THE ROLE OF THE BETAINE/GABA TRANSPORTER IN EPILEPSY, SEIZURE SUSCEPTIBILITY, AND BEHAVIOR by Nicole M. Rowley A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pharmacology and Toxicology The University of Utah May 2011 Copyright © Nicole M. Rowley 2011 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Nicole M. Rowley has been approved by the following supervisory committee members: H. Steve White , Chair 12/10/2010 Date Approved Karen Wilcox , Member 12/10/2010 Date Approved Annette Fleckenstein , Member 12/10/2010 Date Approved J. Gregory Lamb , Member 12/10/2010 Date Approved Arne Schousboe , Member 12/10/2010 Date Approved and by William Crowley , Chair of the Department of Pharmacology and Toxicology and by Charles A. Wight, Dean of The Graduate School. ii ABSTRACT Epilepsy is a chronic neurological disorder characterized by the presence of unprovoked, recurrent, spontaneous seizures. The GABA hypothesis of epilepsy states that a decrease in GABAergic tone leads to an imbalance between excitatory and inhibitory transmission and increases seizure susceptibility. The extracellular levels of GABA are controlled by the GABA transporters (GATs), including the betaine/GABA transporter (BGT1). BGT1 transports both GABA and the organic osmolyte, betaine. Inhibitory tone and osmotic balance both play roles in controlling neuronal excitability, making BGT1 a potential target for the modulation of excitability. Recently, studies utilizing an equipotent inhibitor of GAT1 and BGT1, EF1502, and TGB, a GAT1 selective inhibitor, suggested that BGT1 inhibition is anticonvulsant. Seizures, pain, anxiety, depression, and other neuropsychiatric disorders are influenced by the GABAergic system, including modulation by GAT activity. However, little is known about the involvement of BGT1. Since BGT1 may be involved in the control of osmotic balance and extracellular GABA levels, it has the potential to be involved in the modulation of these disorders. The current dissertation was designed to test the hypothesis that BGT1 expression plays a role in epilepsy, seizure susceptibility, and other neurological disorders, including depression, anxiety, pain, and cognition, as well as motor function. iii To test the hypothesis that BGT1 expression is involved in epilepsy, a model of temporal lobe epilepsy, i.e., status epilepticus (SE), was utilized and BGT1 mRNA expression determined at several time-points following SE. BGT1 mRNA expression was compared to that of the other GATs and osmoprotective genes. From these studies, it appears that the expression of BGT1 may be involved in the pathogenesis of epilepsy. To test whether BGT1 is involved in the regulation of acute seizure susceptibility, four models of seizure threshold were utilized in BGT1 KO and WT mice (minimal tonic extension, minimal clonic, 6 Hz, and i.v.PTZ seizure threshold tests). Furthermore, the rate of corneal kindling acquisition was ascertained. No differences in seizure threshold were observed between genotypes. However, BGT1 KO mice displayed a behavioral profile distinct from that of their WT littermate controls. BGT1 KO mice have increased depressive and anxiety-like behaviors as well as a ‘manic’ phenotype. The results from this dissertation provide evidence in support of continued investigation of BGT1 in a number of distinct CNS disorders. iv TABLE OF CONTENTS ABSTRACT........................................................................................................iii LIST OF FIGURES ...........................................................................................vii LIST OF TABLES...............................................................................................ix ACKNOWLEDGEMENTS .................................................................................. x CHAPTER 1. INTRODUCTION.................................................................................... 1 Epilepsy: a clinical and preclinical challenge........................................... 1 GABA and GABA transporters ................................................................ 2 Osmoregulation and osmolyte transporters........................................... 16 The Betaine/GABA transporter.............................................................. 19 Mouse models....................................................................................... 21 Hypothesis and dissertation overview................................................... 28 References............................................................................................ 30 2. HIPPOCAMPAL BETAINE/GABA TRANSPORTER mRNA EXPRESSION IS NOT REGULATED BY INFLAMMATION OR DEHYDRATION POSTSTATUS EPILEPTICUS................................... 47 Introduction ........................................................................................... 47 Methods ................................................................................................ 52 Results.................................................................................................. 55 Discussion............................................................................................. 62 Acknowledgements............................................................................... 71 References ........................................................................................... 72 v 3. COMPARATIVE ANTICONVULSANT EFFICACY IN THE CORNEAL KINDLED MOUSE MODEL OF PARTIAL EPILEPSY: CORRELATION WITH OTHER SEIZURE AND EPILEPSY MODELS .................................................................... 79 Introduction ........................................................................................... 80 Methods ................................................................................................ 81 Results.................................................................................................. 83 Discussion............................................................................................. 84 Acknowledgements............................................................................... 85 References ........................................................................................... 85 4. PHARMACOLOGICAL PROFILE AND BGT1-SPECIFICITY OF HIT 8 ............................................................................................... 87 Introduction ........................................................................................... 87 Methods ................................................................................................ 90 Results.................................................................................................. 94 Discussion........................................................................................... 100 Acknowledgements............................................................................. 105 References ......................................................................................... 106 5. BEHAVIORAL AND IN VITRO PHENOTYPE OF MICE LACKING THE BETAINE/GABA TRANSPORTER............................. 110 Introduction ......................................................................................... 110 Methods .............................................................................................. 112 Results................................................................................................ 124 Discussion........................................................................................... 133 Acknowledgements............................................................................. 148 References ......................................................................................... 149 6. DISCUSSION...................................................................................... 155 Aim and significance ........................................................................... 155 Summary and conclusions .................................................................. 156 Speculations and future directions...................................................... 163 References ......................................................................................... 169 vi LIST OF FIGURES 1.1 The proposed regulation of BGT1 expression in the kidney .................... 22 2.1 Alterations in GAT and osmoprotective gene mRNA expression 8 h, 24 h, 72 h, 1 wk, and 4 wks postSE ............................... 57 2.2 Effect of SE (24 h postinsult) and 24 h water withdrawal on osmoprotective gene mRNA expression.................................................. 60 2.3 Effect of SE (4 weeks postinsult) and LPS (24 h postinsult) on GAT mRNA expression....................................................................... 63 2.4 Effect of SE (24 h postinsult) and LPS (24 h postinsult) on osmoprotective gene mRNA expression............................................. 65 3.1 Kindling curve .......................................................................................... 83 3.2 Paradigm correlations ............................................................................. 84 4.1 Analgesic action of Hit 8 in the formalin test ............................................ 96 4.2 Hit 8 displays antidepressant activity in the FST...................................... 98 5.1 Generation of the