temperature. After three 15 min washes in TBS and 0.1%
Tween-20, and one wash in TBS, immunocomplexes were detected using a buffered
solution of 250 mM luminol sodium salt and 30% hydrogen peroxide in 1 M
Tris–HCl (pH 8.5) containing 90 mM p-coumaric acid. The signal was visualized
applying the Fujifilm LAS-1000 fluorimager system. Relative levels of immunore-
activity were quantified using Fujifilm software (Image Gauge). As another control
for protein quantity in the samples, some of the membranes were re-probed with
a mouse anti-
b
-actin antibody (1:15 000 dilution, Sigma–Aldrich) after three
10-min washes in TBS and 0.1% Tween-20.
2.7. Statistical analysis
The results were statistically analyzed by one-way analysis of variance (ANOVA).
The Newman–Keuls post-test was used to locate significant differences between
experimental groups.
3. Results
3.1. Influence of alcohol drinking and withdrawal on
a
-synuclein
mRNA levels in the brain
a
-Synuclein mRNA expression in the brainwas studied by in situ
hybridization. The obtained signal distribution and relative ex-
pression levels in discrete brain regions (Fig. 2) were consistent
with those reported previously by us and other authors (Maroteaux
and Scheller,1991; Nakajo et al.,1994; Hong et al., 1998; Ziolkowska
et al., 2005).
a
-Synuclein mRNA signal densities were quantified in
the dorsal striatum, nucleus accumbens core and shell, basolateral
amygdala, substantia nigra and ventral tegmental area. No signifi-
cant changes in
a
-synuclein mRNA levels were found in any brain
region examined at any time point tested (2 h, 24 h or 48 h after
alcohol withdrawal) (Fig. 2).
3.2. Influence of alcohol drinking and withdrawal on
a
-synuclein
protein levels in the brain
a
-Synuclein protein levels were measured by Western blotting
in homogenates of the striatum/accumbens, amygdala and mes-
encephalon of mice withdrawn for 2 h, 24 h or 48 h from chronic
alcohol drinking. No changes were observed in the striatum/
accumbens and mesencephalon at any time point tested (Fig. 3). In
the amygdala, a significant increase by about 80% was found after
24-h abstinence, and the
a
-synuclein levels remained elevated after
48-h abstinence (Fig. 3).
3.3. Influence of alcohol drinking and withdrawal on
a
-synuclein
mRNA levels in the blood
The level of
a
-synuclein mRNA in the whole blood was mea-
sured by quantitative RT-real-time PCR. An increase in
a
-synuclein
expression by about 40% was observed in mice withdrawn for 48 h
from chronic alcohol exposure (Fig. 4). No changes were detected
after 24 h of withdrawal (Fig. 4). Expression of the control gene
Hprt1 did not differ significantly between the experimental groups
(data not shown).
4. Discussion
Previous studies in selectively bred alcohol-preferring rats and
in alcohol-dependent humans have implicated the
a
-synuclein
gene in genetically determined susceptibility to alcoholism. Asso-
ciations of several polymorphic variants of the
a
-synuclein gene
(SNPs or the polymorphic repeat NACP-REP1) with alcohol de-
pendence, craving or preference were found in humans and rats
(Liang et al., 2003; Bonsch et al., 2004, 2005b; Foroud et al., 2007).
Moreover, the sequence variants associated with alcohol-drinking-
prone phenotypes were shown to confer higher
a
-synuclein
expression in the brain or blood (Liang et al., 2003; Bonsch et al.,
2005b).
Whereas potential mechanisms of
a
-synuclein involvement in
alcohol dependence have so far mainly been linked to the influence
of
a
-synuclein on the brain dopaminergic system and other limbic
regions, expression data in primates are limited to measurements
of peripheral blood
a
-synuclein protein or mRNA levels (Bonsch
et al., 2004, 2005b; Walker and Grant, 2006). Interpretation of
these results is confounded by a lack of knowledge of the
B. Ziolkowska et al. / Neuropharmacology 54 (2008) 1239–1246 1241
relationship between
a
-synuclein expression in the brain and in the
blood. Moreover, the only study in which
a
-synuclein expression
was assessed in the brain with reference to alcohol use described
only basal expression of the gene in inbred alcohol-preferring and
-nonpreferring rat strains, without considering effects of exposure
to alcohol (Liang et al., 2003). On the other hand, human studies in
which elevated blood levels of
a
-synuclein protein and mRNA were
demonstrated in alcoholics did not make a distinction between
basal
a
-synuclein expression (possibly related to the
a
-synuclein
gene polymorphisms) and its possible up-regulation resulting from
0
20
40
60
80
100
120
contr alc
24h
alc
48h
alc
2h
α
-synuclein mRNA level
[% of control]
nucleus accumbens shell nucleus accumbens core
dorsal striatum
lateral and basolateral
amygdaloid n.
ventral tegmental area substantia nigra
dStr
AccC AccSh
LA
BLA SNc
VTA
contr alc
24h
alc
48h
alc
2h
contr alc
24h
alc
48h
alc
2h
contr alc
24h
alc
48h
alc
2h
contr alc
24h
alc
48h
alc
2h
contr alc
24h
alc
48h
alc
2h
0
20
40
60
80
100
120
α
-synuclein mRNA level
[% of control]
0
20
40
60
80
100
120
α
-synuclein mRNA level
[% of control]
0
20
40
60
80
100
120
α
-synuclein mRNA level
[% of control]
0
20
40
60
80
100
120
α
-synuclein mRNA level
[% of control]
0
20
40
60
80
100
120
α
-synuclein mRNA level
[% of control]
Fig. 2. The influence of alcohol drinking and withdrawal on
a
-synuclein mRNA in situ hybridization signal densities in subregions of the mouse striatum/accumbens, amygdala and
ventral mesencephalon. The mice drank 8% (v/v) alcohol for 32 days in the two-bottle choice procedure and were withdrawn from alcohol for the indicated periods of time (alc 2 h,
alc 24 h, alc 48 h). The control group (contr) had no access to alcohol at any time. The results are presented as the mean S.E.M. (expressed as % of control) of five to eight animals
per group. One-way ANOVA did not reveal statistically significant differences between the groups. Representative autoradiograms of brain sections hybridized with the
a
-synuclein
probe are shown at the bottom. The outlines show the regions of interest analyzed in the study. dStr – dorsal striatum; AccC – nucleus accumbens core; AccSh – nucleus accumbens
shell; LA – lateral amygdaloid nucleus; BLA – basolateral amygdaloid nucleus; VTA – ventral tegmental area; SNc – substantia nigra pars compacta.
B. Ziolkowska et al. / Neuropharmacology 54 (2008) 1239–12461242
long-term alcohol drinking (Bonsch et al., 2004, 2005a). Altogether,
despite the fairly convincing evidence of
a
-synuclein involvement
in some aspects of alcohol abuse, the actual role of
a
-synuclein
remains obscure, whereas the existing data are difficult to compare
and integrate due to heterogeneity of research approaches.
In the present study, we employed a simple rodent model of
chronic alcohol drinking to assess, in a more systematic way, the
influence of long-term voluntary alcohol intake on
a
-synuclein
expression both in the brain and the blood. We used the C57BL/6J
mice, i.e., the inbred strain showing high preference for alcohol (He
et al., 1997; Peirce et al., 1998). We have demonstrated that with-
drawal from chronic alcohol drinking in the two-bottle choice
procedure results in an increase in
a
-synuclein protein levels in the
amygdala (but not in the striatum/accumbens or mesencephalon).
No corresponding elevation of
a
-synuclein mRNA levels was found
in the amygdala or any other brain region tested (including the
dorsal striatum, nucleus accumbens and dopaminergic cell body
fields in the mesencephalon), which suggests that the increase in
a
-synuclein protein levels was not underlain by transcriptional
regulation of its gene. Since
a
-synuclein is a presynaptic protein, its
up-regulation in the amygdala may have resulted from protein
0
100
200
contr alc
24h
alc
48h
alc
2h
contr alc
24h
alc
48h
alc
2h
contr alc
24h
alc
48h
alc
2h
α
α
-synuclein protein level
[% of control]
α
-synuclein protein level
[% of control]
α
-synuclein protein level
[% of control]
0
100
200 **
0
100
200
striatum / accumbens
amygdala
mesencephalon
contr alc 2h
contr alc 24h alc 48h
contr alc 2h
contr alc 24h alc 48h
contr alc 2h
contr alc 24h alc 48h
Fig. 3. The influence of alcohol drinking and withdrawal on
a
-synuclein protein levels in the mouse striatum/accumbens, amygdala and mesencephalon, as measured by Western
blot analysis. The mice drank 8% (v/v) alcohol for 32 days in the two-bottle choice procedure and were withdrawn from alcohol for the indicated periods of time (2 h, 24 h, 48 h). The
control group (contr) had no access to alcohol at any time. The results are presented as the mean S.E.M. (expressed as % of control) of four to six animals per group and are
representative of several blots for each brain region. *Statistically significant (p<0.05) vs. the control group, one-way ANOVA followed by Newman–Keuls test. Representative
Western blot
a
-synuclein bands are shown on the right panels.
0
50
100
150 *
contr alc
24h
alc
48h
α
-synuclein mRNA level
[% of control]
Fig. 4. The influence of alcohol drinking and withdrawal on the levels of
a
-synuclein
mRNA in the mouse whole blood. The mice drank 8% (v/v) alcohol for 32 days in the
two-bottle choice procedure and were withdrawn from alcohol for the indicated
periods of time (alc 24 h, alc 48 h). The control group (contr) had no access to alcohol
at any time. The results are presented as the mean S.E.M. (expressed as % of control)
of seven to nine animals per group. *Statistically significant (p<0.05) vs. the control
group, one-way ANOVA followed by Newman–Keuls test.
B. Ziolkowska et al. / Neuropharmacology 54 (2008) 1239–1246 1243
accumulation in afferent axon terminals due to alterations of the
protein transport or its degradation rate rather than regulation of
the gene expression. On the other hand, a moderate increase in
a
-synuclein mRNA levels was found in the blood of mice with-
drawn from chronic alcohol for 48 h, but not 24 h.
The latter result is consistent with the observation of elevated
levels of blood
a
-synuclein mRNA (and protein) in alcohol-
dependent humans during early abstinence (Bonsch et al., 2004,
2005a). In contrast to our data, which suggest that the increase in
blood
a
-synuclein expression in mice develops only after some
time of alcohol abstinence, Walker and Grant (2006) reported
a pronounced elevation of blood
a
-synuclein mRNA levels in
monkeys immediately after cessation of a 14-month period of
access to 4% alcohol. This discrepancy between the mouse and
primate models may have resulted from the difference in the time
period of alcohol consumption (1 month vs. 14 months, re-
spectively), although both models may be considered as chronic
when the lifespan of the animal species involved is taken into ac-
count. Whereas further studies would be required to elucidate the
discrepancy in time-courses of the
a
-synuclein expression changes,
data from both the mouse and monkey models clearly indicate that
high blood levels of
a
-synuclein expression may result from pre-
vious long-term alcohol consumption rather than, or in addition to,
its high basal expression in alcoholism-prone individuals. It can be
concluded that the elevation of blood
a
-synuclein mRNA levels is
common to human alcoholism as well as primate and rodent
models of alcohol abuse. Thus, our results further support the
previous suggestion by Walker and Grant (2006) that
a
-synuclein
may serve as a peripheral biomarker of chronic alcohol consump-
tion both in humans and laboratory animals.
On the other hand, on the basis of the present results,
a
-
synuclein expression in blood seems unrelated to its expression in
the brain. The increase in blood
a
-synuclein mRNA level was not
mirrored by any such change in its concentration in several brain
regions under investigation. Although we did detect an elevation of
a
-synuclein protein level in the amygdala, this was not accompa-
nied by a rise in mRNA level. Because of the
a
-synuclein protein
transport into axon terminals, which might be distant from the
mRNA-containing cell bodies, we cannot completely exclude that
the increase in
a
-synuclein protein resulted from up-regulation of
its mRNA in some brain region that we overlooked. Nevertheless,
there was no concurrence between the changes in the blood and
the brain: blood
a
-synuclein mRNA levels peaked 48 h after alcohol
withdrawal, whereas marked elevation of
a
-synuclein protein
levels in the amygdala was already seen 24 h earlier. Therefore,
a
-synuclein expression seems to be regulated independently in the
blood and the brain. This suggests that peripheral changes in
a
-synuclein expression cannot be interpreted in terms of
a
-
synuclein function within the brain.
To the best of our knowledge, this is the first report demon-
strating that chronic alcohol drinking affects
a
-synuclein levels in
the brain. However, the changes in
a
-synuclein mRNA and/or
protein levels in discrete brain areas were previously reported
after treatment with other drugs of abuse. Studies in cocaine
addicts demonstrated up-regulation of
a
-synuclein mRNA and
protein in dopaminergic neurons of the substantia nigra and VTA,
and elevated
a
-synuclein protein levels in the striatum (Mash
et al., 2003; Qin et al., 2005). In animal models, increased
a
-
synuclein protein levels in the substantia nigra and up-regulation
of
a
-synuclein gene expression in the tegmentum, striatum and
hippocampus were demonstrated after administration of am-
phetamines (Brenz Verca et al., 2003; Fornai et al., 2005). These
results suggest that psychostimulants elevate
a
-synuclein levels
by stimulating its transcription, and these effects seem to be
particularly pronounced in dopaminergic neurons. This is in
contrast to the effects of chronic alcohol exposure reported
above, which did not affect
a
-synuclein mRNA levels in the do-
paminergic cells or elsewhere in the brain.
On the other hand, the effects of withdrawal from chronic
alcohol show striking similarity to the regulation of
a
-synuclein
during opiate abstinence. We previously demonstrated that the
a
-synuclein protein level was increased in the mouse amygdala at
48 h after cessation of chronic morphine treatment and remained
elevated for at least 2 weeks (Ziolkowska et al., 2005). As in the case
of alcohol withdrawal, no up-regulation of
a
-synuclein mRNA was
found in the brain of mice withdrawn from chronic morphine.
Moreover, both in alcohol and morphine abstinence, the accumu-
lation of
a
-synuclein protein in the amygdala occurred only after
some time of withdrawal (24–48 h), whereas it was not detected at
short periods (2 h or 4 h) after cessation of the treatment. This
indicates that the increase in
a
-synuclein levels may be a charac-
teristic feature of alcohol and morphine abstinence.
The similarity between the effects of alcohol and morphine,
drugs that act by widely different pharmacological mechanisms,
suggests that accumulation of
a
-synuclein in the amygdala may
contribute to psychological and/or autonomic changes associated
with drug abstinence. Actually, negative affective states, including
dysphoria, anxiety, anhedonia and craving, are common symptoms
of withdrawal from various addictive substances (Markou et al.,
1998), and amygdala has been strongly implicated in generation of
these symptoms (Pandey, 2003;Aston-Jones and Harris, 2004).
They are ascribed mainly to activation of the central amygdaloid
nucleus (CeA), which is observed during withdrawal from chronic
alcohol and opiates, as well as nicotine and cannabinoids (Couceyro
and Douglass, 1995; Rodriguez de Fonseca et al., 1997; Knapp et al.,
1998; Panagis et al., 2000; Gracy et al., 2001; Frenois et al., 2002).
Alcohol withdrawal also activates the basolateral amygdaloid
nucleus (BLA) (Knapp et al., 1998; Borlikova et al., 2006), the
nucleus otherwise strongly implicated in conditioned-stimulus
(cue)-induced craving for alcohol and drugs in animal models and
human addicts (Childress et al., 1999; Ciccocioppo et al., 2001;
Schneider et al., 2001; Bonson et al., 2002; Shalev et al., 2002).
Being a presynaptic protein,
a
-synuclein could participate in the
regulation of CeA and/or BLA activity by influencing neurotrans-
mitter release from their afferent dopaminergic or glutamatergic
fibers. However, understanding the mechanism of
a
-synuclein
action within the amygdaloid complex is precluded at the moment
by the lack of knowledge of the protein distribution in distinct
axonal populations innervating the amygdala. Whereas we were
previously able to demonstrate an abundant innervation of the BLA
output neurons by
a
-synuclein-containing nerve terminals
(Ziolkowska et al., 2005), the origin of these axons remains un-
known. On the other hand, the
a
-synuclein-containing input to the
CeA can only be inferred from the fact that this nucleus receives
afferents from two neuronal populations both expressing
a
-
synuclein mRNA at high levels: dopaminergic neurons of the VTA
and glutamatergic cells of the BLA (Swanson, 1982; McDonald,
1991; Freedman and Cassell, 1994; Zhu and Pan, 2004). Thus, fur-
ther immunolabelling studies are warranted to define the nature of
a
-synuclein-containing input to distinct amygdaloid nuclei.
Nevertheless, the accumulation of
a
-synuclein in the amygdala
occurring selectively during alcohol and opioid abstinence puts
these changes in a position to regulate the emotional states typical
of alcohol and drug abstinence such as negative affect, anxiety and
craving, the generation of which involves the amygdala.
The putative role of
a
-synuclein in drug use disorders has so far
been considered mainly in the context of its propensity to nega-
tively affect dopaminergic neurotransmission in the mesostriatal
pathway (Abeliovich et al., 2000; Perez et al., 2002; Baptista et al.,
2003; Yavich et al., 2004). Actually, our previous study demon-
strated a prolonged elevation of
a
-synuclein protein level in the
striatum/accumbens during withdrawal from chronic morphine
B. Ziolkowska et al. / Neuropharmacology 54 (2008) 1239–12461244
(Ziolkowska et al., 2005). However, in the present experiment, no
such changes were observed in the striatum/accumbens of mice
withdrawn from chronic alcohol. Thus,
a
-synuclein does not seem
to contribute to the decline in the activity of the mesostriatal
projection associated with alcohol abstinence (Acquas et al., 1991;
Diana et al., 1993), which does not exclude its involvement in
striatal neuroadaptations to chronic use of opiates and psychosti-
mulants (Mash et al., 2003; Qin et al., 2005; Ziolkowska et al.,
2005).
In conclusion, we have demonstrated that
a
-synuclein mRNA
level in the blood is significantly elevated during alcohol abstinence
in the mouse, like in humans and monkeys, but seems unrelated to
a
-synuclein expression in the brain. The main effect of prolonged
alcohol drinking on brain
a
-synuclein consisted of accumulation of
a
-synuclein protein in the amygdala after ethanol withdrawal.
Since this phenomenon is common to alcohol and opiate absti-
nence, our observations suggest a role of amygdalar
a
-synuclein in
common neuroadaptations produced by long-term drug and alco-
hol use.
Acknowledgements
This work was supported by the Polish Ministry of Science and
Higher Education Scientific Network fund, grant no. 26/E-40/6.PR
UE/DIE 305/2005–2008, and the EU grant LSHM-CT-2004-005166.
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