Long term Ethanol Consumption Changes the

Expression of Neuronal Nicotinic Receptors:

Implications for treatment strategies.
Masroor Shariff*1, Joan Holgate1, Allison Feduccia2, Maryka Quik3, Selena Bartlett1
1. Translational Research Institute at Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia
2. National institute on Alcohol Abuse and Alcoholism (NIAAA), USA
3. SRI International, San Francisco, USA

Introduction
Varenicline, a nicotinic acetylcholine receptor (nAChR) modulator that is FDA-approved for nicotine addiction, has been previously shown by our laboratory
to be effective in reducing alcohol consumption in rodents [1]. Furthermore, preliminary clinical trials using varenicline have also shown to be effective in
lowering alcohol consumption in humans [2]. The focus of our present study was to investigate possible molecular changes in nAChR expression in the
nucleus accumbens (NAcc) after long-term alcohol consumption in rodents.

Methods
Two bottle choice paradigm Results
Rodents are presented with one bottle of drug (20% ethanol) 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 short-
term (for 4 weeks) and long-term (for 12 weeks) using the above paradigm. For sur-
geries: Male Wistar rats were randomly divided into 3 groups (n=12 per group).
They were trained to consume ethanol using the intermittent access 20% ethanol
two bottle choice drinking paradigm for 10 weeks and then subjected to surgery as
described below For Binding Assay: Male Wistar rats were randomly divided into 4
groups. Two groups (n=10 per group) were trained to consume ethanol for either 4
or 12 weeks respectively using the intermittent access 20% ethanol twov bottle
choice drinking paradigm, two groups (n=10 per group) were offered access to
(A) (B) (C)
water for either 4 or 12 weeks respectively (to control for age dependent changes
Figure 1. Local varenicline infusion into the NAc (core and core-shell border) reduces voluntary etha-
in nAChR expression. nol (20%) consumption in the intermittent-access two-bottle choice paradigm. Values are expressed
as mean ethanol intake (g/kg) +/- SEM 30 mins after the start of the drinking sessions (n=8-12 per
brain region, one-way repeated measures ANOVA followed by Newman-Keuls post hoc tests. *p<0.05,
Surgery **p<0.01 compared to vehicle). Varenicline (1 and 2 μg) microinfusion significantly reduced ethanol
Animals were deeply anesthetized with 2.5% isoflurane. A 26-guage bilateral guide intake (g/kg) in the (A) NAc core and (B) NAc core-shell border, but had no effect on ethanol consump-
cannulae assembly was stereotaxically implanted using the following coordinates tion when infused into the (C) NAc shell.
from bregma with a flat skull: NAc shell (AP: +2.2, ML: +/- 0.75, DV: -7.8); NAc core
(AP: +1.6, ML: +/- 1.5, DV: -7.2); NAc core/shell border (AP: +1.6, ML: +/-1.0, DV:
-7.4). After surgery, rats returned to the two-bottle choice paradigm for at least 2
weeks. On test days, varenicline (1 or 2 μg) or vehicle was infused at a rate of 0.125
μl/min for 4 mins. Rats were placed back in home cages and drinking sessions com-
menced 10 mins after infusions. By Latin square design, subjects received every
dose (balanced order) with 1 week between treatments. The ethanol consumption
data was analyzed using one-way repeated measures ANOVA followed by
Newman-Keuls post hoc tests.

Binding assay
Rats were killed by decapitation and the brains quickly frozen in isopentane on dry
ice and stored at –80°C. Brains were sectioned (8 μm) at -20°C and thaw mounted
onto poly-L-lysine coated slides, dried and stored at –80°C until use. When re- Figure 2. Ethanol increases α4β2* nicotinic receptors in the nucleus accumbens. This experiment inves-
quired, sections were incubated at room temperature for 40 min in 50 mM Tris, pH tigated 125I-epibatidine in the presence of α-CtxMII, a nicotinic receptor antagonist that labels α6β2*
7.0, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.0 mM MgCl2 (Buffer A) plus 0.03 nM nicotinic receptors. α-CtxMII-resistant 125I-epibatidine sites are defined as α4β2* nicotinic receptors.
Nicotinic expression in the NAc of brains from ethanol drinking (4 and 12 weeks), and age-matched
125I-epibatidine (2200 Ci/mmol), a ligand that interacts with high affinity to mul- water control (4 and 12 weeks) rats were analyzed with one-way ANOVAs with Dunnett’s post hoc com-
tiple nicotinic receptors, that is, α2*-α6* nicotinic receptor subtypes (the asterisk parisons. At 4 weeks [F(2,24) = 20.95, p<0.0001] and 12 weeks [F(2,24) = 79.76, p<0.0001], there was a
indicates the presence of other subunits in the receptor complex). Nicotine (0.1 significant change in α-CtxMII-resistant 125I-epibatidine sites, with ethanol rats showing greater α4β2*
expression compared to water controls.
mM) was used to define nonspecific binding. Following incubation, sections were
washed 2 x 5 min in Buffer A at 4°C and 1 x 10 sec in ice-cold water. After drying, sec-
tions were exposed to Kodak MR film for 1-3 days with 125I-standards.

Discussion & Conclusion

nAChR’s endogenous ligand, acetylcholine, is released into the NAcc in response to consumption of various addictive compounds, such as alcohol. Var-
enicline, an nAChR α4β2 modulator, has been shown to reduce alcohol consumption in human clinical trials. Our present study has shown that alcohol
consumption mediates a higher expression of α4β2 receptors. Varenicline and hitherto unknown medications in the same drug family hold potential as
a pharamacotherapeutic agent in treatment of alcohol addiction.

Acknowledgements: This work was supported by grants from NHMRC and ARC. References: 1. STEENSLAND, P., SIMMS, J. A., HOLGATE, J., RICHARDS, J. K. & BARTLETT, S. E. 2007. Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and
seeking. Proc Natl Acad Sci U S A, 104, 12518-23. 2. LITTEN, R. Z., RYAN, M. L., FERTIG, J. B., FALK, D. E., JOHNSON, B., DUNN, K. E., GREEN, A. I., PETTINATI, H. M., CIRAULO, D. A., SARID-SEGAL, O., KAMPMAN, K., BRUNETTE, M. F., STRAIN, E. C., TIOURIRINE, N. A., RANSOM, J., SCOTT, C. & STOUT, R. 2013.
A double-blind, placebo-controlled trial assessing the efficacy of varenicline tartrate for alcohol dependence. J Addict Med, 7, 277-86.