THE EFFECT OF BINGE ALCOHOL BEHAVIOR ON Il-10 IMMUNOREACTIVITY IN THE

VENTRAL TEGMENTAL AREA AND RED NUCLEUS REGIONS OF THE MOUSE BRAIN
Shay Daji
Introduction
Binge drinking is a pattern of behavior in which excessive amounts of alcohol are
consumed in a short period of time. The National Institute on Alcohol Abuse and
Alcoholism defines a binge episode as a 2 hour or less period of time when a persons blood
alcohol concentration (BAC) reaches at least 0.08 grams percent (NIAAA, 2004). For a male
this corresponds to 5 drinks in a two-hour time period and 4 drinks for a female in the
same time period. There are many negative health effects that stem from this sort of binge
drinking behavior such as liver disease, high blood pressure, neurological damage, stroke,
and other cardiovascular diseases. Despite these negative consequences the prevalence of
adults who binge drink on a regular basis is alarmingly high. One in six US adults or over 38
million people binge drink at least 4 times a month (CDC, 2013). With such pressing need,
it is important to investigate the neurobiological mechanisms underlying the behavior of
binge alcohol consumption.
Alcohol addiction is characterized by the compulsive need to obtain alcohol, the
inability to control consumption, as well as the development of tolerance (Mello, 1973).
Current alcohol research focuses on the factors that cause alcoholics to drink excessively as
well as the factors that lead to the development of dependence (Weiss and Porrino, 2002).
Neuronal mechanisms that have reinforcing rewarding effects as well as factors that lead to
a negative affect in alcoholics have been identified and studied to try to understand why
addicts relapse. A number of studies have strongly implied a connection between dopamine
systems and the incentive motivational effects of drugs such as alcohol (Robinson and
Berridge, 1993). Studies have demonstrated that addictive drugs increase dopamine
neurotransmission by affecting the mesolimbic pathway (Di Chiara and Imperato, 1988).
The mesolimbic pathway is a dopaminergic pathway that includes the ventral tegmental
area (VTA) and the red nucleus (RN). Studies have shown that low doses of ethanol activate
and increase the firing of the high concentrations of dopamine containing neurons in the
VTA (Brodie et al., 1990, Young et al., 1992). The sensitivity to low concentrations of

ethanol shown by VTA dopaminergic neurons suggests that they are involved in the
reinforcing effects of alcohol addiction (Gessa et al., 1985).
Interestingly, neuroimmunomodulators such as LPS have been shown to depress
alcohol consumption in the VTA (Blednov et al., 2011). Cytokines are major mediators of
the neuroimmune response and, therefore, may play a role in behavioral maladaptation
within the alcohol reward/aversion system. One potential cytokine of interest present in
this pathway is interleukin-10 (Il-10). Il-10 is an anti-inflammatory cytokine with wide
ranging roles in immunoregulation and inflammatory responses. It neutralizes the
production and/or effects of multiple pro-inflammatory molecules making it critically
important in the regulation of acute and chronic inflammation (Moore et al., 2001).
Common currently used anti-inflammatory molecules, such as TNF- humanized
antibodies, influence one inflammatory mediator. In contrast, Il-10 targets multiple
inflammatory mediators potentially making it more therapeutically potent (Murray, 2006).
Altered expression levels of Il-10 have been linked to various behavioral
maladaptations. Imbalances in the expression of pro-inflammatory and anti-inflammatory
cytokines have been shown to significantly modulate mood and behavior. A 2007 study
published by the Journal of Psychiatric Research demonstrated that altered Il-10
production influences depressive-like behavior in mice (Mesquita et al., 2008). Studies
have shown that increased expression of Il-10 successfully prevented the cognitive and
behavior defects observed in a model of maternal infection. However, the study further
showed that forced overexpression of Il-10 in the absence of prenatal disease also causes
behavioral abnormalities including associative learning and spatial exploration (Meyer et
al., 2008). These studies all show that altered Il-10 levels clearly affect the mechanisms
that underlie mood and behavior.
The Drinking in the dark (DID) method utilized in this study is a procedure that
induces alchohol-preferring strains to consume enough ethanol to reach BECs greater than
100 mg/dl (Thiele and Navarro, 2014). This method is different than common voluntary
ethanol consumption studies in that it produces pharmacologically relevant BECs in a time
frame that can be set by the investigator. This pattern of heavy binge drinking more closely
resembles the human behavior attempting to be modeled, giving the DID model more face
validity than other alcohol disorder models. Another advantage of the DID model is that the

mice are not forced to ingest the alcohol as they are in a voluntary ethanol consumption
experiment. The DID method takes advantage of the increased ingestion behavior that
corresponds with the animals dark cycle, bypassing the need to have physical interaction
between the experimenter and the mouse (Thiele and Navarro, 2014).This interaction,
which is necessary for voluntary administration of ethanol, introduces confounding
variables into the study. This is particularly troubling for this study because stress and
anxiety can be independently affected by these interactions between experimenter and
mouse.
The goal of this study was to determine the effects of binge ethanol consumption on
the levels of expression of Il-10 in the ventral tegmental area and the red nucleus. In this
study an immunohistochemical assessment is used to measure Il-10 expression in the VTA
and red nucleus.
Methods
Animal Handling
Six to eight week old C57BL/6J mice were obtained from the Jackson Laboratory
(Bar Harbor, Maine). The average weight of each mouse upon arrival was between 20 25
grams. Prior to undergoing any procedures, the mice were given a week to become
accustomed to their new environment. Mice were individually housed in an AALAC
certified vivarium. The vivarium was kept at approximately 22C on a 12-hour reversed
light-dark cycle starting with lights off at 8:00am. During all experiments, mice had adlibitum food and water access unless otherwise stated. All procedures used herein adhered
to the National Institute of Health guidelines and were approved by the University of North
Carolina Institutional Animal Care and Use Committee.
Drinking in Dark
A 4-day drinking in the dark procedure was used to model binge alcohol
consumption. This procedure has been previously shown to accurately model binge alcohol
drinking behavior and consistently results in mice with a BEC of least 80mg/dl (NIAAA,
2004). During the DID cycle, the water bottles were removed from the cages of the mice
and replaced with bottles containing 20% (v/v) ethanol or 3% sucrose solution. Coinciding

with the name of the procedure, all bottles were put on 3 hours into the dark cycle. Mice
had access to the ethanol, sucrose, or water for 2 hours before the bottles were replaced
with the homecage water bottles. On the fourth day of the procedure the mice have access
to the ethanol for 4 hours instead of 2. Each 4 day DID procedure is referred to as a cycle.
The mice underwent either 1 or 3 DID cycles, with 3 days of rest in between each. After the
4-hour testing period on the final test day, blood was collected from the mice using
capillary tubes after a small incision was made on the tail. Bloods were centrifuged and the
serum was used to check the blood ethanol concentration (BEC) of each mouse using an
alcohol analyzer (Analox Instruments, Lunenberg, MA) to make sure the mice were
accurately modeling binge consumption behavior. For each mouse, BECs were run in
duplicates and averaged.
Immunohistochemistry
Upon completion of the DID procedure, the mice were anesthetically overdosed with
a 0.15 mL ip injection of a combination of ketamine (66.67 mg/mL) and xylazine (6.67
mg/mL) dissolved in 0.9% saline. They were then transcardially perfused with 1.0 M
Phosphate Buffered Saline (PBS, pH = 7.4), which was immediately followed by a perfusion
of 4% paraformaldehyde/PBS. Extracted brains were postfixed in paraformaldehyde for
twenty-four hours. They were then sectioned coronally into 40 m slices using a Leica VT
1000S vibratome (Leica Microsystems, Nussloch, Germany). The brain sections were
placed in a 1-in-4 series so that every fourth slice was used in the analysis. The slices were
placed in ethylene glycol, a cryoprotectant, at -20C to preserve the tissue
For immunohistochemical analysis, slices were put through three 5 minute washes
of Phosphate Buffered Saline (PBS; pH= 7.4) rinse off the ethylene glycol. To prevent false
positives and high background detection, the brain slices were then soaked in 0.6% 2 2 to
quench naturally occurring endogenous peroxidases. This was immediately followed by
another 3 separate rinses of PBS for 5 minutes each. Next, the slices were put in Standard
Sodium Citrate (SSC) for 1 hour at 65 for antigen retrieval. This was followed by another
round of three 5-minute PBS washes. The slices were then blocked from nonspecific
binding using 4% goat block for 30 minutes. This was followed by another 3 five-minute
PBS washes. Sections were incubated in goat Il-10 (1:1000; R&D Systems; Minneapolis,

MN) primary antibody for 48 hours at 4C. Primary antibody was washed away using 3%
rabbit serum. The sections were then incubated in biotinylated rabbit-anti goat secondary
antibody, which was followed by avidin-biotin-peroxidase complex (ABC elite kit, Vector
Labs). The complex was detected with the chromagen 3,3-diaminobenzidine
tetrahydrochloride (Polysciences; Warrington, PA). Processed sections were mounted onto
glass slides and coverslipped with SHUR/Mount (Triangle Biomedical Sciences; Durham,
NC).
Image Acquisition and Analysis
A Zeiss Axio Zoom.V16 macroscope (Oberkochen, Germany) was used to capture
high definition images of each slide at 100x magnification. Images were coded to ensure
experimenter blindness during analyses. The Zen Pro image processing system was used to
analyze the imaged brain sections. The image-processing suite digitally measures the
immuno-positive area in the region of interest on each brain slice. First, the regions of
interest were determined using the contour drawing tool to trace out the VTA and the red
nucleus. Then a threshold was set by the experimenter to differentiate between immunepositive pixels and the background. The program then determines the Il-10+ area and the
total area of the highlighted brain region. The immunoreactivity is presented as percent
area, the IL10+ area divided by the total regional area.
Statistics
All data were analyzed using Prism (GraphPad Software, Inc. La Jolla CA). One-way
ANOVA tests were conducted comparing the water and ethanol groups and the water and
sucrose groups for both the VTA and RN regions. If a significant effect of treatment was
determined, post-hoc Dunnetts Multiple Comparison tests were used to compare sucrose
or ethanol to the control water group. All data are reported as the mean standard error of
the mean and analyses considered significantly different if p < 0.05.
Results
Binge Alcohol Exposure Increased Il-10 Immunoreactivity in the Red Nucleus
For the VTA, one-way ANOVA testing showed that sucrose had no effect on the Il-10

immunoreactivity ([F = 1.020, p = 0.3750]; Figure 1A), which was expected. However,
ANOVA testing also indicated that EtOH levels had no effect on Il-10 expression for either
the 1 or 3 DID cycle groups ([F = 1.352, p = 0.2762]; Figure 1B). Although no significant
differences were found within the VTA, a one-way ANOVA of the red nucleus region
comparing mice that received water or ethanol showed a significant effect of treatment ([F
= 3.503, p = 0.0450]; Figure 2B). A post-hoc Dunnetts test indicated that animals that
underwent three cycles with ethanol had increased Il-10 immunoreactivity compared with
the water control (p<0.05). The control group within the RN comparing water and sucrose
groups did not show any significance effect of treatment ([F = 2.152, p = 0.1373]; Figure
2A). This data set demonstrates that changes in Il-10 immunoreactivity is specific to
ethanol in the red nucleus and does not generalize to rewarding solutions.
Figure 1. Il-10 immunoreactivity was visualized at 10x magnification in the VTA and can be seen as the
numerous dark spots on the tissue. Il-10, a naturally occurring cytokine in the brain was visible in both the
control and experimental groups.

B
Water v Sucrose

Water v EtOH

0.06

IL-10 Immunoreactivity
(% Area)

IL-10 Immunoreactivity
(% Area)

0.06

0.04

0.02

0.04

0.02

0.00

0.00
Water

Suc-1

Suc-3

Water

EtOH-1

EtOH-3

Figure 2. The effects of binge-like alcohol consumption on Il-10 immunoreactivity in the ventral tegmental area for
water control groups, 1-DID cycle groups, and 3-DID cycle groups. Although the figures show trending, ANOVA
testing showed no significant correlation between EtOH and Il-10 immunoreactivity.

Figure 3. Il-10 immunoreactivity was visualized in the RN regions at 10x magnification and can be seen as
the numerous dark spots on the tissue. Il-10, a naturally occurring cytokine in the brain was visible in both
the control and experimental groups.

B
Water v Sucrose

Water v EtOH

0.05

0.04

IL-10 Immunoreactivity
(% Area)

IL-10 Immunoreactivity
(% Area)

0.05

0.03

0.02

0.01

0.00

0.04

0.03

0.02

0.01

0.00

Water

Suc-1

Suc-3

Water

EtOH-1

EtOH-3

Figure 4. The effects of binge-like alcohol consumption on Il-10 immunoreactivity in the red nucleus. ANOVA
testing of Water v EtOH group showed significance for the mice that underwent the 3 DID cycles ([F = 3.503, p =
0.0450]. Testing of the water v sucrose groups showed no significance proving that this response was specific to
EtOH, not other calorie-rich reinforcers.

Table 1. Ethanol consumption and blood ethanol concentrations for both the 1-week and 3-week ethanol and
sucrose groups. Values shown are averages and standard deviations of each test group.

Treatment
Group
Ethanol 1 week

Consumption
(g/kg/day)
4.5 0.4

BEC
(mg/dL)
54.1 13.3

Ethanol 3 weeks

4.6 0.3

91.3 16.7

Sucrose 1 week

7.8 0.4

Sucrose 3 weeks

7.7 0.6

Discussion
In this study, immunohistochemical assessment was used to determine the effects of
binge ethanol consumption on the levels of expression of Il-10 in the ventral tegmental
area and in the red nucleus. Previous research has established that alcohol can alter the
expression of cytokines in the brain. Imbalances in the expression levels of these
inflammatory cytokines have been shown to significantly modulate mood and behavior in
other paradigms. Past research has shown that repeated ethanol exposure results in
persistent alterations of cytokines and significantly increases the magnitude and duration
of central and peripheral proinflammatory cytokines in the brain (Qin et al, 2008).
Concerning anti-inflammatory cytokines, it was found that ethanol depresses Il-10
immunoreactivity throughout the brain. This study goes a step further and looks at
cytokine levels in specific brain regions and how they are affected by binge behavior.
Previous studies have found that seven days after ethanol exposure, there is a significant
increase in Il-10 expression levels (Marshall et al, 2013). There was not an immediate
change in Il-10 expression after ethanol consumption, similar to what was found in the VTA
for this study. Future studies should look at other time points to see how Il-10
immunoreactivity fluctuates after a period of abstinence in the brain regions of interest.
Prior to the experiments, it was expected that binge behavior would have a
significant effect on Il-10 immunoreactivity in the VTA because alcohol is known to disrupt
various neurobiological systems within the VTA. For example, studies have found that

ethanol can increase the activity of the many dopamine-releasing neurons in the region.
Interestingly, studies have demonstrated that TNF- and Il-10 can be released by
dopaminergic neurons following stimulation of their own receptors (Besser et al., 2005).
Studies have also shown that behavioral maladaptations are linked to irregular ratios of
pro-inflammatory and anti-inflammatory molecules. Specifically, altered Il-10 expression
has been linked to behavioral maladaptations and may be involved in the reward and
reinforcement pathways that are central to alcohol addiction (Mesquita et al., 2008).
Furthermore, dopaminergic neurons have been demonstrated to have high sensitivity to
ethanol ingestion suggesting that they might be partly responsible for some of the addictive
properties of the substance (Gessa et al., 1985).
The data concludes that binge EtOH behavior in mice leads to a significant increases
in Il-10 immunoreactivity in the red nucleus. However, only the 3-DID mouse group
showed significant effects implying the greater the duration of binge behavior, the greater
the effect on Il-10 immunoreactivity. The data suggests that the ethanol is responsible for
this effect because neither of the sucrose control groups had an effect on Il-10
immunoreactivity.
One of the most commonly associated side effects of binge alcohol consumption is
impaired motor skills. Interestingly, numerous previous studies have demonstrated that
the RN is involved in motor learning, regulating muscle tension, triggering conditioned
motor responses, and postural corrections (Kuchler et al., 2002, Muir and Whishaw, 2013,
Zhang et al, 2013). Future studies should investigate whether imbalances in cytokines in
the RN are responsible for the reduced motor skills side effect that accompanies heavy
alcohol consumption. Alternate future studies should also further explore how longer
durations of EtOH binge behavior affects the immunoreactivity of Il-10. The study could
have groups that undergo even more than 3 DID cycles to see how prolonged binge
behavior effects Il-10 immunoreactivity in the VTA and RN.

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