Stress Abolishes the Effect of Previous Chronic Ethanol Consumption on Drug Place Preference and on the Mesocorticolimbic Brain Pathway
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ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH Vol. 38, No. 5
May 2014
Stress Abolishes the Effect of Previous Chronic Ethanol
Consumption on Drug Place Preference and on the
Mesocorticolimbic Brain Pathway
Daniel Moreira-Silva, Gessynger Morais-Silva, Juliana Fernandes-Santos,
Cleopatra S. Planeta, and Marcelo T. Marin
Background: Conditioned place preference (CPP) to ethanol (EtOH) is an important addiction-
related alteration thought to be mediated by changed neurotransmission in the mesocorticolimbic brain
pathway. Stress is a factor of major importance for the initiation, maintenance, and reinstatement of
drug abuse and modulates the neurochemical outcomes of drugs. Thus, the aim of this study was to
investigate the effects of concomitant exposure to chronic EtOH and stress on CPP to this drug and
alterations of dopaminergic and serotonergic neurotransmission in mice.
Methods: Male Swiss mice were chronically treated with EtOH via a liquid diet and were exposed
to forced swimming stress. After treatment, animals were evaluated for conditioning, extinction, and
reinstatement of CPP to EtOH. Also, mice exposed to the same treatment protocol had their prefrontal
cortex (PFC), nucleus accumbens (NAc), and amygdala dissected for the quantitation of dopamine,
serotonin, and their metabolites content.
Results: Data showed that previous chronic exposure to EtOH potentiated EtOH conditioning and
increased dopaminergic turnover in PFC. Exposure to stress potentiated EtOH conditioning
and decreased dopaminergic turnover in the NAc. However, animals exposed to both chronic EtOH
and stress did not display alterations of CPP and showed an elevated content of dopamine in amygdala.
No treatment yielded serotonergic changes.
Conclusions: The present study indicates that previous EtOH consumption as well as stress expo-
sure induces increased EtOH conditioning, which can be related to dopaminergic alterations in the
PFC or NAc. Interestingly, concomitant exposure to both stimuli abolished each other’s effect on con-
ditioning and PFC or NAc alterations. This protective outcome can be related to the dopaminergic
increase in the amygdala.
Key Words: Ethanol, Stress, Addiction, Place Preference, Dopamine.
E THANOL (ETOH) CONSUMPTION is one of the
major factors of morbidity and mortality worldwide
with an annual prevalence of 42% (World Health Organiza-
a 2-bottle choice model (Quebec et al., 1997) and can trigger
the reinstatement of EtOH self-administration (Funk et al.,
2006) in experimental animals. Moreover, chronic stress can
tion, 2011). Individual risk factors for the development of enhance conditioned place preference (CPP) to cocaine
alcohol abuse and addiction are not completely understood. (McLaughlin et al., 2003), while acute stress promotes CPP
Acute and chronic stress play an important role in the to nicotine (Brielmaier et al., 2012). There are few data
motivation to the abuse of addictive substances and addicted linking stress effects on CPP to EtOH in spite of critical effect
individuals often cite the exposure to aversive stimuli and of conditioned responses to drug seeking and maintenance of
negative mood states as the reason for the initiation, mainte- EtOH consumption (Gremel and Cunningham, 2009).
nance, and reinstatement of drug seeking (Sinha, 2001). Nevertheless, Funk and colleagues (2004) have shown the
Repeated exposure to stress increases EtOH consumption in inhibition of EtOH place aversion induced by stress exposure
during the conditioning period.
The CPP model is widely used as all abuse substances that
From the Institute of Biomedical Sciences (DM-S, GM-S, JF-S, promote addiction in humans also develop conditioning
MTM), Federal University of Uberlândia (UFU), Uberl^ andia, Brazil;
and Laboratory of Pharmacology (CSP, MTM), School of Pharmaceu-
between environmental associated characteristics and drug
tical Sciences, Univ. Estadual Paulista (UNESP), Araraquara, Brazil. effects in rodents (Sanchis-Segura and Spanagel, 2006).
Received for publication June 27, 2013; accepted January 10, 2014. Studies have demonstrated that memory acquired by the
Reprint requests and Present address: Marcelo T. Marin, Depto. association between drug effects and the environment is also
Princıpios Ativos Naturais e Toxicologia (PANT), Faculdade de Ci^ encias subject to extinction, caused for example by drug-free succes-
Farmac^ euticas, Universidade Estadual Paulista (UNESP), Rod. Ara-
raquara-Ja u km 01, CEP 14801-902, Araraquara, SP, Brazil; Tel.: +55-
sive exposures to the environment previously paired with the
16-3301-6984; Fax: +55-16-3322-0073; E-mail: marcelo@fcfar.unesp.br psychoactive substance (Mueller and Stewart, 2000). Using
Copyright © 2014 by the Research Society on Alcoholism. CPP procedure, it is also possible to evaluate the reinstate-
DOI: 10.1111/acer.12388
ment of drug conditioning after the behavior has been
Alcohol Clin Exp Res, Vol 38, No 5, 2014: pp 1227–1236 12271228 MOREIRA-SILVA ET AL.
extinct. Reinstatement can be induced by the administration Thus, the aim of this study was to investigate the effects of
of the drug or stress exposure (Sanchis-Segura and Spanagel, previous chronic EtOH consumption and exposure to stress
2006). on EtOH CPP and dopaminergic and serotonergic altera-
The neurochemical substrate for CPP as well as most tions in the PFC, NAc, and amygdala.
behaviors related to drug addiction includes the mesocortico-
limbic brain pathway and their dopaminergic and serotoner-
MATERIALS AND METHODS
gic circuits (Kreek and Koob, 1998). Studies using strains of
mice with different preferences to EtOH showed differences Subjects
in the content of dopamine, serotonin, and their metabolites andia – MG, Brazil;
Male Swiss mice (Vallee Institute, Uberl^
in brain regions such as the nucleus accumbens (NAc), 30 to 35 g) were transferred to our animal depository at least
amygdala, and prefrontal cortex (PFC) between the strains, 5 days before the start of the experiments and were housed 4
per cage. The room was maintained at a temperature of
which corroborate a role of this neural circuitry in preference 23 2°C on a 12:12 hour light/dark cycle with ad libitum
to EtOH (Strother et al., 2005). Tests showed that EtOH water and food access (except during the liquid diet procedure).
stimulates neuronal activation in the ventral tegmental area All experiments were performed during the light phase and ani-
in vivo and in vitro and that this action is blocked by the co- mals were randomly tested across this time period. The experi-
administration of dopamine D2 receptor agonists. There are mental protocol was approved by the Ethical Committee for the
Animal Utilization of the Federal University of Uberl^ andia
also reports that lesions of dopaminergic terminals in NAc (CEUA 04/11 and 120/11) and the experiments were conducted
are able to reduce EtOH drinking and self-administration according to the principles of the Brazilian College of Animal
(Morikawa and Morrisett, 2010) and evidence that reduced Experimentation (COBEA), based on NIH Guidelines for the
dopaminergic release in the shell of NAc is able to predispose Care and Use of Laboratory Animals.
to EtOH seeking and preference (Bustamante et al., 2008;
Quintanilla et al., 2007). While the self-administration of Chronic EtOH Administration
EtOH acutely increases serotonin release within the NAc, Chronic EtOH administration procedure was adapted from
drug withdrawal causes progressive suppression of serotonin experiments with forced liquid diet intake performed previously
release in this brain region (Yoshimoto et al., 2012). Studies by Bonassoli and colleagues (2011) that induced EtOH with-
have shown that the extracellular concentration of dopamine drawal syndrome. Animals allocated in plastic cages (19
[width] 9 30 [length] 9 13 [height] cm, 4 to 5 animals per
in the NAc is approximately 25% higher in alcohol-preferring cage) had free access to bottles containing a solution composed
(P) rats with prior access to 10% EtOH, even after 2 weeks of of Sustagen Mâ (chocolate flavor; Mead Johnson, S~ ao Paulo –
EtOH withdrawal, compared with animals that only had SP, Brazil) at 28.5 g/100 mL (1.1 kcal/mL) and no chow. The
access to water. On the other hand, rats given 8 weeks of con- liquid diet was the only source of food available to the animals,
tinuous access to EtOH presented a serotonin content that and it was prepared fresh on a daily basis and presented to the
animals at the same time each day (12:00 AM). This liquid diet
was 35% lower than water controls (Thielen et al., 2004). provided all of the necessary nutrients to rodents and presented
Chronic EtOH exposure also alters the expression of dopa- them at higher concentrations than are normally consumed to
mine transporters (DAT) in the reward pathway of C57BL/ ensure ad libitum consumption. Bottles were weighed before
6J mice. Tests with chronic intermittent EtOH vapor expo- and after exposure to mice to evaluate the diet consumption
sure showed increased DAT expression in the NAc, while per animal group; any remaining diet was removed following
24-hour exposure. One bottle of drinking water was available,
chronic continuous EtOH vapor treatment reduced DAT in together with the liquid diet. Mice were kept in the groups with
the NAc (Healey et al., 2008). Although there are few studies no restricted volumes of the diet to reduce the stress of isola-
showing the direct effects of EtOH consumption on PFC tion or food restriction.
activity, data demonstrated lower basal concentrations of Liquid diet was administered for 15 days, distributed in 3 cycles
dopamine in strains of animals which prefer EtOH (Engl- of 5 days, with a 2-day interval without the diet when the mice
received conventional mice chow, between each diet cycle. Groups
eman et al., 2006). However, studies still showed a main role treated with EtOH had EtOH 6% (v/v) added to their solution on
of amygdala on CPP to alcohol. Bilateral lesions in this brain the first 2 days and EtOH 8% (v/v) on the remaining 13 days, while
region after EtOH administration eliminated acquired drug the control group received the same liquid diet without EtOH
conditioning (Gremel and Cunningham, 2008). Moreover, throughout the experiment and was named vehicle.
dopaminergic release seems to be related to this phenomenon.
Studies reported that prior exposure to EtOH increased CPP Stress
to cocaine and effects mediated by dopamine D1 receptors The stress model was developed based on forced swimming pro-
(Sidiropoulou et al., 2009). The action of amygdala mediat- cedure, described by McLaughlin and colleagues (2003), which has
ing stress influence on behaviors related to EtOH addiction been shown to be effective in altering the effects of EtOH as well as
has also been reported, as this brain region has remarkable other abuse psychoactive substances. During 5 consecutive days,
mice from the stress group were placed in a cylindrical 5 l container
involvement in responses to environmental stimuli. An filled with 3.5 l of water, from which the animal was not able to
altered serotonergic release within the amygdala of rats escape. On days 1, 3, and 5, the mice remained in the water for a sin-
exposed to psychosocial stress situations — such as maternal gle trial of 15 minutes, while on days 2 and 4, the mice underwent 3
separation — that also predispose to addiction-related behav- trials of 6 minutes with intervals of 5 minutes. After each swimming
iors has been shown (Oreland et al., 2011). session, mice were towel-dried and put back in their home cages.STRESS AND CHRONIC ETHANOL EFFECTS 1229
The control group consisted of animals not exposed to stress, named PFC Bregma1.98 mm
the nonstress group.
Conditioned Place Preference
PrL
This was based on the tests by Brenhouse and Andersen IL
(2008). CPP boxes (Insight Ltd, Ribeir~ao, Preto-SP, Brazil)
consisted of a rectangular acrylic apparatus (45 9 14 9 15)
with 3 compartments. The 2 end compartments were larger
and with different floor textures and wall coloring. The middle
gray compartment was smaller and had 2 doors which could
be opened to allow the animals to walk freely between the
compartments. NAc Bregma1.54 mm
On day 1, mice were placed inside the central compartment,
with open doors to establish an initial preference of each sub-
ject for 20 minutes. This phase was named the preconditioning
test. The conditioning phase was performed from days 2 to 9,
when animals received intraperitoneal (i.p.) injections of EtOH
(0.8 mg/kg; days 2, 4, 6, and 8) before being confined for
20 minutes in the nonpreferred compartment, and saline (days
3, 5, 7, and 9), before being confined for 20 minutes in the aca
AcbC
preferred compartment. The frequency of animals preferring 1 AcbSh
compartment of CPP boxes was not superior to 60% within the I
groups. Then, on day 10, a 20-minute session was performed by
animals in a drug-free state, with open doors again so that their Amygdala Bregma-1.22 mm
EtOH-paired side preference could be assessed. This phase was
named the postconditioning test. From days 11 to 19, the ani-
mals were exposed daily to the CPP box with the doors open;
the animals received no injections to induce the extinction of
EtOH conditioning. On days 13, 16, and 19, the extinction of
conditioning was evaluated. On day 20, a reinstatement test was
performed, where the subjects were submitted to a new forced
swimming procedure for 15 minutes, 10 minutes before being
placed in the CPP box again with the doors open.
All exposures to CPP boxes were recorded by cameras placed BLA
CeL
CeM
above the apparatus and connected to a computer for video record- CeC
ing. Videos were analyzed posteriorly, and the time spent by animals
in each compartment of the box was quantified. EtOH dose of
0.8 mg/kg was chosen because previous (unpublished) data from
our laboratory using the same experimental procedure and appara- Fig. 1. Brain location of regions extracted for the quantitation of neuro-
tuses have shown conditioning for both 0.8 and 1.6 g/kg EtOH i.p. transmitters and their metabolites. The circles represent the regions dis-
injections but not for 0.4 g/kg. sected. Figures were adapted from Paxinos and Franklin (2001).
Neurochemical Measurements
The potential difference was set at 800 mV versus an Ag/AgCl
Following decapitation, the brains were rapidly removed from reference electrode. The mobile phase containing 50 mM citric acid,
the skull and frozen in liquid nitrogen ( 196°C) and then kept at 2 mM KCl, 0.1 mM EDTA, 9.86% methanol, and 2.11%
20°C until dissection. In a cryostat at 20°C, brains were coronal- acetonitrile was adjusted to pH 3.2, vacuum filtered and degassed
ly sectioned to find target areas according to stereotaxic coordinates by ultrasound before being pumped through the system. The cali-
from the Atlas of Paxinos and Franklin (2001) as follows: PFC, bration curve was constructed with standard solutions of 1, 2.5, 5,
Bregma 1.98 mm; NAc, Bregma 1.54 mm, and Amygdala, Bregma 10, 25, 50, 100, 200, 400, and 600 ng/mL of dopamine, DOPAC,
1.22 mm. Samples of 1 mm thickness were then removed with a HVA, serotonin, and 5-HIAA, which were injected into the chro-
flat-tipped 15-gauge needle, as represented in Fig. 1. matograph in triplicate. The limit of detection and quantitation, as
The technique for quantitation of dopamine, serotonin, already standardized in this apparatus, was for dopamine: 0.5 and
homovanillic acid (HVA), 3,4 dihydroxyphenylacetic acid 1.66, for DOPAC: 0.7 and 2.4, for HVA: 1.9 to 6.4, for serotonin:
(DOPAC), and 5-hydroxyindolacetic acid (5-HIAA) was based on 1.0 to 3.5, and for 5-HIAA: 1.3 and 4.26 ng/mL. Finally, the con-
assays described by Cannazza and colleagues (2005). In summary, centrations of the substances were corrected according to the mass
the dissection samples were homogenized in 0.1 M perchloric acid, of tissue from the dissected samples and were expressed as ng of sub-
and centrifuged at 13,0009g for 20 minutes at 4°C. The volumes of stance per milligram of tissue. Based on the concentration of these
perchloric acid were as follows: 80 ll for PFC and amygdala and substances, dopamine turnover was calculated as the ratio
100 ll for NAc. DOPAC + HVA/dopamine and serotonin turnover as 5-HIAA/
Thirty microliters of the supernatant were injected automatically serotonin.
into the high-performance liquid chromatography (HPLC) Alli-
ance 2465 system (Waters, Milford, MA). The HPLC system was
equipped with a reverse-phase column (Symmetry C18, Statistics
150 9 4.6 mm, 5 lm and 100 A pore-diameter particle size; Statistical analysis was performed using Statistica software (Stat-
Waters), coupled with glassy-carbon electrochemical detector. Soft Inc., Tulsa, OK). The results of experiment 1 were analyzed by1230 MOREIRA-SILVA ET AL.
3-way analysis of variance (ANOVA) considering the factors ment. There were no significant differences between groups
chronic EtOH (EtOH vs. vehicle), stress (stress vs. no stress) and in these measures.
phases (pre conditioning, post conditioning, extinction, and
A 3-way ANOVA revealed a significant difference for
reinstatement). In cases where ANOVA showed significant differ-
ences (p ≤ 0.05), the planned comparison test between the groups of phases factor, F(3, 102) = 2.8, p ≤ 0.05, and interaction of
interest was performed. The results of experiment 2 were analyzed stress and chronic EtOH factors, F(1, 34) = 4.7, p ≤ 0.05.
by 2-way ANOVA considering the factors chronic EtOH (EtOH vs. As shown in Fig. 4, planned comparison analysis revealed
vehicle) and stress (stress vs. no stress). In cases where ANOVA that there was a significant intragroup difference between
showed significant differences (p ≤ 0.05), the Duncan post hoc test
the preconditioning and postconditioning test in the
was performed.
groups EtOH non stress (p ≤ 0.001) and vehicle stress
(p ≤ 0.001), while the groups vehicle nonstress and EtOH
Experiment 1 — Effects of Stress and Chronic EtOH Administration
on EtOH Conditioning, Extinction, and Reinstatement stress did not express conditioning (p > 0.05 for difference
intragroup between pre- and postconditioning tests). CPP
Animals were submitted to a liquid diet containing EtOH or just
observed in the group EtOH nonstress and vehicle stress
vehicle and, in the last 5 days of chronic administration, half of the
subjects were exposed to forced swim stress (FSS) daily. After the was not due to preconditioning difference between groups,
last day of FSS exposure, animals were started on to the exposure as the preference to each CPP box compartment was not
to CPP to assess the effects of this previous treatment on EtOH significantly different between groups for the precondition-
conditioning, extinction, and reinstatement. The experimental ing test.
groups were: vehicle nonstress, EtOH nonstress, vehicle stress, and
For extinction (also Fig. 4), measured on day 9 after the
EtOH stress (N = 9 to 10/group). Fig. 2A,B show the experimental
procedure timeline. postconditioning test, planned comparison showed a signifi-
cant difference between permanence on preconditioning and
Experiment 2 — Effects of Stress and Chronic EtOH Administration extinction days only in the EtOH nonstress group (p ≤ 0.05).
in the Content of Dopamine, Serotonin, and Their Metabolites Then, chronic EtOH pretreatment hampered CPP extinction.
However, there was CPP extinction in the vehicle stress
Animals were submitted to a liquid diet containing EtOH or
just vehicle; in the last 5 days of chronic administration, half of group. The extinction was also measured on days 3 and 6,
the subjects were exposed to FSS daily. Following the last day of but no significant difference was shown for these days com-
chronic EtOH and FSS exposure, all experimental groups had pared to postconditioning. Reinstatement results using the
the liquid diet replaced by the diet with no EtOH and were FSS prior the CPP box exposure revealed that this stress
decapitated by a guillotine 14 hours later; brains were removed
model was not efficient to induce EtOH reinstatement. How-
from skulls (within 60 to 90 seconds) and frozen in liquid nitro-
gen for later neurochemical measurements on the samples of ever, a very similar protocol promptly yielded morphine rein-
PFC, NAc, and amygdala. Figure 2A shows the experimental statement (Ma et al., 2007).
procedure for this test. There were 4 experimental groups: vehicle To better understand the neuronal substrate that promotes
nonstress, EtOH nonstress, vehicle stress, and EtOH stress the changes observed in behavioral tests, dopamine, seroto-
(N = 8 to 9/group).
nin, and their metabolites were quantified from samples of
PFC, NAc, and amygdala after chronic exposure to EtOH
RESULTS and stress (Figs 5, 6, and 7 and Table 1).
A 2-way ANOVA revealed no differences between
Figure 3 shows liquid diet consumption, mice body weight
groups regarding serotonin and 5-HIAA content or seroto-
and EtOH consumption during the chronic liquid diet treat-
nergic turnover in PFC, NAc, or amygdala (p > 0.05), as
described in Table 1. Likewise, when dopamine activity in
the PFC was analyzed, ANOVA showed no differences in
responses of dopamine, DOPAC, or HVA content to both
EtOH and stress treatments (p > 0.05 for stress and EtOH
factors). However, ANOVA showed significant interaction
between chronic EtOH and stress for dopaminergic turn-
over, F(1, 29) = 4.1, p ≤ 0.05, and Duncan post hoc test
revealed an increase of dopaminergic turnover in the EtOH
non stress group compared to the vehicle non stress and
EtOH stress groups (p ≤ 0.05), indicating that only chronic
exposure to EtOH had an effect on this parameter in the
PFC (Fig. 5).
In the NAc, there were also no significant differences
according to ANOVA in the concentration of dopamine,
DOPAC, or HVA (p > 0.05 for factors stress and EtOH)
between groups, as demonstrated in Fig. 6. However, ANO-
Fig. 2. (A) Experimental protocol used for chronic exposure to ethanol
and stress, and (B) phases of conditioned place preference (CPP) proce- VA revealed significant effect of stress for dopaminergic
dure. turnover, F(1, 29) = 3.9, p ≤ 0.05, and the Duncan post hocSTRESS AND CHRONIC ETHANOL EFFECTS 1231
Fig. 3. Liquid diet consumption (A), mice body weight (B), and ethanol consumption (C) during the chronic diet exposure. Values are expressed as
mean SEM.
DISCUSSION
test revealed a lower dopamine turnover in the vehicle stress
group compared to vehicle nonstress (p ≤ 0.05), indicating Results presented here demonstrate that previous chronic
that only stress interfered with dopaminergic activity in the EtOH consumption or exposure to stress was sufficient to
NAc. facilitate Pavlovian EtOH conditioning, but that previous
Moreover, concerning the amygdala (Fig. 7), a 2-way concomitant EtOH consumption and stress exposure abol-
ANOVA revealed significant differences for chronic EtOH ished each other’s effect. Similar to behavioral alterations,
factor, F(1, 29) = 4.2, p ≤ 0.05). Duncan post hoc test dopaminergic changes in the PFC and NAc emerged only
showed that the EtOH stress group had elevated dopaminer- after chronic EtOH consumption or exposure to stress, but
gic content compared with vehicle nonstress and vehicle not after concomitant EtOH consumption and stress expo-
stress groups (p ≤ 0.05). Nevertheless, there was no change sure. Additionally, increased dopamine content in the amyg-
in concentration of DOPAC and HVA or dopaminergic dala was found after concomitant EtOH consumption and
turnover (p > 0.05). stress exposure.1232 MOREIRA-SILVA ET AL.
Fig. 4. Effects of chronic exposure to stress and ethanol (EtOH) on conditioned place preference. Values are expressed as mean SEM of perma-
nence in seconds of each group in the nonpreferred compartment: Vehicle NonStress, EtOH NonStress, Vehicle Stress and EtOH Stress. *p ≤ 0.05
when difference between pre-and postconditioning is significant; #p > 0.05 when difference between preconditioning and extinction is significant. N = 9
to 10 animals per group.
A - DA B - DOPAC C - HVA D - Turnover of DA
0,05 4,0
*
0,12 0,08
Concentration of DA (ng/mg of tissue)
Vehicle
Concentration of HVA (ng/mg of tissue)
Concentration of DOPAC (ng/mg of
0,10 0,04 Ethanol
0,06 3,0
HVA+DOPAC/DA
0,08
0,03
tissue)
0,06 0,04 2,0
0,02
0,04
0,02 1,0
0,01
0,02
0,00 0,00 0,00 0,0
Non stress Stress Non stress Stress Non stress Stress Non stress Stress
Fig. 5. Effect of chronic exposure to stress and ethanol (EtOH) on the concentration of dopamine and its metabolites in the prefrontal cortex (PFC).
Values are expressed as mean SEM of the ratio between the concentration in nanograms of (A) dopamine (DA), (B) DOPAC, or (C) HVA and mass in
milligrams of tissue sample from the prefrontal cortex removed from animals of each experimental group. In (D), data are expressed as mean SEM of
the ratio between the amount in nanograms of DOPAC + HVA and the amount in nanograms of dopamine present in samples from each experimental
group. *p ≤ 0.05: different from vehicle nonstress and EtOH stress groups. N = 8 to 9 animals per group.
The present data confirmed that previous chronic EtOH Our data show that 5 days of FSS prior to CPP procedure
consumption increase the acquisition of EtOH-induced CPP. also increases place conditioning to this drug, suggesting a
Other studies have shown that previous EtOH injections long-term enhancement of rewarding properties of EtOH
(Camarini and Hodge, 2004) or vapor exposure (Carrara- induced by stress. This result corroborates the recent study
Nascimento et al., 2013) increases EtOH drinking. Also, of Bahi (2013), which demonstrated that chronic psychoso-
chronic EtOH consumption in a liquid diet has enhanced mor- cial stress exposure increased EtOH-CPP acquisition. It is
phine-induced CPP (Shibasaki et al., 2013). The group previ- also similar to FSS enhancement of CPP to other abuse
ously exposed to EtOH consumption but not stress showed an drugs such as nicotine (Brielmaier et al., 2012).
absence of CPP extinction. However, the same alteration was Interestingly, despite the fact that sole exposure to FSS or
not observed in the vehicle stress group, which extinguished chronic EtOH consumption enhanced EtOH place condi-
the acquired conditioning. Then, the previous consumption of tioning, animals previously exposed to both (chronic EtOH
EtOH appears to be more robust in inhibiting the extinction consumption and swim stress) showed no change in condi-
of EtOH-induced CPP than previous stress exposure. tioning in the present study. In spite of most of the investiga-STRESS AND CHRONIC ETHANOL EFFECTS 1233
A - DA B - DOPAC C - HVA D - Turnover of DA
2,5 1,2 8,0
Concentration of DA (ng/mg of tissue)
Concentration of HVA (ng/mg of tissue)
1,2
Vehicle
Concentration of DOPAC (ng/mg of
1,0
1,0 2,0 Ethanol
HVA+DOPAC/DA
6,0
0,8
0,8 1,5
tissue)
*
0,6 4,0
0,6
1,0
0,4
0,4
2,0
0,5
0,2
0,2
0,0 0,0 0,0
0,0
Non stress Stress Non stress Stress Non stress Stress
Non stress Stress
Fig. 6. Effect of chronic exposure to stress and ethanol on the concentration of dopamine and its metabolites in the nucleus accumbens (NAc). Values
are expressed as mean SEM of the ratio between the concentration in nanograms of (A) dopamine (DA), (B) DOPAC or (C) HVA and mass in milli-
grams of tissue sample from NAc removed from animals of each experimental group. In (D), the data are expressed as mean SEM of the ratio between
the amount in nanograms of DOPAC + HVA and the amount in nanograms of dopamine present in samples from each experimental group. *p ≤ 0.05:
different from vehicle non stress group. N = 8 to 9 animals per group.
A - DA B - DOPAC
Concentration of HVA (ng/mg of tissue)
C - HVA D - Turnover of DA
0,8 0,6 0,6 6,0
Concentration of DA (ng/mg of tissue)
Vehicle
Concentration of DOPAC (ng/mg of
*
0,5 0,5 5,0 Ethanol
HVA+DOPAC/DA
0,6
0,4 0,4 4,0
tissue)
0,4 0,3 0,3 3,0
0,2 0,2 2,0
0,2
0,1 0,1 1,0
0,0 0,0 0,0 0,0
Non stress Stress Non stress Stress Non stress Stress Non stress Stress
Fig. 7. Effect of chronic exposure to stress and ethanol on the concentration of dopamine and its metabolites in the amygdala. Values are expressed
as mean SEM of the ratio between the concentration in nanograms of (A) dopamine (DA), (B) DOPAC, or (C) HVA and mass in milligrams of tissue
sample from amygdala removed from animals of each experimental group. In (D), data are expressed as mean SEM of the ratio between the amount
in nanograms of DOPAC + HVA and the amount in nanograms of dopamine present in samples from each experimental group *p ≤ 0.05: different from
the vehicle nonstress and vehicle stress groups. N = 8 to 9 animals per group.
Table 1. Effects of Chronic EtOH and Stress Exposure on Serotonin (5-HT) and Serotonin Metabolite (5-HIAA) Concentration (ng/mg of Tissue) and
Turnover of 5-HT within the Prefrontal Cortex (PFC), Nucleus Accumbens (NAc) and Amygdala
Brain region Treatment 5-HT 5-HIAA Turnover of 5-HT
PFC Vehicle nonstress 0.06 0.02 0.23 0.04 8.51 4.46
EtOH nonstress 0.03 0.01 0.18 0.03 11.64 3.18
Vehicle stress 0.04 0.01 0.16 0.03 4.00 0.97
EtOH stress 0.05 0.01 0.25 0.04 6.04 0.64
NAc Vehicle nonstress 0.16 0.05 0.44 0.04 4.98 0.69
EtOH nonstress 0.15 0.01 0.32 0.05 3.85 1.01
Vehicle stress 0.23 0.07 0.31 0.03 2.63 0.73
EtOH stress 0.21 0.09 0.37 0.03 3.62 0.78
Amygdala Vehicle nonstress 0.20 0.05 0.54 0.06 4.32 1.52
EtOH nonstress 0.22 0.04 0.48 0.04 3.32 0.88
Vehicle stress 0.20 0.06 0.49 0.08 4.39 1.60
EtOH stress 0.36 0.11 0.35 0.03 2.16 0.71
EtOH, ethanol.
Data are expressed as mean SEM. (N = 7 to 9). Turnover of 5-HT = 5-HIAA/5-HT.1234 MOREIRA-SILVA ET AL. tions revealing that stress increases addiction-related behav- Following stress exposure, a reduction of dopaminergic iors (Sinha, 2001), some stress protocols do not change or turnover in the NAc was found. Although there are few even decrease behavioral, hormonal, or neurochemical reports of experiments involving only chronic stress and effects of abuse drugs (Miczek et al., 2008). Chester and col- dopamine changes in the NAc, enhanced dopamine release leagues (2004) showed, for example, that stress moderately into the NAc during acute tail-pinch (Rouge-Pont et al., reduced EtOH intake in P strains during the 10 days of stress 1998) and social defeat (Miczek et al., 1999) exposure is application, but increased EtOH intake in the first 5 days reported, similar to the administration of abuse drugs. In after stress termination. Also recent study has shown that spite of the fact that we did not measure dopaminergic altera- FSS reduce the acquisition of morphine-induced CPP when tions during acute stress exposure, our results suggest that stress is applied 10 minutes before morphine injections (At- chronic stress drives a decrease of mesolimbic dopaminergic tarzadeh-Yazdia et al., 2013). Our result is intriguing, activity similar to the effect of withdrawal following chronic because it shows that the same stress protocol that increases abuse drugs administration. Corroborating the behavioral the acquisition of EtOH CPP, when applied individually, is data of CPP acquisition, animals previously treated with able to abolish the increased CPP induced by previous both chronic EtOH and FSS showed no dopaminergic alter- chronic EtOH consumption. We hypothesized that stress ation in the PFC or NAc 14 hours after the last drug or and EtOH decrease each other effects when applied at the stress exposure. This indicates that dopaminergic alterations same time or during a short time interval but stress increases in the PFC or NAc predispose animals to EtOH place condi- drug effect when applied long before. This idea is corrobo- tioning, and they are absent following treatments that did rated by results that stress increased CPP to EtOH (Bahi, not change CPP. Additionally, the reciprocal modulation 2013) and nicotine (Brielmaier et al., 2012) when applied between NAc and PFC can explain the suppression of the 24 hours or longer before drug injections, but it decreases effects of chronic EtOH consumption on PFC dopaminergic CPP when applied few minutes before drug administration alterations induced by stress exposure. Lesions of dopami- (Attarzadeh-Yazdia et al., 2013). The inhibition of stress and nergic terminals in the PFC increase the dopaminergic turn- EtOH effects when they are in place at the same time occurs over in the NAc (King and Finlay, 1997), which is an effect also in the central nervous system. It has been shown that that is evident only under stress conditions. On the other EtOH revert some of the stress-induced changes in brain cat- hand, Pascucci and colleagues (2007) showed that the sus- echolamine levels (DeTurck and Vogel, 1982). tained activation of dopamine release in the PFC induced by Data from our neurochemical assays contribute to the stress leads to a profound inhibition of NAc dopamine understanding of behavioral changes induced by stress expo- release. Therefore, the opposite changes induced by EtOH sure and EtOH consumption. Most of the microdialysis consumption and stress on dopaminergic turnover in the studies have revealed an increase of dopamine release in the PFC and NAc could abolish each other’s effect on the subse- NAc right following EtOH administration (Kiianmaa et al., quent EtOH conditioning. 1995; L€ of et al., 2007) and a decrease of dopamine release in Dopaminergic alterations in the amygdala were evident the same area upon withdrawal of chronic EtOH treatment only in animals exposed to both EtOH consumption and (Fadda and Rossetti, 1998). This decrease of basal mesolim- stress. It is possible that the increased storage of dopamine bic dopaminergic activity appears to be related to behavioral somehow inhibited EtOH-seeking behavior due to the par- alterations of abstinence syndrome (George et al., 2012). ticipation of amygdala on the development of CPP to Our results showed no alterations of dopaminergic neuro- EtOH. Amygdala is involved in associative drug addiction transmission in the NAc following chronic EtOH exposure learning processes (Luo et al., 2013) and lesions of amyg- but revealed increased dopaminergic turnover in the PFC. dala disrupt EtOH CPP (Gremel and Cunningham, 2008). The difference between our and others’ results regarding Dopaminergic neurotransmission in the amygdala is impor- NAc may be related to the sampling technique, as we mea- tant to EtOH conditioning because intra-amygdalar infu- sured the tissue content of the neurotransmitter and metabo- sions of a dopaminergic antagonist blocked CPP to EtOH lites, while others have measured extracellular dopamine by (Gremel and Cunningham, 2009). Amygdala has long been microdialysis. However, our results revealed that EtOH attributed an important role in negative emotional states withdrawal also induces dopaminergic changes in the PFC. that define alcohol dependence and withdrawal (Gilpin, This effect corroborates the tests of Carlson and Stevens 2012). The role of the dopaminergic system in the amygdala (2006), which also showed an increased dopaminergic turn- mediating the interaction between stress and EtOH expo- over within the PFC in EtOH withdrawal rats administered sures has also been reported. A study performed by Ma- with a liquid diet containing EtOH. Moreover, the revealed tsuzawa and colleagues (1999) has shown that both D1 and changes in the PFC could be important in drug-seeking D2 receptor antagonists reduced the effects of stress on behavior during abstinence syndrome based on evidences CPP to EtOH. As a result, dopamine increases in the amyg- from the study of Samson and Chappell (2003). They dala could act as a protective factor against EtOH condi- described that dopamine action in the PFC is involved in the tioning. Nevertheless, functional studies with dopamine onset and offset of EtOH drinking, while the NAc is involved alterations in the amygdala are necessary to confirm this in maintaining the ongoing drug administration behavior. hypothesis.
STRESS AND CHRONIC ETHANOL EFFECTS 1235
Therefore, these data bring the relevance of studying the dopamine and serotonin in the medial prefrontal cortex and amygdala.
environmental influence of addiction-related behaviors and Alcohol Clin Exp Res 30:1678–1692.
Carrara-Nascimento PF, Olive MF, Camarini R (2013) Ethanol pre-expo-
neuroadaptations. This study demonstrated that both prior sure during adolescence or adulthood increases ethanol intake but
exposure to stress and the chronic consumption of EtOH ethanol-induced conditioned place preference is enhanced only when pre-
promote changes in the mesocorticolimbic dopamine system, exposure occurs in adolescence. Dev Psychobiol 56:36–48.
which seem to be related to increased drug conditioning. Chester JA, Blose AM, Zweifel M, Froehlich JC (2004) Effects of stress on
When there was prior exposure to both EtOH consumption alcohol consumption in rats selectively bred for high or low alcohol drink-
ing. Alcohol Clin Exp Res 28:385–393.
and stress, an abolishment of the observed behavioral and DeTurck KH, Vogel WH (1982) Effects of acute ethanol on plasma and
dopaminergic changes seems to occur, indicating a role of brain catecholamine levels in stressed and unstressed rats: evidence for an
stress in reducing previous chronic EtOH effects. Increase of ethanol-stress interaction. J Pharmacol Exp Ther 223:348–354.
dopamine content in the amygdala is suggested as a protec- Engleman EA, Ingraham CM, McBride WJ, Lumeng L, Murphy JM (2006)
tive factor against EtOH conditioning. Further studies are Extracellular dopamine levels are lower in the medial prefrontal cortex of
alcohol-preferring rats compared to Wistar rats. Alcohol 38:5–12.
needed to understand the molecular mechanisms responsible Fadda F, Rossetti ZL (1998) Chronic ethanol consumption: from neuroad-
for these interactions between stress and EtOH administra- aptation to neurodegeneration. Prog Neurobiol 56:385–431.
tion as this process seems complex and the mesocorticolimbic Funk D, Li Z, L^e AD (2006) Effects of environmental and pharmacological
pathway is altered in different ways depending on the stressors on c-fos and corticotropin-releasing factor mRNA in rat brain:
sequence of challenge. relationship to the reinstatement of alcohol seeking. Neuroscience
138:235–243.
Funk D, Vohra S, L^e AD (2004) Influence of stressors on the rewarding
ACKNOWLEDGMENTS effects of alcohol in Wistar rats: studies with alcohol deprivation and place
conditioning. Psychopharmacology 176:82–87.
This work was supported by “Fundacß~ ao de Amparo a George O, Le Moal M, Koob GF (2012) Allostasis and addiction: role of the
Pesquisa do Estado de Minas Gerais-FAPEMIG” (CBB- dopamine and corticotropin-releasing factor systems. Physiol Behav
106:58–64.
APQ-04545-10). The authors appreciate the technical assis- Gilpin NW (2012) Corticotropin-releasing factor (CRF) and neuropeptide Y
tance provided by Elisabete Z. P. Lepera during the measure- (NPY): effects on inhibitory transmission in central amygdala, and anxi-
ment of neurotransmitters. ety- & alcohol-related behaviors. Alcohol 46:329–337.
Gremel CM, Cunningham CL (2008) Roles of the nucleus accumbens and
amygdala in the acquisition and expression of ethanol-conditioned behav-
REFERENCES ior in mice. J Neurosci 28:1076–1084.
Gremel CM, Cunningham CL (2009) Involvement of amygdala dopamine
Attarzadeh-Yazdia G, Karimib S, Azizib P, Yazdi-Ravandib S, Hesamb S, and nucleus accumbens NMDA receptors in ethanol-seeking behavior in
Haghparastb A (2013) Inhibitory effects of forced swim stress and cortico- mice. Neuropsychopharmacol 34:1443–1453.
sterone on the acquisition but not expression of morphine-induced condi- Healey JC, Winder DG, Kash TL (2008) Chronic ethanol exposure leads to
tioned place preference: involvement of glucocorticoid receptor in the divergent control of dopaminergic synapses in distinct target regions.
basolateral amygdala Behav. Brain Res 252:339–346. Alcohol 42:179–190.
Bahi A (2013) Increased anxiety, voluntary alcohol consumption and etha- Kiianmaa K, Nurmi M, Nytinen I, Sinclair JD (1995) Effect of ethanol on
nol-induced place preference in mice following chronic psychosocial stress. extracellular dopamine in the nucleus accumbens of alcohol-preferring AA
Stress 16:441–451. and alcohol-avoiding ANA rats. Pharmacol Biochem Behav 52:
Bonassoli VT, De Oliveira W, Milani H, De Oliveira RMW (2011) Ethanol 29–34.
withdrawal activates nitric oxide-producing neurons in anxiety-related King D, Finlay JM (1997) Loss of dopamine terminals in the medial prefron-
brain areas. Alcohol 45:641–652. tal cortex increased the ratio of DOPAC to DA in tissue of the nucleus
Brenhouse HC, Andersen SL (2008) Delayed extinction and stronger rein- accumbens shell: role of stress. Brain Res 767:192–200.
statement of cocaine conditioned place preference in adolescent rats, com- Kreek MJ, Koob GF (1998) Drug dependence: stress and dysregulation of
pared to adults. Behav Neurosci 122:460–465. brain reward pathways. Drug Alcohol Depend 51:23–47.
Brielmaier J, McDonald CG, Smith RF (2012) Effects of acute stress on L€
of E, Chau PP, Stomberg R, S€ oderpalm B (2007) Ethanol-induced dopa-
acquisition of nicotine conditioned place preference in adolescent rats: a mine elevation in the rat–modulatory effects by subchronic treatment with
role for corticotropin-releasing factor 1 receptors. Psychopharmacology nicotinic drugs. Eur J Pharmacol 555:139–147.
219:73–82. Luo YX, Xue YX, Shen HW, Lu L (2013) Role of amygdala in drug mem-
Bustamante D, Quintanilla ME, Tampier L, Gonzalez-Lira V, Israel Y, ory. Neurobiol Learn Mem 105:159–173.
Herrera-Marschitz M (2008) Ethanol induces stronger dopamine release in Ma Y, Chu N, Guo C, Han J, Cui C (2007) NR2B-containing NMDA recep-
nucleus accumbens (shell) of alcohol-preferring (bibulous) than in alcohol- tor is required for morphine-but not stress-induced reinstatement. Exp
avoiding (abstainer) rats. Eur J Pharmacol 591:153–158. Neurol 203:309–319.
Camarini R, Hodge CW (2004) Ethanol pre-exposure increases ethanol self- Matsuzawa S, Suzuki T, Misawa M, Nagase H (1999) Involvement of dopa-
administration in C57BL/6J and DBA/2J mice. Pharmacol Biochem mine D(1) and D(2) receptors in the ethanol-associated place preference in
Behav 79:623–632. rats exposed to conditioned fear stress. Brain Res 835:298–305.
Cannazza G, Di Stefano A, Mosciatti B, Braghiroli D, Baraldi M, Pinnen F, McLaughlin JP, Marton-Popovici M, Chavkin C (2003) Kappa opioid
Sozio P, Benatti C, Parenti C (2005) Detection of levodopa, dopamine and receptor antagonism and prodynorphin gene disruption block stress-
its metabolites in rat striatum dialysates following peripheral induced behavioral responses. J Neurosci 23:5674–5683.
administration of L-DOPA prodrugs by mean of HPLC-EC. J Pharm Bio- Miczek KA, Mutschler NH, Van Erp M, Blank D, McInerney SC (1999)
med Anal 36:1079–1084. D-amphetamine “cue” generalizes to social defeat stress: behavioral
Carlson JN, Stevens KD (2006) Individual differences in ethanol self-admin- sensitization and attenuated accumbens dopamine. Psychopharmacology
istration following withdrawal are associated with asymmetric changes in 147:190–199.1236 MOREIRA-SILVA ET AL. Miczek KA, Yap JJ, Covington HE (2008) Social stress, therapeutics and Samson HH, Chappell A (2003) Dopaminergic involvement in medial pre- drug abuse: preclinical models of escalated and depressed intake. Pharma- frontal cortex and core of the nucleus accumbens in the regulation of etha- col Ther 120:102–128. nol self-administration: a dual-site microinjection study in the rat. Physiol Morikawa H, Morrisett RA (2010) Ethanol action on dopaminergic neurons Behav 79:581–590. in the ventral tegmental area: interaction with intrinsic ion channels and Sanchis-Segura C, Spanagel R (2006) Behavioural assessment of drug rein- neurotransmitter inputs. Int Rev Neurobiol 91:235–288. forcement and addictive features in rodents: an overview. Addict Biol Mueller D, Stewart J (2000) Cocaine-induced conditioned place preference: 11:2–38. reinstatement by priming injections of cocaine after extinction. Behav Shibasaki M, Watanabe K, Takeda K, Itoh T, Tsuyuki T, Narita M, Mori Brain Res 115:39–47. T, Suzuki T (2013) Effect of chronic ethanol treatment on l-opioid recep- Oreland S, Raudkivi K, Oreland L, Harro J, Arborelius L, Nylander I tor function, interacting proteins and morphine-induced place preference. (2011) Ethanol-induced effects on the dopamine and serotonin systems in Psychopharmacology 228:207–215. adult Wistar rats are dependent on early-life experiences. Brain Res Sidiropoulou K, Lu F-M, Fowler MA, Xiao R, Phillips C, Ozkan ED, Zhu 1405:57–68. MX, White FJ, Cooper DC (2009) Dopamine modulates an mGluR5- Pascucci T, Ventura R, Latagliata EC, Cabib S, Puglisi-Allegra S (2007) The mediated depolarization underlying prefrontal persistent activity. Nat medial prefrontal cortex determines the accumbens dopamine response to Neurosci 12:190–199. stress through the opposing influences of norepinephrine and dopamine. Sinha R (2001) How does stress increase risk of drug abuse and relapse? Cereb Cortex 17:2796–2804. Psychopharmacology 158:343–359. Paxinos G, Franklin KBJ (2001) The Mouse Brain in Stereotaxic Coordi- Strother WN, Lumeng L, Li T-K, McBride WJ (2005) Dopamine and seroto- nates. 3rd ed. Academic Press, San Diego. nin content in select brain regions of weanling and adult alcohol drinking Quebec M, Bowers WJ, Sabongui AG, Amit Z (1997) The role of ethanol rat lines. Pharmacol Biochem Behav 80:229–237. availability on stress-induced increases in ethanol consumption. Alcohol Thielen RJ, Engleman EA, Rodd ZA, Murphy JM, Lumeng L, Li T, 14:551–556. McBride WJ (2004) Ethanol drinking and deprivation alter dopaminergic Quintanilla ME, Bustamante D, Tampier L, Israel Y, Herrera-Marschitz M and serotonergic function in the nucleus accumbens of alcohol-preferring (2007) Dopamine release in the nucleus accumbens (shell) of two lines of rats. J Pharmacol Exp Ther 309:216–225. rats selectively bred to prefer or avoid ethanol. Eur J Pharmacol 573: World Health Organization (2011) Global Status Report on Alcohol and 84–92. Health. World Health Organization, Geneva. Rouge-Pont F, Le Moal M, Piazza PV (1998) Individual differences in stress- Yoshimoto K, Watanabe Y, Tanaka M, Kimura M (2012) Serotonin2C induced dopamine release in the nucleus accumbens are influenced by receptors in the nucleus accumbens are involved in enhanced alcohol- corticosterona. Eur J Neurosci 10:3903–3907. drinking behavior. Eur J Neurosci 35:1368–1380.
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