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Dopamine D1 and D2 Receptor Gene Expression

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Dopamine D1 and D2 Receptor Gene Expression DOPAMINE D1 AND D2 RECEPTOR GENE EXPRESSION AND cGMP, IP3 AND Ca2+ REGULATION IN THE BRAIN REGIONS OF HYPOXIC NEONATAL RATS: ROLE OF GLUCOSE, OXYGEN AND EPINEPHRINE SUPPLEMENTATION THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY UNDER THE FACULTY OF SCIENCE OF COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY BY BINOY JOSEPH DEPARTMENT OF BIOTECHNOLOGY COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY COCHIN - 682 022, KERALA, INDIA. NOVEMBER 2008 Dedicated To My Beloved Parents and Sisters . DECLARATION I hereby declare that the thesis entitled “Dopamine D1 and D2 Receptor Gene Expression and cGMP, IP3 and Ca2+ Regulation in the Brain Regions of Hypoxic Neonatal Rats: Role of Glucose, Oxygen and Epinephrine Supplementation” is based on the original research carried out by me at the Department of Biotechnology, Cochin University of Science and Technology, under the guidance of Dr. C. S. Paulose, Director, Centre for Neuroscience, Professor & Head, Department of Biotechnology and no part thereof has been presented for the award of any other degree, diploma, associateship or other similar titles or recognition. Cochin - 682 022 Binoy Joseph 10-11-2008 Reg. No. 2940 Department of Biotechnology Cochin University of Science and Technology ACKNOWLEDGEMENT “A fervent prayer to the Almighty Power, blessings that I need forever.” I am very much indebted to my beloved guide and mentor Prof. C. S. Paulose, Director, Centre for Neuroscience and Head, Department of Biotechnology, Cochin University of Science and Technology for his keen interest in me and brightening my ideas, dreams of research during the tenure of my Ph.D. His exemplary guidance, inspiring encouragement and timely advice will always be remembered with deep sense of gratitude. I solemnly submit my honest and humble thanks to him for bringing my dreams into reality. I offer my profound gratitude to Dr. E Vijayan, ICMR Emeritus Professor, Centre for Neuroscience, Department of Biotechnology, Cochin University of Science and Technology for his timely help and encouragement during the period of my research. Also, I wish to extend my gratitude to Dr. Babu Philip, Department of Marine Biology, Microbiology and Biochemistry, Dr. KE George and Dr. Rani Joseph, Department of Polymer Science and Rubber Technology, CUSAT for their help throughout my Ph.D. I would like to acknowledge Prof. M Chandrasekaran, Dr. Sarita G Bhat, Dr. Padma Nambisan teachers of our Department for their help and encouragement throughout my work. Dr. Elyas KK, was more a friend than a teacher. I remember the days we played badminton together and thank him. It will be a great injustice if I do not thank Ms. Savithri and Ms. Flora Crasta who were my first teachers. Without their blessings I would have been nowhere. I also wish to thank all my teachers at Jawahar Navodaya Vidyalaya, Periye, Kasaragod especially, Mr. Shambhu Nampoodiri, Mr. Joseph, Ms. Vineetha K, Mr. NP Rajan, Mr. V Sreekumar and Ms. Mohanakumari Amma who instilled in me many virtues and interest in Biology. I fondly remember my Principal Mr. George Joseph and Ms. Annamma George who showed great love towards me and without whose timely advice; my life would have taken a different turn. It gives me pleasure to thank my teachers during M.Sc. at Kasturba Medical College, Mangalore. I take this occasion to thank Prof. Narendra Nayak, Prof. Vivian D’Souza, and Dr. Benedicta D’Souza for introducing me to scientific thinking. I am nobody sans my friends. I cherish the friendship I had and take this opportunity to thank each one of them. I would like to thank Mr. Anil Johny and Ms. Siya Anil for their constant support, advice, care and love. I take this opportunity to acknowledge my dearmost friends Mr. Girish C, Mr. Harish C, Mr. Suneesh GS, Mr. Bineesh GS, Mr. Suraj K George, Mr. Pavithran P, Mr. Anoop Kumar B who have been a source of inspiration, encouragement and support. I fondly remember the great times we shared as children, as teenagers and now as grownups. I would like to thank all my Navodaya batch mates for their love and support. I especially cherish those days as a member of Yamuna House. It was very soothing to have friends like Dr. Bernard Rajeev SW and Dr. Madhu K Mani who were always there for me. I thank Dr. Remya Robinson for her love and support. I wish to thank Mr. Sreekumar MD, Mr. Anil Kumar K, Mr. Thapanjith T, Dr. Manoj Mathews and all my friends at Mathikere for their help and making many of my evenings memorable. I also thank Ms. Suni Ann Thomas, Ms. Nitha Shajeev and Ms. Merin Thankam Shaju for their friendship, love and support. I would like to thank my friend Ms. Amee Krishnakumar Nair whose constant support was there in shaping this work. I also wish to thank my friends Ms. Poppy Rajan, Ms. Jitha B and Ms. Cilla Abraham for their love and support. I sincerely acknowledge my senior colleagues Dr. Pyroja S, Dr. Mohanan VV, Dr. Balarama Kaimal S, Dr. Akash K George, Dr. Gireesh G, Dr. Finla Chathu, Dr. Reas Khan S and Dr. Savitha Balakrishnan for their immense help and encouragement. I specially thank Dr. Santhosh Thomas K who taught me a lot in life. My colleagues, more than that, my friends Ms. Anu Joseph, Ms. Pretty Mary Abraham, Mr. Jobin Mathew, Mr. Peeyush Kumar, Ms. Sherin Antony, Ms. Anju TR, Ms. Athira Babu, Mr. Jes Paul, Mr. Nandhu MS and Mr. Korah P Kuruvilla had always been there with a helping hand throughout the period of my Ph.D. work. I thank them for the affection they showered on me. I also thank Dr. Sreeja Chellappan, Dr. Soorej M Basheer, Dr. Jasmin C, Dr. Archana Kishore, Dr. Bijna B, Dr. Lailaja VP, Dr. Jissa G Krishna, Ms. Beena PS, Mr. P Karthikeyan, Mr. Cikesh PC, Mr. Sajan, Mr. Sijo Joseph, Mr. Siju M Varghese, Ms. Jina Augustine, Mr. Raghul Subin S, Ms. Helvin Vincent, Ms. Smitha S, Mr. Satheesh Kumar MK, Mr. Manzur Ali PP, Ms. Sapna K, Mr. Abraham Mathew, Mr. Ramesh Kumar S, Mr. Prasanth S, Ms. Jasmine Koshy, Ms. Anita Pinheiro, Ms. Jikku Jose, Ms. Elizabeth Mathew, Ms. Thresia Regimol TT and M.Sc. students of this Department for their friendship, help and co-operation. Mr. Ajay Abraham will be in my thoughts for all the help and happiness he created around. Words fail me in expressing my gratitude to my dear friends Dr. Ajimsha RS, Mr. Radhakrishnan R, Mr. Manu Punnen John, Dr. Jijo PU and Mr. Rajesh Krishnan for their help and support. I am also thankful to the authorities of Small Animal Breeding Centre, KAU, Mannuthy and Amrita Institute of Medical Sciences, Cochin for readily providing animals for my work. I express my sincere gratitude to all the past and present office staff in the Department of Biotechnology for their help and co-operation. I thank the authorities and staffs of Cochin University of Science and Technology for their help and co-operation. I would like to extend my sincere thanks to Cochin University of Science and Technology and Dept. of Biotechnology, Govt. of India for supporting this work with fellowship. I wish to thank Very Rev. Msgr. Mathew M Chalil who took great care in my spiritual growth. Thanks to my cousins Fr. Jose Pandiyamparampil, Fr. Jacob Pandiyamparampil, Sr. Shalini, Sr. Rosamma, Sr. Christa and Sr. Reema who had me in all their prayers. Words fail me to express my heartfelt thanks to my beloved parents. They have been selfless in giving me the very best of every thing and I express my deep gratitude for their love, sacrifice and support without which this work would not have been complete. Also, I am deeply indebted to my dearest sisters Ms. Bincy Theressa and Ms. Ancy Susanna for their love, prayers and constant motivation throughout my life. My Grandmothers had always been my well wishers. I take this moment to express my never ending love and respect towards them for their blessings. I thank my fiancée Ms. Sumy Joseph for her love and support. I also o thank Mr. MPC Nair, Ms. Valsa C Nair, Mr. Gopi CH and Ms. Rani Gopi for their love and support. There are so many others whom I may have inadvertently left out and I sincerely thank all of them for their help. Binoy Joseph CONTENTS INTRODUCTION 1 OBJECTIVES OF THE PRESENT STUDY 7 LITERATURE REVIEW 9 Impact of Hypoxia 9 Dopamine 10 Biosynthesis of dopamine 11 Dopamine reuptake and metabolism 12 Dopamine receptors 12 Dopamine D1 like family 14 Dopamine D1 receptor 15 Dopamine D5 receptors 16 Dopamine D2 like family 17 Dopamine D2 receptors 17 Dopamine D3 receptors 19 Dopamine D4 receptors 19 Second Messengers 23 Inositol 1,4,5-trisphosphate 23 Cyclic Adenosine Monophosphate (cAMP) 24 Cyclic Guanosine Monophospahte (cGMP) 27 Neurotransmitter Receptors and their Role in Hypoxia 28 Adrenergic Receptors 29 Glutamate Receptors 30 GABA Receptors 36 Adenosine Receptors 38 Serotonin Receptors 39 Acetylcholine Receptors 40 Muscarinic Receptors 41 Hypoxia and Medications 41 Neonatal Resuscitation during Hypoxia 43 Role of Glucose in Regulating Energy Demand 46 Role of ATP in Regulating Energy Demand 47 Developmental Changes due to Hypoxia 48 Effect of Hyper Oxygenation 49 Free Radical Release and Toxicity 52 Behavioural Changes Associated with Hypoxia 53 Brain Wave Activity and Seizures as a Result of Hypoxia 55 Calcium Imaging 57 MATERIALS AND METHODS Chemicals used and their sources 59 Biochemicals 59 Radiochemicals 59 Molecular Biology Chemicals 60 Animals 60 Induction of Acute Hypoxia in Neonatal Rats 60 Tissue preparation 61 Quantification of brain monoamines and their metabolites in the experimental groups of neonatal rats 61 Dopamine receptor binding
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