University of Kentucky UKnowledge Theses and Dissertations--Neuroscience Neuroscience 2013 EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11 ON THE CENTRAL DOPAMINE SYSTEM OF NORMAL AND PARKINSONIAN FISCHER 344 RATS James H. Sonne University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Sonne, James H., "EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11 ON THE CENTRAL DOPAMINE SYSTEM OF NORMAL AND PARKINSONIAN FISCHER 344 RATS" (2013). Theses and Dissertations-- Neuroscience. 5. https://uknowledge.uky.edu/neurobio_etds/5 This Doctoral Dissertation is brought to you for free and open access by the Neuroscience at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Neuroscience by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained and attached hereto needed written permission statements(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine). I hereby grant to The University of Kentucky and its agents the non-exclusive license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless a preapproved embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s dissertation including all changes required by the advisory committee. The undersigned agree to abide by the statements above. James H. Sonne, Student Dr. Don Marshall Gash, Major Professor Dr. Wayne A. Cass, Director of Graduate Studies EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11 ON THE CENTRAL DOPAMINE SYSTEM OF NORMAL AND PARKINSONIAN FISCHER 344 RATS _______________________________________ DISSERTATION _______________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By James William Hendry Sonne Lexington, Kentucky Director: Don Marshall Gash, Ph.D., Professor of Anatomy and Neurobiology Lexington, Kentucky 2013 Copyright © James William Hendry Sonne ABSTRACT OF DISSERTATION EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11 ON THE CENTRAL DOPAMINE SYSTEM OF NORMAL AND PARKINSONIAN FISCHER 344 RATS Due to the blood-brain barrier, delivery of many drugs to the brain has required intracranial surgery which is prone to complication. Here we show that Dopamine Neuron Stimulating Peptide 11 (DNSP-11), following non-invasive intranasal administration, protects dopaminergic neurons from a lesion model of Parkinson’s disease in the rat. A significant and dose-dependent increase in an index of dopamine turnover (the ratio of DOPAC to dopamine) was observed in the striatum of normal young adult Fischer 344 rats by whole-tissue neurochemistry compared to vehicle administered controls. Among animals challenged with a moderate, unilateral 6-hydroxy- dopamine (6-OHDA) lesion of the substantia nigra, those treated repeatedly with intranasally administered DNSP-11 exhibited greater numbers of tyrosine hydroxylase (TH) positive dopaminergic neuronal cell bodies in the substantia nigra and greater TH+ fiber density in the striatum when compared to animals treated intranasally with vehicle only or a scrambled version of the DNSP-11 sequence. Lesioned animals that received intranasal DNSP-11 treatment did not exhibit abnormal, apomorphine-induced rotation behavior, contrasted with animals that received only vehicle or scrambled peptide that did exhibit significantly greater rotation behavior. In addition, the endogenous expression of DNSP-11 from the pro-region of GDNF was investigated by immunohistochemistry with a custom, polyclonal antibody. Signal from the DNSP-11 antibody was found to be differentially localized from the mature GDNF protein both spatially and temporally. While DNSP-11-like immunoreactivity extensively colocalizes with GDNF immunoreactivity at post-natal day 10, the day of maximal GDNF expression, DNSP-11-like signal was found to be present in the 3 month old rat brain with signal in the substantia nigra, ventral thalamic nucleus, dentate gyrus of the hippocampus, with the strongest signal observed in the locus ceruleus where GDNF is not expressed. Results from immunoprecipitation of brain homogenate were not consistent with the synthetic, amidated 11 amino-acid rat DNSP-11 sequence. However, binding patterns in the literature of NPY, the only homologous sequence present in the CNS, do not recapitulate the immunoreactive patterns observed for the DNSP-11 signal. This study provides evidence for a potential easy-to-administer intranasal therapeutic using the DNSP-11 peptide for protection from a 6-OHDA lesion rat model of Parkinson’s disease. KEYWORDS: Parkinson’s disease, GDNF, 6-OHDA, aging, non-invasive James William Hendry Sonne May 3rd, 2013 EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11 ON THE CENTRAL DOPAMINE SYSTEM OF NORMAL AND PARKINSONIAN FISCHER 344 RATS By James William Hendry Sonne Don Marshall Gash, Ph.D. Director of Dissertation Wayne A. Cass, Ph.D. Director of Graduate Studies May 3rd, 2013 For my grandparents, Jody Tharp and Lloyd Gould Hendry ACKNOWLEDGEMENTS I am truly grateful for the help and invaluable assistance I have received, without which this work would not have been feasible. My success is truly not my own. First and foremost, I am indebted to my mentor, Don Marshall Gash, Ph.D., for his support, faith and confidence in me, as well as his friendship. Before beginning my endeavors, when I envisioned a translational research laboratory, it was the goals and motivations that he shares which were the core of my vision. It is difficult to imagine how I can take a step upward from his laboratory. My committee members have provided excellent guidance, and I appreciate them providing me with resources and taking the time out of their productive schedules to support my progress. They are Michael Bardo, Ph.D., Luke H. Bradley, Ph.D., Wayne A. Cass, Ph.D., James Geddes, Ph.D., Greg A. Gerhardt, Ph.D., Zhiming Zhang, M.D., my outside examiner John T. Slevin, M.D., and Susan Barron, Ph.D. for acting as a substitute committee member for my defense. I would like to especially thank Yi Ai, M.D., Wayne Cass, Ph.D., Richard Grondin, Ph.D., and Zhiming Zhang, M.D. They are fantastic investigators and sincere and caring people who have provided technical assistance and a deep interest in my development as a scientist. I would also like to thank the teaching faculty, especially April D. Richardson-Hatcher, Ph.D. and Bruce E. Maley, Ph.D., for helping me to become a better instructor and exposing me to the joy of helping students succeed. The members of Dr. Gash’s laboratory and his collaborating laboratories have been more than simply invaluable resources, but true friends that I know I can trust. My fellow denizens of the laboratory have been tremendous in their technical assistance and comic relief, especially Ramsey Edwards, M.B.A., Eric Forman, Hamed Haghnazar, M.P.H., April Evans, Ryan Weeks, Daisy Ramos, Ofelia Meagan Littrell, Ph.D. and Jennifer Moorehead. Most of all, Mallory J. Stenslik, M.S. has provided countless hours of laughs and teamwork. These people have kept me going during tough times. iii I am indebted to my grandparents, Jody T. and Lloyd G. Hendry, J.D., for teaching me so much about life. They instilled in me the meaning of hard work, devotion, and the importance of taking joy in a job well done, and taught me that without these things success is not possible. I believe that every act they performed was directed at making life better for their grandchildren. Their way of looking forward is what made them successful, and it has influenced my life beyond measure. They are my role models in every aspect of life, while my parents, Mary C. H. Sonne, J.D., and Kenneth G. Sonne, Jr., have provided the support to allow me to pursue my dreams wherever they lead. Above all of these, I owe my wife, Stephanie Lynn Sonne, C.P.A., everything. During my long hours and tribulations, she has stood by me, supporting me when I needed it the most. Without her love, I would be nothing. These people have been my fuel, the oil in my engine, and the tread on my tires. My successes and accomplishments are because of them, while my missteps are purely mine own. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ..................................................................................... iii LIST OF TABLES .................................................................................................ix LIST OF FIGURES ..............................................................................................