Introduction

Varenicline, a nicotinic acetylcholine receptor (nAChR) antagonist that is FDA-

approved for nicotine addiction, has been previously shown by our laboratory to be
effective in reducing alcohol consumption in rodents. Furthermore, preliminary
clinical trials using Varenicline have also shown to be effective in lowering alcohol
consumption in humans. The focus of our present study was to investigate possible
molecular changes to nAChR expression in the nucleus accumbens (NAcc) after long-
term alcohol consumption in rodents.

Materials and Methods

Male Wistar rats were randomly divided into 7 groups. One naïve control group
(n=10) was incorporated to determine initial levels of nAChR expression. Two groups
(n=10 per group) were trained to consume ethanol for either 4 or 12 weeks
respectively using the intermittent access 20% ethanol two bottle choice drinking
paradigm, two groups (n=10 per group) were offered access to water for either 4 or
12 weeks respectively (to control for age dependent changes in nAChR expression),
and two groups were trained to consume 5% sucrose for 4 or 12 weeks (n=10) to
determine whether the changes are specific to ethanol. Finally,

Two bottle choice paradigm

Rodents were presented with one bottle of choice of drug (20% ethanol / 5%
sucrose) and another bottle of water simultaneously for a period of 24 hours on
alternating days (Mondays, Wednesdays and Fridays). On other days, rats are given
access to water only and food ad libitum. For our experiment, Wistar rats were
exposed to ethanol and sucrose short-term (for 4 weeks) and long-term (for 12
weeks) using the above paradigm.

Binding assay
At each respective time point, the rats were killed by decapitation and the brains
were quickly frozen in isopentane on dry ice and stored at –80oC. When required,
brains were sectioned (8 μm) at -20C using a Leica cryostat. The sections were then
thaw mounted onto poly-L-lysine coated slides, dried and stored at –80oC until use.
When required, sections were thawed and incubated at room temperature for 40
min in 50 mM Tris, pH 7.0, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.0 mM MgCl2
(Buffer A) plus 0.03 nM 125I-epibatidine (2200 Ci/mmol), a ligand that interacts with
high affinity to multiple nicotinic receptors, that is, α2*-α6* nicotinic receptor
subtypes (the asterisk indicates the presence of other subunits in the receptor
complex). Nicotine (0.1 mM) will be used to define nonspecific
binding. Following incubation, sections will be washed 2 x 5 min in Buffer A at 4°C
and 1 x 10 sec in ice-cold water. After drying, sections will be exposed to Kodak MR
film for 1-3 days with 125I-standards. 2

Results
Fig 1

Methods

Rats will be randomly divided into 7 groups. Two groups (n=10 per group) will be
trained
to consume ethanol for either 4 or 12 weeks respectively using the intermittent
access 20% ethanol two bottle
choice drinking paradigm, two groups (n=10 per group) will consume water for
either 4 or 12 weeks
respectively (to control for age dependent changes in nAChR expression), and one
naïve control group (n=10)
to determine initial levels of nAChR expression and two groups will consume 5%
sucrose for 4 or 12 weeks
(n=10) to determine whether the changes are specific to ethanol.