Alcoholism and the Brain Reward
Pathway
Mentor: Dr. Michael Miles
PhD Candidate: Jennifer Wolstenholme-Faison
Shauna Spanos
July 23, 2007
Goals

To Drink or not to Drink?


Determine molecular and behavioral variations
My project
Identify areas of the brain that are
activated/excited upon alcohol consumption
 Looking for verification


Ultimately:

Develop drug to prevent relapse in alcoholics
Alcoholism

Seeking alcohol
despite negative
effects




In the work place
Socially and personally
Excessive intake of
alcohol
Altered Brain Reward
Pathway

Allostasis versus
Homeostasis
http://aboutalcoholism.biz/drunk.jpg
Brain Reward Pathway






Ventral Tegmental Area (VTA)
releases dopamine
Nucleus Accumbens contains
dopamine sensitive cells
Causes feelings of pleasure
Amygdala and Hippocampus
(not shown) plays role in
memory and whether
experience is desirable
Prefrontal Cortex coordinates
all the information and
determines behavior of
individual
Pathway the same for
opiates, methamphetamines,
and nicotine
What We’re Looking For

Transcription Factors



Specifically interested in the c-Fos TF
c-FOS is an Immediate Early Gene (IEG)



Proteins that up-regulate or down-regulate the
expression of other genes
Expressed rapidly after cellular stimuli
Looking for region of brain where c-FOS most
abundant
Our Expectation

To find differences in cFOS expression between high
drinking mice and low drinking mice
My Project

20 C57BL/6NCrl Mice from
Charles Rivers Laboratory



2 choices – 10% ethanol or
water
Monitored drinking
behavior for 2 weeks



because they like to drink
Unlimited access to alcohol
Model without influences of
stress
Highly Inbred

variation in drinking behavior
http://www.uwo.ca/biology/Faculty
/singh/images/drinkingmouse.jpg
The Sacrifice

Day 14
Recorded Data
 Sacrificed Mice

Gassed mice with CO2
 Fixed brains in 4% Paraformaldehyde in
phosphate-buffered saline (PBS)

The Data
CR#8 Avg EtOH intake by mouse
16
14
12
g/kg
10
8
6
4
2
0
1
9
13
5
15 20
2
3
16 Don’t
7 14
Peek 11
mouse#
18 12
4
17
10
8
6
19
Slices for Slides

Cryostat slices brain




Temp ~ -10 to -20 C
40 μm slices
Stored in PBS
(Phosphate Based
Saline)
Dispose Olfactory
Bulbs
Staining




Stain every 6th slice
Single cell is
approximately 20 μm
deep
40 μm slices, every 6th
slice
Viewing new cell in
each slice
Analysis - Immunohistochemistry





Goat Serum for blocking
Rabbit polyclonal primary antibody attaches to Nterminus of cFOS protein
Goat anti-rabbit antibody attaches
Avidin-biotin-horseradish peroxidase complex attaches
Result is brown staining visible on the slice
NH2-------COOH
Results
Results
20x, Lateral Septum
Results
20x, Hippocampus
What Now. . .
Quantify Data
 Is there a difference in cFOS expression
between high drinkers and low drinkers?
 If there is a difference

Did it cause the drinking variation?
 Or, was it a result of the drinking variation?


Redo Experiment

Change variables
Acknowledgements

Thank you Dr. Miles and Jennifer

Thank you Nate, Sean, Aaron, Alex and Ryan
References




Wightman, Mark. New insights on the neural basis of brain
reward and alcohol drinking. UNC Bowles Center for Alcohol
Studies Center Line, Volume 11, Number 2, 2000. Retrieved
June 12, 2007 from
http://www.med.unc.edu/alcohol/cenline/11_2_1.htm.
Koob, George F. (2003). Alcoholism: Allostasis and Beyond.
Alcoholism: Clinical and Experimental Research, Vol.27, No.
2, February 2003.
Hill, Katherine G., et al. FOS expression induced by an
ethanol-paired conditioned stimulus. Pharmacology
Biochemistry and Behavior (2007), doi:10.1016/j.
Pbb.2007.04.017
Jackson Laboratory. http://jaxmice.jax.org/info/index.html