A ROLE FOR MPDZ IN ETHANOL WITHDRAWAL BUT NOT BINGE-LIKE ETHANOL DRINKING By Lauren C. Kruse A DISSERTATION Presented to the Department of Behavioral Neuroscience and Oregon Health & Science University School of Medicine In partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2015 School of Medicine Oregon Health & Science University ___________________________________________ CERTIFICATE OF APPROVAL ___________________________________________ This is to certify that the Ph.D. dissertation of Lauren C Kruse has been approved ___________________________________________ Advisor – Kari Buck, Ph.D. ___________________________________________ Committee Chair – John Crabbe, Ph.D. ___________________________________________ Committee Member – Robert Hitzemann, Ph.D. ___________________________________________ Committee Member – Tamara Phillips, Ph.D. ___________________________________________ Committee Member – Jodi McBride, Ph.D. ___________________________________________ Committee Member – Deb Finn, Ph.D. TABLE OF CONTENTS LIST OF FIGURES AND TABLES ....................................................................... iii LIST OF ABBREVIATIONS ................................................................................. vi CONTRIBUTIONS OF AUTHORS & ACKNOWLEDGEMENTS ........................ xii ABSTRACT ......................................................................................................... xv CHAPTER 1: Introduction General Introduction .......................................................................................... 1 Alcohol withdrawal in humans ........................................................................... 3 Alcohol withdrawal in mice ................................................................................ 4 Mapping genetic determinants of risk ................................................................ 9 Genetic mouse models .................................................................................... 10 Identification of Mpdz as a QTG ...................................................................... 14 PDZ domain proteins ....................................................................................... 17 MUPP1 ............................................................................................................ 17 MUPP1 and GABABRs .................................................................................... 22 Neural circuitry associated with EWD .............................................................. 24 The SNr and its involvement in CNS hyperexcitability .................................... 25 Neural circuitry associated with EWD in an Mpdz dependent manner ............ 30 GABABRs and EWD ........................................................................................ 31 Binge alcohol drinking in humans .................................................................... 32 Animal models of ethanol drinking ................................................................... 33 Ethanol consumption and EWD in mice .......................................................... 34 Mpdz and ethanol drinking .............................................................................. 36 GABABRs in alcohol drinking ........................................................................... 38 Central focus of dissertation ............................................................................ 40 i CHAPTER 2: Role of Mpdz expression in the caudolateral substantia nigra pars reticulata on acute ethanol withdrawal Introduction ...................................................................................................... 41 Materials and Methods .................................................................................... 43 Results ............................................................................................................. 48 Discussion ....................................................................................................... 59 CHAPTER 3: The role of GABABRs on ethanol withdrawal and MUPP1 effects Introduction ...................................................................................................... 65 Materials and Methods .................................................................................... 68 Results ............................................................................................................. 82 Discussion ..................................................................................................... 112 CHAPTER 4: The role of Mpdz on binge-like ethanol drinking Introduction .................................................................................................... 119 Materials and Methods .................................................................................. 121 Results ........................................................................................................... 132 Discussion ..................................................................................................... 156 CHAPTER 5: Discussion Overview ........................................................................................................ 163 Summary of Major Findings ........................................................................... 165 The role of Mpdz in predisposition to EWD but no binge-like ethanol drinking ................................................................................................... …168 Mpdz, the clSNr, and GABABRs associated with EWD ................................. 174 Conclusions ................................................................................................... 181 Future Directions ........................................................................................... 181 REFERENCES .................................................................................................. 184 ii LIST OF FIGURES AND TABLES CHAPTER 1: Introduction Table 1-1. HIC scale ........................................................................................ 8 Figure 1-1. MUPP1 structure and known association partners ...................... 20 Figure 1-2. Input and output projections of the SNr ........................................ 27 CHAPTER 2: Role of Mpdz expression in the caudolateral substantia nigra pars reticulata in ethanol withdrawal Table 2-1. Mpdz knockdown in NS20Y cells .................................................. 49 Figure 2-1. Bilateral Mpdz RNAi targeting in the clSNr .................................. 51 Figure 2-2. Mpdz shRNA mice demonstrated more severe EWD compared to controls ..................................................................................... 54 Figure 2-3. Mpdz shRNA and control mice did not differ in PTZ-enhanced HICs ............................................................................................. 56 Figure 2-4. Mpdz expression in the clSNr was reduced in Mpdz shRNA compared to scrambled control mice ........................................... 58 CHAPTER 3: Role of GABABRs in ethanol withdrawal and MUPP1 effects on GABABR function Figure 3-1a. Microinjection placements for intra-clSNr baclofen administration...84 Figure 3-1b. Intra-clSNr baclofen enhanced HICs in ethanol-withdrawn and control mice…………………………………………………………..…85 Figure 3-2a. Microinjection placements for intra-clSNr CGP55845 administration .............................................................................. 88 Figure 3-2b. Intra-clSNr CGP55845 attenuated EWD .................................... 89 Figure 3-3a. Microinjection placements for intra-clSNr CGP55845 administration .............................................................................. 91 iii Figure 3-3b. Intra-clSNr CGP55845 did not affect PTZ-enhanced HICs ........ 92 Figure 3-4. MUPP1 protein expression in whole brain is reduced in Mpdz+/- compared to WT mice… ............................................................... 94 Figure 3-5. Baclofen-enhanced HICs (10 mg/kg) were greater in Mpdz+/- compared to WT littermates ......................................................... 98 Figure 3-6. Baclofen-enhanced HICs were lower in MpdzTg homozygotes compared to WT mice ................................................................ 100 Figure 3-7. Mpdz+/- and WT mice did not differ in locomotor in response to baclofen ...................................................................................... 102 Figure 3-8. The response of Mpdz+/- and WT SNr GABAergic neurons to baclofen………………………………………………………………..105 Figure 3-9. [3H]CGP54626 binding in Mpdz+/- and WT whole brain .............. 108 Figure 3-10. Gene expression in Mpdz+/- and WT in (a) whole brain and (b) LCMD-clSNr .......................................................................... 111 CHAPTER 4: Role of Mpdz on binge-like ethanol drinking Table 4-1. Summary of the methods for 2 h 1B-DID……………………….…123 Table 4-2. Summary of the methods for 1B-DID…..………………...……..…127 Table 4-3. Summary of the methods for 2B-DID……………………….…..…129 Figure 4-1. Mpdz+/- and WT mice did not differ in 2 h 1B-DID ............. .……134 Figure 4-2. Baclofen administration reduced ethanol intake comparably in Mpdz+/- and WT mice (2 h 1B-DID) ............................................. 137 Figure 4-3. Baclofen (5 mg/kg) administration reduced water intake comparably in Mpdz+/- and WT mice (2 h 1B-DID) ..................... 138 Figure 4-4. Mpdz+/- and WT mice did not differ in binge-like ethanol