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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

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alcoholism and the brain reward pathway

Alcoholism and the Brain Reward Pathway

Mentor: Dr. Michael Miles

PhD Candidate: Jennifer Wolstenholme-Faison

Shauna Spanos

July 23, 2007

goals
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
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
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
What We’re Looking For
  • Transcription Factors
    • Proteins that up-regulate or down-regulate the expression of other genes
  • Specifically interested in the c-Fos TF
  • c-FOS is an Immediate Early Gene (IEG)
    • 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
My Project
  • 20 C57BL/6NCrl Mice from Charles Rivers Laboratory
    • because they like to drink
  • 2 choices – 10% ethanol or water
  • Monitored drinking behavior for 2 weeks
    • 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
The Sacrifice
  • Day 14
    • Recorded Data
    • Sacrificed Mice
  • Gassed mice with CO2
  • Fixed brains in 4% Paraformaldehyde in phosphate-buffered saline (PBS)
the data
The Data

Don’t Peek

slices for slides
Slices for Slides
  • Cryostat slices brain
    • Temp ~ -10 to -20 C
  • 40 μm slices
  • Stored in PBS (Phosphate Based Saline)
  • Dispose Olfactory Bulbs
staining
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
Analysis - Immunohistochemistry
  • Goat Serum for blocking
  • Rabbit polyclonal primary antibody attaches to N-terminus of cFOS protein
  • Goat anti-rabbit antibody attaches
  • Avidin-biotin-horseradish peroxidase complex attaches
  • Result is brown staining visible on the slice

NH2-------COOH

results13
Results

20x, Lateral Septum

results14
Results

20x, Hippocampus

what now
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
Acknowledgements
  • Thank you Dr. Miles and Jennifer
    • Thank you Nate, Sean, Aaron, Alex and Ryan
references
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