1. Alcoholism and the Brain Reward Pathway Mentor: Dr. Michael Miles PhD Candidate: Jennifer Wolstenholme-Faison Shauna Spanos July 23, 2007

2. 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

3. 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

4. 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

5. 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

6. 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

7. The Sacrifice Day 14 Recorded Data Sacrificed Mice Gassed mice with CO 2 Fixed brains in 4% Paraformaldehyde in phosphate-buffered saline (PBS)

8. The Data Don’t Peek

9. Slices for Slides Cryostat slices brain Temp ~ -10 to -20  C 40 μm slices Stored in PBS (Phosphate Based Saline) Dispose Olfactory Bulbs

10. Staining Stain every 6 th slice Single cell is approximately 20 μm deep 40 μm slices, every 6 th slice Viewing new cell in each slice

11. 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 NH 2 -------COOH

12. Results

13. 20x, Lateral Septum

14. Results 20x, Hippocampus

15. 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

16. Acknowledgements Thank you Dr. Miles and Jennifer Thank you Nate, Sean, Aaron, Alex and Ryan

17. 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. 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