Replenishing what is Lost: Using Supplementation to Enhance Hippocampal Function in Fetal Alcohol Spectrum Disorders by Anna Ruth Patten Bachelor of Science, University of Otago, 2006 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in the Department of Biology (Neuroscience) Anna Ruth Patten, 2013 University of Victoria All rights reserved. This dissertation may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author. ii Supervisory Committee Replenishing what is lost: Using Supplementation to Enhance Hippocampal Function in Fetal Alcohol Spectrum Disorders by Anna Ruth Patten Bachelor of Science (Honours), University of Otago, Dunedin, New Zealand Supervisory Committee Dr. Brian R. Christie (Division of Medical Sciences, Department of Biology) Supervisor Dr. Leigh Anne Swayne (Division of Medical Sciences, Department of Biology) Departmental Member Dr. Francis Choy (Department of Biology) Departmental Member Dr. Robert Burke (Department of Biochemistry and Microbiology) Outside Member iii Abstract Supervisory Committee Dr. Brian R. Christie (Division of Medical Sciences, Department of Biology) Supervisor Dr. Leigh Anne Swayne (Division of Medical Sciences, Department of Biology) Departmental Member Dr. Francis Choy (Department of Biology) Departmental Member Dr. Robert Burke (Department of Biochemistry and Microbiology) Outside Member Fetal Alcohol Spectrum Disorders (FASD) are the most common cause of cognitive impairment in the United States (Sokol et al., 2003). In young school children in North America and some Western European countries, recent reports have estimated the prevalence of FASD to be as high as 2-5% (May et al., 2009). Currently there are no widely accepted treatment options for FASD, mainly due to the fact that the underlying neurological deficits that occur with prenatal ethanol exposure (PNEE) are still largely unknown. This thesis examines the long-lasting changes that occur in the hippocampus following PNEE using biochemical and electrophysiological techniques. We find that PNEE produces a reduction of the endogenous antioxidant glutathione (GSH), resulting in an increase in oxidative stress that is accompanied by long-lasting reductions in long- term potentiation (LTP) of synaptic efficacy. Interestingly, males exhibited greater deficits in synaptic plasticity than females, despite similar reductions in GSH in both sexes. By depleting GSH in control animals we determined that LTP in the DG of female animals is more resistant to changes in GSH, which may explain the sexual dichotomy observed in these studies of PNEE. Based on these findings, ethanol-exposed animals received postnatal dietary supplementation with either a precursor of GSH, N- iv Acetylcysteine (NAC) or Omega-3 fatty acids. These supplements helped to counteract the effects of PNEE and improved hippocampal function. The findings in this thesis support the hypothesis that increasing antioxidant capacity can enhance hippocampal function, which in turn may improve learning and memory in FASD, providing a therapeutic avenue for children suffering with these disorders. v Table of Contents Supervisory Committee .................................................................................................... ii Abstract ............................................................................................................................. iii Table of Contents .............................................................................................................. v List of Tables .................................................................................................................... ix List of Figures .................................................................................................................... x List of Abbreviations ....................................................................................................... xi Acknowledgments .......................................................................................................... xiii Dedication ........................................................................................................................ xv 1. Introduction ............................................................................................................... 1 1.1 Fetal Alcohol Spectrum Disorders ........................................................................ 1 1.1.1 Cognitive symptoms .......................................................................................... 2 1.1.2 Rodent models of FASD .................................................................................... 3 1.1.3 Ethanol and the developing brain ...................................................................... 4 1.1.4 Underlying mechanisms of PNEE damage ........................................................ 7 1.2 Oxidative stress and PNEE .................................................................................... 8 1.2.1 Reactive oxygen species and reactive nitrogen species ..................................... 8 1.2.2 Lipid peroxidation ............................................................................................ 12 1.2.3 Protein oxidation .............................................................................................. 13 1.2.4 Antioxidants ..................................................................................................... 14 1.2.4.1 Glutathione (GSH) .................................................................................... 15 1.2.5 Oxidative stress and antioxidants in the brain ................................................. 19 1.2.6 PNEE and oxidative stress ............................................................................... 20 1.3 Learning and memory deficits following PNEE ................................................ 23 1.3.1 The hippocampal formation ............................................................................. 23 1.3.1.1 Anatomy of the DG ................................................................................... 23 1.3.1.2 Information flow in the hippocampus ....................................................... 25 1.3.1.3 Hippocampal development ....................................................................... 27 1.3.1.4 PNEE and the hippocampus...................................................................... 28 1.3.2 The role of the hippocampus in learning and memory .................................... 29 1.3.3 PNEE and learning and memory...................................................................... 32 1.4 Synaptic plasticity in the hippocampus............................................................... 35 1.4.1 Synaptic plasticity ............................................................................................ 36 1.4.2 The NMDA receptor ........................................................................................ 37 1.4.3 Long-term potentiation .................................................................................... 38 1.4.3.1 Mechanism of LTP ................................................................................... 39 1.4.3.2 Experimental induction of LTP ................................................................ 42 1.4.3.3 LTP in the DG ........................................................................................... 43 1.4.4 LTP and learning and memory ........................................................................ 44 1.4.5 Synaptic plasticity and PNEE .......................................................................... 44 1.5 Therapeutic interventions to treat hippocampal deficits associated with FASD ....................................................................................................................................... 46 1.5.1 Omega-3 fatty acids ......................................................................................... 46 1.5.1.1 Omega-3 fatty acids and FASD ............................................................. 47 vi 1.6 Summary and objectives ...................................................................................... 48 2. General Methods ..................................................................................................... 50 2.1. Animals and breeding .......................................................................................... 50 2.1.1 Prenatal diets .................................................................................................... 51 2.1.2 Litters and weaning .......................................................................................... 52 2.2. Blood samples to determine blood alcohol concentration ................................ 52 2.3 In vivo electrophysiology ...................................................................................... 53 2.4 Preparation of samples for biochemical analysis ............................................... 55 2.5 General statistical analysis ................................................................................... 56 3. Examining the Differences in Synaptic Plasticity in Males and Females Following PNEE .............................................................................................................. 57 3.1 Background ........................................................................................................... 57 3.1.1 The effects