Alcohol 48 (2014) 471e476

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Alcohol
journal homepage: http://www.alcoholjournal.org/

Differential sensitivity of ethanol-elicited ERK phosphorylation in

nucleus accumbens of Sardinian alcohol-preferring and -non
preferring rats
Michela Rosas a,1, Alessandro Zaru b,1, Marta Sabariego c, 2, Valentina Giugliano d, g, Ezio Carboni b, d, e, f,
Giancarlo Colombo b, Elio Acquas a, e, f, *
a
Pharmaceutical, Pharmacological and Nutraceutical Sciences Section, Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy
b
Neuroscience Institute, Section of Cagliari, National Research Council of Italy, Monserrato, Cagliari, Italy
c
Department of Psychology, University of Jaèn, Campus Las Lagunillas s/n Building C-5, 23071 Jaèn, Spain
d
Department of Biomedical Sciences, Division of Neuropsychopharmacology, University of Cagliari, Cagliari, Italy
e
Centre of Excellence on Neurobiology of Addiction, University of Cagliari, Cagliari, Italy
f
National Institute of Neuroscience e INN, University of Cagliari, Cagliari, Italy
g
Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Sardinian alcohol-preferring (sP) and -non preferring (sNP) rats have been selectively bred for opposite
Received 24 October 2013 ethanol preference and consumption; sP rats represent a validated experimental tool to model several
Received in revised form aspects of excessive ethanol drinking in humans. Phosphorylated Extracellular signal-Regulated Kinase
30 April 2014
(pERK) in dopamine-rich terminal areas plays a critical role in several psychopharmacological effects of
Accepted 30 April 2014
addictive drugs, including ethanol. This study was aimed at investigating whether ethanol-elicited ERK
activation may differ in key brain areas of ethanol-naïve sP and sNP rats. To this end, the effects of
ethanol (0, 0.5, 1, and 2 g/kg, administered intra-gastrically [i.g.]) on ERK phosphorylation were
Keywords:
Ethanol
assessed by pERK immunohistochemistry in the shell (AcbSh) and core (AcbC) of the nucleus accum-
Phosphorylated extracellular signal bens (Acb) as well as in the prelimbic (PrL) and infralimbic (IL) prefrontal cortex (PFCx), in the bed
regulated kinase (pERK) nucleus of stria terminalis (BSTL) and in the central nucleus of the amygdala (CeA). Ethanol (1 g/kg)
Nucleus accumbens (Acb) significantly increased pERK immunoreactivity in AcbSh and AcbC of sP but not sNP rats. Conversely,
Sardinian alcohol-preferring (sP) and -non ethanol failed to affect pERK expression in PrL and IL PFCx as well as in BSTL and CeA of both sP and sNP
preferring rats (sNP) rats. These results suggest that selective breeding of these rat lines results in differential effects of acute
ethanol on ERK phosphorylation in brain regions critical for the psychopharmacological effects of
ethanol.
Ó 2014 Elsevier Inc. All rights reserved.

Introduction Association, 1994; Feuerlein, 1977). Alcoholism can also be viewed

Ethanol consumption is the third leading risk factor for disease
and mortality after tobacco and high blood pressure in Western
countries (World Health Organization, 2009). Such ethanol misuse
is related to premature death and is a major avoidable risk factor for
cardiovascular diseases, liver cirrhosis, cancer (Anderson, Møller, &
Galea, 2012), and neuropsychiatric disorders (American Psychiatric
* Corresponding author. University of Cagliari, Department of Life and Environ-
mental Sciences, Via Ospedale, 72, I-09124, Cagliari, Italy. Tel./fax: þ39 070
675 8623.
E-mail address: acquas@unica.it (E. Acquas).
1
These authors contributed equally to this work.
2
Present address: Department of Psychiatry, University of California, San Diego,
La Jolla, USA.
0741-8329/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.alcohol.2014.04.002
as a complex neuroadaptive disorder that cannot be modeled fully
in animals. However, some features of alcoholism, such as initiation
and maintenance of high ethanol intake, ethanol seeking during
abstinence, and relapse-like drinking can be effectively reproduced
in laboratory animals (Bell et al., 2012; Colombo, Lobina, Carai, &
Gessa, 2006; Martin-Fardon & Weiss, 2013; Sanchis-Segura & Spa-
nagel, 2006). Interestingly, genetic factors are responsible for about
50e60% of the risk of developing alcoholism, despite the fact that a
prolonged ethanol exposure is an essential precondition to develop
the disease (Ciccocioppo, 2013). Accordingly, one of the most effi-
cacious approaches to model several aspects of alcoholism is rep-
resented by the use of rat lines selectively bred for high ethanol
preference or excessive ethanol drinking (Bell et al., 2012; Colombo,
Lobina, et al., 2006); these rat lines represent well-suited
472 M. Rosas et al. / Alcohol 48 (2014) 471e476
experimental models to investigate the biological bases of alco-
holism by virtue of their predictive, face, and construct validity (Bell
et al., 2012).
Sardinian alcohol-preferring (sP) rats constitute one of these rat
lines selectively bred for excessive ethanol intake. When given a
choice between 10% (v/v) ethanol and water, under the standard,
home cage 2-bottle regimen, with unlimited access for 24 h/day, sP
rats display a clear preference for the ethanol solution and daily
consume 6e7 g/kg pure ethanol. Voluntary ethanol intake in sP rats
gives rise to relatively high blood ethanol levels (BELs) and pro-
duces measurable psychopharmacological effects, including
amelioration of genetically based anxiety-related behaviors and
locomotor stimulation (Colombo, Lobina, et al., 2006). Accordingly,
sP rats meet all the fundamental requirements posed when
defining an animal model of alcoholism, namely: a) oral ingestion of
ethanol, b) attainment of psychopharmacologically relevant BELs, c)
willingness to “work” for ethanol (e.g., lever-respond to access to
ethanol), d) development of tolerance to a given effect of ethanol
induced by its voluntary consumption, and e) development of
physical or “behavioral” dependence upon ethanol voluntary con-
sumption (Colombo, Lobina, et al., 2006; Loi et al., 2010).
Conversely, selectively bred Sardinian alcohol-non preferring (sNP)
rats avoid ethanol virtually completely (Colombo, Lobina, et al.,
2006), even when exposed for relatively long periods of time to
mixtures containing ethanol and palatable tastants (Brunetti et al.,
2003; Orrù et al., 2007).
The psychopharmacological effects of ethanol involve an
extended range of brain structures and processes whose interac-
tion with personality traits may, ultimately, result in behavioral
abnormalities and alcoholism development (Koob et al., 1998). In
particular, the brain structures critically involved in the reinforc-
ing, motivational, and rewarding properties of ethanol are part of
the mesocorticolimbic system (Vilpoux, Warnault, Pierrefiche,
Daoust, & Naassila, 2009) and extended amygdala (Alheid et al.,
1998). These include the ventral tegmental area (VTA) (Gessa,
Muntoni, Collu, Vargiu, & Mereu, 1985) and the nucleus
accumbens (Acb) (Di Chiara & Imperato, 1988), responsible for
drug-mediated reward processes (Di Chiara et al., 2004), and the
prefrontal cortex (PFCx) (Kauer & Malenka, 2007), implicated in
attentional processes, working memory, behavioral flexibility
(George & Koob, 2010; Goldstein & Volkow, 2011), and ethanol-
seeking behavior related to environmental cues (Wedzony et al.,
2003).
Extracellular signal-Regulated Kinase (ERK) is a ubiquitously
expressed kinase that participates in the Ras-Raf-MEK-ERK signal
transduction cascade. ERK activation takes place by dual phos-
phorylation on threonine and tyrosine residues and acts in
sequence following extracellular signals and/or increased intracel-
lular Ca2þ concentrations (Sweatt, 2004). Phosphorylated ERK
(pERK) is involved in short- and long-term consequences of expo-
sure to addictive drugs (Girault, Valjent, Caboche, & Hervé, 2007;
Valjent, Pagès, Hervé, Girault, & Caboche, 2004), including
ethanol, that activates ERK in Acb of SpragueeDawley rats (Ibba
et al., 2009).
The present study was aimed at investigating whether acute
administration of ethanol produces differential effects in Acb, PFCx,
BSTL, and CeA of ethanol-naïve sP and sNP rats. We hypothesized
that the assessment of ethanol-elicited ERK phosphorylation in
these brain regions might contribute to characterize the molecular
bases of their differential sensitivity to acute ethanol (Agabio et al.,
2001; Colombo et al., 2000). In addition, elucidation of the
involvement of ERK cascade in the acute actions of ethanol in sP and
sNP rats might contribute to posit the grounds for future in-
vestigations on the role of this kinase in excessive ethanol drinking
in sP and/or ethanol avoidance in sNP rats.
Materials and methods
This study was approved by the ethical committee of the Univer-
sity of Cagliari and was carried out in accordance with Italian law, D.L.
116, 1992, which allows experiments on laboratory animals provided
that competent authorities have approved the submitted research
project, and to the European Commission Recommendation n. 2007/
526/CE. All possible efforts were made to minimize animal pain and
discomfort and to limit the number of experimental subjects.
Animals
Male sP (n ¼ 24) and sNP (n ¼ 24) rats, from the 75th and 80th
generation, respectively, 70 days old and weighing 300e325 g,
were used. Rats were housed 4 per cage in standard plastic cages
with wood chip bedding. The animal facility was under an inverted
12 h:12 h light/dark cycle (lights on at 7:00 PM), at a constant
temperature of 22  2  C and relative humidity of approximately
60%. In order to avoid any possible interference of stress due to
gavage administration procedure, rats had been extensively
handled (once a day for at least 5 days before the experiment) to
habituate them to intragastric and intraperitoneal administrations.
Standard rat chow (Mucedola, Settimo Milanese, Italy) and tap
water were always available, except as indicated below.
Experimental procedure
Rats of each line were divided into 4 groups of n ¼ 6 each and
treated acutely with 0, 0.5, 1, and 2 g/kg ethanol. Ethanol was
diluted in tap water (20% v/v) and administered i.g. (by a reusable
stainless-steel feeding needle); ethanol solutions were obtained by
dilution of ethanol (96%) (Medicamenta, 1991e1992). Five hours
before ethanol administration, rats were food-deprived to ensure
they had empty stomachs at the time of ethanol infusion.
Fifteen minutes after ethanol administration, rats were deeply
anesthetized with ketamine HCl (100 mg/kg, intra-peritoneally;
Ketavet 50, Gellini International, Aprilia, Italy) before being trans-
cardially perfused with ice-cold PBS (phosphate-buffered saline:
137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4)
and 4% paraformaldehyde (PFA). The time interval between ethanol
administration and anesthesia for sacrifice was selected in accor-
dance with Ibba et al. (2009) and Neasta, Ben Hamida, Yowell,
Carnicella, and Ron (2011). After being perfused, the brains were
removed and post-fixed in 4% PFA (4  C) overnight. Brain sections
(40 mm) of the regions of interest were cut, in accordance with the
Paxinos and Watson (1998) rat brain atlas, in ice-cold PBS with a
vibratome (Leica VT1000, Leica, Germany), kept in ice-cold PBS or
stored in a cryoprotective solution [40% PBS, 30% ethylene glycol
(Carlo Erba, Milan, Italy), 30% glycerol (Carlo Erba, Milan, Italy)] for
immediate or future immunohistochemistry processing, respec-
tively. After being incubated 30 min in 1% H2O2, sections were
incubated again for 1 h with 3% bovine serum albumin. The incu-
bation with the primary antibody [anti-pERK (1:300), Cell Signaling
Technology, Beverly, MA, USA] was conducted overnight at 4  C.
On the following day, after rinsing, the sections were incubated
for 1 h with the biotinylated secondary antibody (1:800; Sigma-
eAldrich, Milan, Italy) at 4  C. After rinsing again, the sections were
incubated in an avidin-biotin peroxidase complex which was pre-
pared following the manufacturer’s suggestions (Vectastain ABC
kit; Vector Laboratories, Burlingame, CA, USA) and a 3,30 -dia-
minobenzidine solution (10 mg/mL) was added until brown stain
developed. Sections were rinsed and mounted onto gelatin-coated
slides and processed through alcohol-xylene for light microscopy
examination. pERK-positive neurons were identified at the lowest
magnification (10) and quantitative analysis was performed using
 M. Rosas et al. / Alcohol 48 (2014) 471e476 473

a light microscope (Zeiss Axioskop 40, Carl Zeiss Spa, Varese, Italy) 300
equipped with PL Floutar 10 (na ¼ 0.3), and coupled with a digital sP AcbSh

% pERK-positive neurons/area
sNP
camera (PixeLink, PL-A686C, 6.6 MPixels). Image analysis and
quantification was conducted by 2 experimenters blind to phar-
macological treatments in at least 2 slices for all evaluated brain
200 *
regions of each experimental subject of sP and sNP rats. This **
analytical approach was validated by its application to negative
controls. Data  SEM of pERK-positive neurons/area following each
treatment were expressed as the average of % changes with respect
to the average number of pERK-positive neurons/area of the saline 100
groups set as 100% and were used for statistical purposes. pERK
immuno-positive cells in the regions of interest were automatically
counted by application of the software ImageJ in conjunction with
automated background subtraction, to avoid experimenter bias,
0
and with the application of the algorithm entropy threshold (Kapur, 0 0.5 1 2
Sahoo, & Wong, 1985). 300
AcbC
*

% pERK-positive neurons/area
Statistics **
Immunohistochemical data were analyzed by 2-way analysis 200
of variance (ANOVA) with % average numbers of pERK-positive
neurons/area as the dependent variable and with rat line
(n ¼ 2: sP and sNP) and ethanol doses (n ¼ 4: 0, 0.5, 1, and 2 g/kg)
as the independent variables. When allowed by significant main
effects and interactions, LSD post hoc analyses were applied for 100
multiple comparisons, with the statistical significance set at
p < 0.05.

Results
Effects of ethanol on pERK immunoreactivity in AcbSh and AcbC of
sP and sNP rats
Fig. 1 shows the effects of ethanol administration to sP and sNP
rats on % pERK-positive neurons/area in AcbSh (top panel) and
AcbC (bottom panel). Two-way ANOVA of the data collected in
AcbSh revealed significant main effects of dose [F(3,40) ¼ 2.92,
p ¼ 0.045] and line [F(1,40) ¼ 4.25, p ¼ 0.045] but not a significant
dose by line interaction [F(3,40) ¼ 0.88, NS]. LSD post hoc test
revealed that administration of 1 g/kg ethanol increased % pERK-
positive neurons with respect to water (0 g/kg ethanol) in AcbSh
of sP rats; conversely, post hoc analysis revealed that ethanol
administration did not affect % pERK-positive neurons/area in sNP
rats.
Two-way ANOVA of the data collected in AcbC revealed signif-
icant main effects of dose [F(3,40) ¼ 3.21, p ¼ 0.03] and line
[F(1,40) ¼ 7.17, p ¼ 0.01], and a significant dose by line interaction
[F(3,40) ¼ 3.43, p ¼ 0.026]. LSD post hoc test revealed that
administration of 1 g/kg ethanol increased % pERK-positive neu-
rons with respect to water in AcbC of sP rats; in contrast, ethanol
administration failed to affect % pERK-positive neurons/area in sNP
rats.
Effects of ethanol on pERK immunoreactivity in PrL and IL PFCx of sP
and sNP rats
0
0 0.5 1 2
Ethanol (g/kg)
Fig. 1. Top panel: Effect of administration of ethanol (0, 0.5, 1, and 2 g/kg i.g.) (n ¼ 6/
experimental group) on % pERK-positive neurons/area in AcbSh of sP and sNP rats.
Bottom panel: Effect of administration of ethanol (0, 0.5, 1, and 2 g/kg i.g.) (n ¼ 6/
experimental group) on pERK-positive neurons/area in AcbC of sP and sNP rats. Histo-
grams represent the % average  SEM of counts from at least 2 slices/rat from AP þ1.7
to AP þ1.2 mm from bregma, according to Paxinos and Watson (1998) rat brain atlas. *
indicates p < 0.05 with respect to ethanol 0 g/kg same area and same rat line; ** in-
dicates p < 0.05 with respect to ethanol 1 g/kg of sNP rat line (LSD post hoc test).
[F(1,40) ¼ 3.33, NS], as well as a significant dose by line interaction
[F(3,40) ¼ 0.574, NS].
Effects of ethanol on pERK immunoreactivity in BSTL and CeA of sP
and sNP rats
Table 1 shows the effects of ethanol administration to sP and sNP
rats on % pERK-positive neurons/area in BSTL and CeA. Two-way
ANOVA of data collected in BSTL revealed a significant effect of
line [F(1,40) ¼ 6.28, p < 0.016] but not of dose [F(3,40) ¼ 0.54, NS],
nor a significant dose by line interaction [F(3,40) ¼ 1.28, NS].
Similarly, 2-way ANOVA of data collected in CeA failed to reveal
significant effects of line [F(1,40) ¼ 0.03, NS], dose [F(3,40) ¼ 1.94,
NS], and a significant dose by line interaction [F(3,40) ¼ 0.85, NS].
Discussion

Fig. 2 shows the effects of ethanol administration to sP and sNP
rats on % pERK-positive neurons/area in PrL (top panel) and IL
(bottom panel) PFCx. Two-way ANOVA of the data collected in PrL
PFCx revealed a significant main effect of line [F(1,40) ¼ 4.17,
p ¼ 0.047] but not of dose [F(3,40) ¼ 1.095, NS], nor a significant
dose by line interaction [F(3,40) ¼ 0.85, NS].
In addition, 2-way ANOVA of the data collected in IL PFCx failed
to reveal significant effects of dose [F(3,40) ¼ 0.49, NS] and line
In a previous study (Ibba et al., 2009) conducted using Spra-
gueeDawley rats, we found that ethanol, similarly to other addic-
tive substances (Acquas et al., 2007; Valjent et al., 2004), elicits ERK
phosphorylation in Acb and other nuclei of the extended amygdala,
an anatomical continuum deeply involved in distinct stages of the
addiction cycle (Koob & Volkow, 2010). The present research was
aimed at testing the hypothesis that acute ethanol administration
may differentially affect pERK expression in rats belonging to 2 lines
474
200
sP
% p E R K -p o s i ti v e n e u r o n s / a r e a
sNP
100
0
0 0.5
200
% pERK-positive neurons/area
100
0 cedures, sP/sNP and AA/ANA rat lines have been found to differ for
0 0.5
Ethanol (g/kg)
Fig. 2. Top panel: Effect of administration of ethanol (0, 0.5, 1, and 2 g/kg i.g.) (n ¼ 6/
experimental group) on % pERK-positive neurons/area in PrL PFCx of sP and sNP rats.
Bottom panel: Effect of administration of ethanol (0, 0.5, 1, and 2 g/kg i.g.) (n ¼ 6/
experimental group) on % pERK-positive neurons/area in IL PFCx of sP and sNP rats.
Histograms represent the % average  SEM of counts from at least 2 slices/rat from
AP þ3.2 to AP þ2.2 mm from bregma, according to Paxinos and Watson (1998) rat
brain atlas.
selectively bred for opposite ethanol preference and consumption,
possibly providing some clues on the mechanism(s) by which one
rat line (sP) is prone to develop excessive ethanol intake, while the
other (sNP) tends to avoid ethanol. In particular, the study was
undertaken to assess whether acute ethanol administration to
ethanol-naïve sP and sNP rats would result in a differential ethanol-
regulated ERK phosphorylation in Acb and PFCx, two brain regions
involved in the control of motivated behaviors (Di Chiara et al.,
2004; Salamone & Correa, 2012), and behavioral flexibility and
impulsiveness (George & Koob, 2010; Goldstein & Volkow, 2011),
respectively. In addition, the study also aimed at characterizing the
effects of ethanol in two nuclei of the extended amygdala, the BSTL
and CeA, also involved in abnormal ethanol consumption (Kash,
2012; Koob, 2003). We found that administration of ethanol
differentially affected pERK expression in a line- and brain region-
dependent fashion. Specifically, 1 g/kg ethanol increased the
expression of pERK in AcbSh and AcbC of sP rats while sparing ERK
phosphorylation in AcbSh and AcbC of sNP rats (Figs. 1 and 3); in
contrast, ethanol failed (at all doses) to affect ERK phosphorylation
in both PrL and IL sub-regions of PFCx (Fig. 2) as well as in BSTL and
CeA (Table 1) of both sP and sNP rats.
The findings in Acb of sP rats appear to be in agreement with
those of our previous studies conducted with SpragueeDawley
(Ibba et al., 2009) and Wistar (this laboratory, unpublished obser-
vations) rats, although the present ones slightly differ in that
M. Rosas et al. / Alcohol 48 (2014) 471e476
Table 1
Effect of administration of ethanol (0, 0.5, 1, and 2 g/kg i.g.) (n ¼ 6/experimental
PrL PFCx
group) on % pERK-positive neurons/area in BSTL and CeA of sP and sNP rats.
Ethanol 0 g/kg Ethanol 0.5 g/kg Ethanol 1 g/kg Ethanol 2 g/kg
BSTL
SP 100  19 101  21 129  26 150  39
sNP 100  12 72  11 90  15 70  10
CeA
SP 100  13 79  14 129  14 67  12
sNP 100  21 85  13 103  9 95  25
Data are % average  SEM of counts from at least 2 slices/rat from AP 0.26 to
AP 0.40 mm and from AP 3.6 to AP 4.16 mm from bregma, according to Paxinos
and Watson (1998) rat brain atlas, for BSTL and CeA, respectively.
1 2 ethanol increased the number of pERK-positive neurons in AcbSh
and AcbC of sP rats only at the dose of 1 g/kg. Notably, the present
IL PFCx results appear in contrast with those by Davis, Szarowski, Turner,
Morrisett, and Shain (1999) (PN5 pups), Sanna, Simpson, Lutjens,
and Koob (2002) (Wistar rats), Neznanova et al. (2009) (AA rats),
and Neasta et al. (2011) (C57BL6J mice), who reported that ethanol
decreases the expression of pERK in Acb, PFCx, and hippocampus.
These discrepancies might be justified by different experimental
conditions (i.e., different brain regions, routes of administration,
doses and animal species or rat strain/line). Even the discrepancies
between the present data and those by Neznanova et al. (2009), the
latter collected in Acb and PFCx of selectively bred alcohol-
preferring Alko Alcohol (AA) and -non preferring Alko Non
Alcohol (ANA) rats, are not fully surprising: although selectively
bred for the same phenotype and using similar breeding pro-
1 2
multiple alcohol-related responses and behaviors (e.g., Maccioni
et al., 2012; Roman et al., 2012), likely because of the specific ge-
netic make-up of each single line and consistent with the polygenic
nature of alcohol preference (e.g., McBride et al., 2012; 2013). Thus,
divergent results between sP and AA rats might indicate that se-
lective breeding programs for opposite ethanol preference and
consumption have brought about different sensitivities to ethanol
in terms of ERK phosphorylation.
In the attempt to possibly link the present results with the
behavioral phenotype for which sP rats have been selectively bred
(i.e., high alcohol preference and consumption), it might be
considered that the ethanol dose (1 g/kg) found to increase the
expression of pERK in AcbSh and AcbC of sP rats is about the
amount of ethanol that sP rats usually consume in each drinking
bout when exposed to the home cage, 2-bottle “ethanol vs. water”
regimen with unlimited access for 24 h/day, i.e., the standard
ethanol-drinking procedure used in their selective breeding pro-
gram (Colombo, Lobina, et al., 2006); specifically, the 1 g/kg dose is
approximately the amount of ethanol that a) sP rats voluntarily
consume any time they access the ethanol bottle and b) produces,
in sP rats, measurable psychopharmacological effects, including
anxiolysis and locomotor-stimulation (Colombo, Lobina, et al.,
2006). Additionally, BELs resulting from experimenter-
administered 1 g/kg ethanol (i.g.), 15 min post-administration
(i.e., the time point when rats were sacrificed in the present
study), average 40e50 mg% (e.g., Colombo, Serra, et al., 2006) and
correspond to those produced by voluntary ethanol drinking at
each drinking bout (Colombo, Lobina, et al., 2006).
The results in the ACb of sP rats are also in agreement with the
view that points to activated ERK in AcbSh as a possible biochemical
and neuroanatomical marker of the acute effects of ethanol (Ibba
et al., 2009) and, more generally, of addictive drugs including ec-
stasy, amphetamine, nicotine, and cocaine (Acquas et al., 2007;
Beninger & Gerdjikov, 2004; Girault et al., 2007; Valjent et al.,
2004).
 M. Rosas et al. / Alcohol 48 (2014) 471e476
Fig. 3. Representative, low (5) and high (40) magnification images of pERK immunostaining from control (ethanol 0 g/kg, panels A and C) and treated (ethanol 1 g/kg, panels B
and D) in the Acb of sP and sNP rats, respectively. The images on the right, taken from Paxinos and Watson (1998) rat brain atlas, indicate the antero-posterior gradient (mm) used to
cut the brains and to select the slices for immunostaining. Abbreviations: AcbSh, nucleus accumbens shell; AcbC, nucleus accumbens core; aca, anterior commissure, anterior part.
Scale bar indicates 100 mm.
The finding that ethanol is much less effective in eliciting ERK
activation in the Acb of ethanol-naïve sNP than sP rats, in agree-
ment with ethanol’s ability to increase pERK expression in the Acb
of SpragueeDawley (Ibba et al., 2009) and Wistar rats (unpub-
lished observations), depicts sNP rats as a rat line with lower
sensitivity, in terms of pERK expression in Acb, to acute ethanol
administration.
A critical role has been attributed, in particular to the ERK2
isoform, in reward-mediated intracellular signaling (Girault et al.,
2007; Mazzucchelli et al., 2002), and drug-elicited expression of
phosphorylated ERK in Acb has been suggested as a post-synaptic
marker of drug-induced dopamine transmission in this area
(Acquas et al., 2007; Ibba et al., 2009). Our results for ethanol-
elicited pERK expression in Acb appear in agreement with the
suggestion that a higher dopamine neuronal spontaneous activity
may be associated with ethanol preference and increased vulner-
ability to develop excessive ethanol intake in sP rats (Melis et al.,
2009).
Data collected in PrL and IL PFCx, as well as in BSTL and CeA,
reveal that administration of ethanol to both sP and sNP rats failed
to affect ERK phosphorylation. In particular, while the results in the
PFCx are in agreement with those of Nuutinen, Kiianmaa, and
Panula (2011) in AA and ANA rats, they appear in contrast with
the observation made by Neznanova et al. (2009) that ethanol
decreases pERK expression in AA rats, but a reasoning similar to
that followed for the Acb might apply to discuss results in PFCx.
Similarly, the finding that ethanol fails to elicit ERK phosphoryla-
tion in BSTL and CeA in both sP and sNP rats contrasts with our
previous study (Ibba et al., 2009), and we might overall interpret
the present results as an indication of different consequences of
selective breeding for opposite ethanol preference and
consumption.
In conclusion, whatever is the mechanism at the basis of the
changes of pERK expression detected in the present study, the most
parsimonious explanation to interpret these results is to hypothe-
size that ethanol, at the dose of 1 g/kg, acts differentially on
475
common mechanism(s), resulting in differential line- and brain
region-dependent pERK expression, or acts on distinct mechanisms
able to affect pERK expression differentially.
As previously pointed out, in this study ethanol was
experimenter-delivered. While this approach prevented investi-
gating the effect of voluntarily consumed ethanol on pERK
expression, it allowed the inclusion of sNP rats in the experimental
design and led to identifying the activation of MAPK-ERK cascade in
Acb as a pathway that may contribute to differentiate biochemically
between sP and sNP rats.
In summary, at least two main observations can be drawn: on
one hand, sP rats display a sensitivity to ethanol in terms of pERK
expression (increase in the Acb); on the other, these results indi-
cate that sNP rats do appear insensitive to ethanol effects in the
Acb. The selective breeding of sP and sNP rats may likely involve
multiple genes, resulting in a number of different phenotypes
(Colombo, Lobina, et al., 2006); the present data add pERK
expression in Acb but not in PFCx, BSTL, and CeA, in response to
ethanol, to this list.
Acknowledgments
This study was supported by a grant from Regione Autonoma
della Sardegna (RAS), Italy, L.R. 7/2007, n CRP2_537-CUP
F71J090006200002 to EA, GC and EC.
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