ADRENERGIC AND GLUTAMERGIC RECEPTOR GENE EXPRESSION AND THEIR FUNCTIONAL REGULATION IN THE CEREBRAL CORTEX OF HYPOXIA INDUCED NEONATAL RATS: ROLE OF OXYGEN, EPINEPHRINE AND GLUCOSE SUPPLEMENTATION THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY UNDER THE FACtJLTY Of. SCIENCE OF COCHlN llNIVERSITY OF SCIENCE AND TECHNOLOGY BY FINLA CHATHU DEPARTMENT OF BIOTECHNOLOGY COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY COCHIN - 682 022. KERALA, INDIA. FEBRUARY 2007 DEPARTMENT OF BIOTECHNOLOGY COCHIN UNIVERSITY Of' SCIENCE AND TECHNOLOGY COCHIf- 6IZ GZJ, ItID&A ....: 04I4-U7ut7 tol. oe-Ul1CH (I.) IIrlb: M41V 114ZJ r-t: It tell. et K In, pet ........OD... fa: " ......157U61. J577595 DR. C.S._LOSE HEAD DIRECTOR, CENTRE FOR NWROSCIEHCE This is 10 certify that the thesis entitled "ADRINERGIC AND GLUT.uR.RGIC UCEI'TOR GRNE IXPRl:SSION AND THEIR J'UNCflONAL RlGULA.'nON IN mE Cl.UBRAL coarex OF HYPOXIA INDUCltD NWNATAL RATS: ROLE O' OXYGEN. UINIPHRlNE AND GUJCOSE SUPPLEMJ:NTATION'" is a bonafide record of the resean::h work carried out by Ms. Fial. Cbathu.. under my guidance and supervision in the Department of Biotec:bnology. Cochin Uaiversity of Science and Tecb.nology and that DO pan thereof has been presented for theaward ofany other degree. Cochin 682 022 2-02-07 Or. C.S. PAUlO SE M se. Ph.D. FIM$A. FGSI DIRECTOR, CE NTRF t · <;r-IENCE HEAD. DEFT. C ..... LOGY Cochin Unlvers .yor ~ ... Technology Cochin·682 012, Ke rclc, India DECLARATION I hereby declare that this thesis entitled "ADRENERGIC AND GLUTAMERGIC RECEPTOR GENE EXPRESSION AND THEIR FUNCTIONAL REGULATION IN THE CEREBRAL CORTEX OF HYPOXIA INDUCED NEONATAL RATS: ROLE OF OXYGEN, EPINEPHRINE AND GLUCOSE 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 and supervision of Dr. C. S. Paulose, Director. Centre for Neuroscience, Reader & 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 FINLA CHATHU. Date: February 2. 2007. Reg. No. 2858 Acknowledgement I wish to express my sincere heartfelt gratitude and indebtedness to my supervising guide Dr. C. S. Paulose, Director, Centre for Neuroscience, Reader and Head, Department ofBiotechnology for his sustained interest, excellent guidance and motivation throughout the course ofthis work. His encouragement and timely advice will always be remembered. I am thankful to Pro! M Chandrasekaran, former Head, Department of Biotechnology, for all his help and encouragement and providing me the necessary facilities to pursue my Ph.D. I thank Dr. E. Vijayan, ICMR Emeritus Professor, Dr. Sarita G. Bhat, Dr. Padma Nambisan and Dr. Elyas KK, lecturers of this Department for their help and encouragement throughout my work. I take this opportunity to thank Pro! V. Unnikrishnan Nair, Dean, Faculty of Science, Cochin University of Science & Technology for his encouragement and support to me. I would like to extend my profound thanks to Dr. Babu Philip, Department of Marine Biology, Microbiology and Biochemistry for his valuable suggestions and encouragement I wish to express my sincere thanks to my senior colleagues Dr. Ani Das V. Dr. Pyroja. S, Dr. Eswar Shankar P.N, Dr. Renuka T.R, Dr. Mohanan V. V. Dr. Balarama Kaimal. S, Dr. Santbosh Thomas K., Mr. Akash K. George and Mr. Gireesh G.for their immense help and encouragement. My dearest friends and colleagues, Mr. Reas Khan. S, Ms. Remya Robinson, Ms. Savitha Balakrishnan, Mr. Binoy Joseph, Ms. Anu Joseph, M\·. Amee Krishnakumar, Ms. Pretty Mary Abraham, Mr. Jobin Mathew, Mr. Peeyush and Ms. Sherine Binoy were always been there with a helping hand throughout the period ofmy work. I thank them for all their affection. friendship and help which I shall always cherish. I would like to thank Mr. Soorej M Basheer, Mr. Madhu K. M, Ms. Archana Kishore, Mr. Bernard Rajeev S W. Mrs. Lailaja V. P, Mrs. Bijna; Mrs. Manjusha. Ms. Jissa, Mr. Siju M Varghese, Ms. Jina Augustine, Mrs. Swapna Kesav and Ms. Beena, research scholars ofthe Department ofBiotechnology. for their kind help and co-operation during the period (llmy Ph. D. I also thank all the M. Se. students in the Department ofBiotechnologyfor their affection, help and co-operation. I thank all the non-teaching office staffofour department-present and past­ for the care and help extended to me. I thank the authorities of Amrita Institute of Medical Science, Cochin [or providing animalsfor my research work. My special thanks to the authorities and staffofCochin University ofScience and Technologyfor their help and co-operation. 1 am grateful to Cochin University ofScience and Technology for providing me fellowshipfor the research. I thank all the teachers of my School days and graduation for their kind leading and showing light in the path of my life and motivating me to the field of science. I am wordless in expressing my deep gratitude to my beloved parents and my dear brother, Mr. Finu V Chathu, for their constant encouragement, affection and support. I thank my family for their blessings and prayers that made my dreams come true. There are so many others whom I may have inadvertently left out and I sincerely thank all ofthem/or their help andprayers. Thanksfor being with me. (Finla Chathu) Dedicated To My Beloved Parents and Dearest Brother . ABBREVIATIONS USED IN THE TEXT AADC Aromatic amino acid decarboxylase 5-HIAA 5-Hydroxy indole - 3 acetic acid 5-HT 5-Hydroxy tryptamine 5-HTP 5-Hydroxy tryptophan ACh Acety lcholine AMPA Amino-3-hydroxy-5-methysoxazole-4-propionic acid AR Adrenergic receptor ATP Adenosine triphosphate Bmax Maximal binding bpm Beats per minute cAMP cyclic Adenosine monophosphate CAT Catalase CNS Central nervous system COPD Chronic obstructive pulmonary disease CP Cerebral palsy CPR Cardia pulmonary resuscitation CT Threshold cycle DA Dopamine DEPC Diethyl pyro carbonate DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate DTT Dithiothreitol ECD Electrochernical detector EDTA Ethylene diamine tetra acetic acid EEG Electro encephalogram EPI Epinephrine GABA Gamma aminobutyric acid GDB Glutamate dehydrogenase Glu Glutamate GOD Glucose oxidase GPCRs G protein-coupled receptors GPx Gluthatione peroxidase H/H Hypercapnic hypoxia H202 Hydrogen peroxide HBOT Hyperbaric oxygen therapy HI Hypoxic-ischemia HIE Hypoxic-ischemic encephalopathy HIF Hypoxia-induced factor HPLC High performance liquid chromatography HVA Homovanillic acid i.p Intra-peritone ally ICU Intensive care units IP:, Inositol triphosphate KA Kainate Kd Dissociation constant Michaelis Constant mRNA Messenger Ribonucleic acid NADH Nicotinamide adenine dinucleotide (reduced form) NADPH Nicotinamide adenine dinucleotide phosphate, reduced form NBM Nucleus basalis rnagnocellularis NE Norepinephrine NICU Neonatal intensive care units NMDA N-methyl-D-aspartic acid NO Nitric oxide O.D Optical density P Level ofsignificance PEA Pulseless electrical activity PFC Prefrontal cortical PKA Protein kinase A p0 2 Partial pressure ofOxygen POD Peroxidase PTX Pertussis toxin PVL Periventricular leukomalacia RNA Ribonucleic acid ROS Reactive oxygen species RT-PCR Reverse-transcription-polymerase chain reaction S.E.M. Standard error ofmean SIDS Sudden infant death syndrome SOD Superoxide dismutase Maximal Velocity CONTENTS INTRODUCTION Objectives 9 LITERATURE REVIEW 11 Impact of Hypoxia 11 Functional Neuropharmacology ofthe Sympathetic Nervous System 12 Neurotransmitters and Their Role in Hypoxia 14 Adrenergic Receptors 15 a-Adrenergic Receptors 15 ClI_ Adrenergic Receptors 16 arAdrenergic Receptors 16 f}-Adrenergic Receptors 18 Role ofCatecholamines in Macronutrient Metabolism 19 Hypoxia and Adrenergic Neurotransmitters 21 Glutamate Receptors 23 GABA Receptors 25 Dopamine Receptors 26 Adenosine Receptors 28 Serotonin Receptors 31 Acetylcholine Receptors 31 Muscarinic Receptors 32 Hypoxia & Medications 33 Neonatal Resuscitation During Hypoxia 35 Role ofGlueose in Regulating Energy Demand 39 Role of ATP in Regulating Energy Demand 40 Developmental Changes due to Hypoxia 42 Effect of Hyper Oxygenation 42 Free Radical Release and Toxicity 45 Behavioural Changes Associated With Hypoxia 47 Brain Wave Activity and Seizures as a Result of Hypoxia 49 MATERIALS AND METHODS 53 Biochemicals and Their Sources 53 Chemicals used in the Study 53 Biochemicals 53 Radiochemicals 53 Animals 53 Induction of Acute Hypoxia in Neonatal Rats 54 Tissue preparation 54 Estimation of Blood Glucose 55 Quantification of neurotransmitters and its metabolites in the cerebral cortex and adrenals of experimental groups of neonatal rats. 56 Glutamate Dehydrogenase Assay 57 Acetylcholine Esterase Assay 58 Adrenergic Receptor Binding Studies using 13 HI radioligands 58 13 HlEpinephrine Binding Studies 59 13HIYohimbine Binding Studies 59 13 HIPropranolol Binding Studies 60 Glutamate Receptor Binding Assay using 13 HI Radioligand 60 Protein Determination 60 Analysis of the Receptor Binding Data 61 Linear Regression Analysis for Scatchard Plots 6t Nonlinear Regression Analysis for Displacement Curve 61 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 62 Isolation of RNA 62 cDNA Synthesis 63 Quantitative Real-Time Assay 63 Assay of cyclic Adenosine Monophosphate (cAMP) 64 Tissue preparation for cAMP assay 65 cAMP Assay Protocol 65 EEG Analysis 66 Behavioural Study 67 Elevated Plus-Maze 67 Open field test