ELUCIDATING THE ROLE OF ALPHA1-CONTAINING GABA(A) RECEPTORS IN ETHANOL ACTION by David Francis Werner Bachelor of Science, Ashland University, 2001 Submitted to the Graduate Faculty of School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2007 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This thesis was presented by David Francis Werner It was defended on September 12th, 2007 and approved by ----------------------------------------- ---------------------------------------- Edwin S. Levitan, Ph.D. Yan Xu, Ph.D. Professor Professor Department of Pharmacology Department of Anesthesiology Committee Chair Committee Member ---------------------------------------- ---------------------------------------- A. Paula Monaghan-Nichols, Ph.D. William R. Lariviere, Ph.D. Assistant Professor Assistant Professor Department of Neurobiology Department of Anesthesiology Committee Member Committee Member ---------------------------------------- Gregg E. Homanics, Ph.D. Associate Professor Department of Anesthesiology Major Advisor ii ------------------------------ Gregg E. Homanics, Ph.D. ELUCIDATING THE ROLE OF ALPHA1-CONTAINING GABA(A) RECEPTORS IN ETHANOL ACTION David Francis Werner University of Pittsburgh, 2007 Alcohol (ethanol) has a prominent role in society and is one of the most frequently used and abused drugs. Despite the pervasive use and abuse of ethanol, the molecular mechanisms of ethanol action remain unclear. What is well known is that ethanol intoxication elicits a range of behavioral effects. These effects most likely occur through the direct action of ethanol on targets in the central nervous system. By studying behavioral effects, the role of individual targets can be determined. The function of γ-amino butyric acid type A (GABAA) receptors is altered by ethanol, but due to multiple receptor subunits the exact role of individual GABAA receptor subunits in ethanol action is not known. This dissertation focused on the role of α1-containing GABAA receptors in ethanol action using gene knockin mice with ethanol insensitive α1 GABAA receptors. In the second chapter, knockin mice were molecularly characterized and ethanol- induced behavioral effects were assessed. α1 was found to mediate acute tolerance to the motor ataxic effects of ethanol. In the third chapter, α1 involvement in ethanol induction of neuronal activity was assessed in discrete neuroanatomic regions using the immediate early gene c-fos. Specifically, c-fos immunohistochemistry was characterized after acute ethanol exposure, after chronic ethanol exposure, and finally during the ethanol withdrawal phase. α1 was found to be involved in ethanol-mediated effects in the dentate gyrus. In the fourth chapter, α1 involvement in chronic tolerance to ethanol as well as physical dependence on ethanol was characterized. Results demonstrated that α1-GABAA-Rs play a role in the development of tolerance to chronic ethanol in motor ataxia. Intriguingly, α1 was implicated in dependence as assessed with ethanol withdrawal-related hyperexcitability. Knockin mice were more sensitive to ethanol’s withdrawal-related hyperexcitability effects. iii In summary, this dissertation further supports α1 GABAA-Rs in the mechanism of ethanol action. By chiseling away at the various components of ethanol action we are beginning to elucidate the mechanism of ethanol action. Further elucidation of the mechanism of action of α1 GABAA-Rs in tolerance and dependence could deepen our understanding of the molecular mechanisms behind alcohol abuse and alcoholism. By understanding the molecular mechanisms of ethanol, alcohol abuse may be lessened and alcoholism could potentially be cured. iv FORWARD “Never never never give up.” -Winston Churchill “Nothing in the world can take the place of persistence. Talent will not; nothing is more common than unsuccessful men with talent. Genius will not; unrewarded genius is almost a proverb. Education will not; the world is full of educated derelicts. Persistence and determination alone are omnipotent. The slogan ‘press on’ has solved and always will solve the problems of the human race.” -Calvin Coolidge v ACKNOWLEDGEMENTS I take great pride in this thesis, of which it is part of the work I have done in Dr. Gregg Homanics’ lab for the past six years of my graduate school career. The University of Pittsburgh is a tremendous environment with fantastic professors, centers and departments that are too numerous to list here. Nonetheless, I would like to express my appreciation and gratitude to all else whom are not mentioned below. First and foremost, I need to give a huge amount of appreciation for my mentor and advisor, Dr. Gregg Homanics. I feel truly lucky to be able to work with someone who has made my journey through graduate studies and onto a science career all the more enjoyable. He truly has a gift for acknowledging everyone needs different advising and being able to match his teaching style accordingly. Gregg has helped me realize my potential and always been encouraging. In allowing me to explore numerous projects and allowing me to work independently, he has helped me learn the joy of science and to always keep preservering, be dedicated, work hard, and have patience, and all else, enjoy what you do. I am endebted to my thesis committee for their guidance and support. They have kept me on track with my thesis and always been encouraging with my project. First thanks is to my committee chair, Dr. Ed Levitan. I’m in awe by his knowledge base and encourage any student to sit down and visit with him. His passion for discussing science is contagious. Next, I’d like to thank Dr. Paula Monaghan-Nichols. I’m truly inspired by her knowledge and dedication to her students and trainees. Her encouragement and support always left me feeling ready to get back to the bench for my next experiment. Next, I’d like to thank Dr. Yan Xu. His ability to see the whole picture and has been truly insightful. As a scientist, his hard-working attitude and dedication to the field is truly eye-opening. Last but definitely not least, Dr. Wiliam Lariviere. Will is truly a person dedicated to the quest for knowledge. It is with his spirit and open-mindedness that I feel everyone in the sciences should mimic. I hope that I am able to able to exemplify many of the qualities of my committee members in my own career. I also need to thank everyone in the Homanic’s Lab – past and present, and all the camaraderie within the lab. First off are Carolyn Ferguson and Ed Mallick. Their patience in teaching and always being there for anything are a lifeline for everyone who walks into W1311. They really helped make Pittsburgh feel like home the last few years. I need to give a special appreciation to one specific graduate student in Gregg’s lab as we’ve basically gone through everything together in the lab – Dev Chandra. By having each other to keeping encouraging and to keep working has made even some of the longest days seem short. To Bryan Sloat, who was always there. To Kristen Skvorak and Sangeetha Iyer as they have also made work enjoyable and helped me out when needed. Special thanks to Steve Hardy, who really took the time and to instruct me on various other projects. A special thanks goes to Andrew Swihart and Rodrigo Benevides. Their hard work and dedication with the their projects gives me that much more satisfaction watching the projects come into fruition. Lastly, I need to thank Michelle Darabante for all her help with everything. vi I also need to thank individuals from other labs and institutions. I’d like to thank Dr. Jean-Marc Fritschy for the use of his α1, α2 and α3 antibodies. I’d also like to thank to Drs. Neil Harrison and Adron Harris for their intellectual contributions and thoughtful discussions. Through my Ph.D. training I have receieved immence help and guidance from a number of people. I am endebted to Dr. Steve Phillips. His guidance and advising have helped me when I needed it the most. To Dr. Michael Zigmond. Through his training and additional advising has aided me in realizing my academic potential. To Dr. Don DeFranco whose encouragement always came at the right time. And special thanks to the rest of the Pharmacology faculty who are too numerous to mention – I am endebted to your teaching and support. To the staff of the Graduate Studies office – Dr. John Horn, Sandra Honick, Jen Walker and especially Cindy Duffy along with the rest of the staff members for always being there. I am also especially greatful to the Department of Pharmcacoogy Staff – especially Jim Kaczynski and Jen Wong, Pat Smith, Kris Fertig, Rich Smith, Holly Gergely, Jeanette McDew, Rich Smith, Linda Sorch, Virgina Reiner, Charlotte McKinnon, Melenie McCain, Georgeanna Williams, and Lisa McGreal. On a personal level, first and foremost I need to acknowledge and thank my wife, Kelly. She has been by me through my graduate career and I feel it would have been all the much harder without her, her encouragement, and just being there – especially down the finishing stretch as thoughts of finishing consumed me. She’s truly been my rock. I need to thank my parents for always being there for me, being supportive in all my endeavors. Without their support I would not be where I am today. To my sister Kate and her husband Aaron, for making Pittsburgh feel more like home with family around. Thanks to my sister Becky, her husband Howard, and my brother Andy and the rest of my family for their never-ending support. Also to my in-laws, who have made life all the more enjoyable. I’d like to thank all the friends I’ve met in graduate school and around Pittsburgh especially Marc Uy, Susan Harrison, Anne Stetler, David Levinthal, Trevor Lester, Basak Isin, Amie Eisfeld- Fenney, as well as everyone I bike and do outdoor activities with.