THE DEVELOPMENT OF ANIMAL MODELS FOR AUTISM: A GENE-ENVIRONMENT APPROACH BY PETER RANGER A thesis submitted to the Victoria University of Wellington in fulfilment of the requirements for the degree of Doctor of Philosophy Victoria University of Wellington 2016 2 Acknowledgements I would like to thank all the friends, family members, and people from the lab who helped me along the way. Special thanks must go to Bart Ellenbroek – the best supervisor I could ever have imagined. “When nothing seems to help, I go and look at a stonecutter hammering away at his rock perhaps a hundred times without as much as a crack showing in it. Yet at the hundred and first blow it will split in two, and I know it was not that blow that did it, but all that had gone before.” – Jacob August Riis. 3 Contents Acknowledgements .......................................................................................................... 3 List of Abbreviations ........................................................................................................ 8 Abstract .......................................................................................................................... 10 CHAPTER 1: GENERAL INTRODUCTION .................................................................. 11 Autism ............................................................................................................................ 11 Genetics and the 5-HTTLPR .......................................................................................... 15 Serotonin ........................................................................................................................ 15 Serotonin Transporter ..................................................................................................... 16 The 5-HTTLPR .............................................................................................................. 16 The 5-HTTLPR and ASD .............................................................................................. 17 Explanations for Heterogeneity ...................................................................................... 18 Animal Research and SERT ........................................................................................... 21 Other Genetic Candidates............................................................................................... 22 The Environment and ASD ............................................................................................ 23 Maternal Infection and the Immune System .................................................................. 23 Maternal Infection and Human Research ....................................................................... 24 Maternal Infection and Animal Research....................................................................... 26 Poly (I:C) and LPS ......................................................................................................... 26 Maternal Immune Activation Mechanism...................................................................... 28 Prenatal Exposure to Valproate ...................................................................................... 29 Prenatal VPA as an Animal Model for ASD.................................................................. 30 Experimental Variables in VPA Models ........................................................................ 33 Mechanisms of Action ................................................................................................... 35 Histone Deacetylase Inhibition ...................................................................................... 35 Reactive Oxygen Species and Oxidative Stress ............................................................. 37 Animal Models ............................................................................................................... 40 4 Modelling ASD in Rats .................................................................................................. 41 Combining the Risk Factors ........................................................................................... 42 Objectives and Hypotheses ............................................................................................ 43 CHAPTER 2: MATERNAL IMMUNE ACTIVATION AND SOCIAL BEHAVIOUR . 45 Animals ....................................................................................................................... 46 Breeding ...................................................................................................................... 46 Data analysis ............................................................................................................... 47 Experiment I (EXP-I): Social approach and novelty seeking in juveniles ..................... 47 Method ........................................................................................................................ 47 Results ........................................................................................................................ 49 Experiment II (EXP-II): Social motivation in adolescents ............................................ 53 Method ........................................................................................................................ 53 Results ........................................................................................................................ 55 Experiment III (EXP-III): Social approach and novelty seeking in adults .................... 57 Method ........................................................................................................................ 57 Results ........................................................................................................................ 57 Experiment IV (EXP-IV): Olfactory-choice paradigm in adults ................................... 61 Method ........................................................................................................................ 61 Results ........................................................................................................................ 62 Discussion ...................................................................................................................... 65 CHAPTER 3: PRENATAL VALPROATE AND BEHAVIOUR .................................... 72 Animals ....................................................................................................................... 73 Breeding ...................................................................................................................... 73 Data analysis ............................................................................................................... 74 Experiment V (EXP-V): Ultrasonic vocalisations in pups............................................. 74 Method ........................................................................................................................ 75 Results ........................................................................................................................ 76 5 Experiment VI (EXP-VI): Social interaction following isolation in adults ................... 78 Method ........................................................................................................................ 78 Results ........................................................................................................................ 79 Experiment VII (EXP-VII): Scent marking in adults..................................................... 83 Methods ...................................................................................................................... 83 Results ........................................................................................................................ 85 Experiment VIII (EXP-VIII): Social approach and novelty seeking in adolescents ...... 87 Method ........................................................................................................................ 87 Results ........................................................................................................................ 88 Experiment IX (EXP-IX): Repetitive behaviour in juveniles ........................................ 91 Methods ...................................................................................................................... 91 Results ........................................................................................................................ 92 Discussion ...................................................................................................................... 95 CHAPTER 4: THE BIOCHEMICAL AND IMMUNOLOGICAL CONSEQUENCES OF PRENATAL EXPOSURE TO VALPROATE ................................................................ 103 BDNF........................................................................................................................ 103 IL-6 ........................................................................................................................... 105 Breeding.................................................................................................................... 106 Experiment X (EXP-X): BDNF mRNA expression in the hippocampus and frontal lobe ...................................................................................................................................... 107 Methods ...................................................................................................................