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Evaluating the effects of variable NR2E1 levels on gene expression, behaviour, and neural and ocular.. Wong, Bibiana (Ka Yan) 2009

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    Evaluating the effects of variable NR2E1 levels on gene expression, behaviour, and neural and ocular development by Bibiana Ka Yan Wong B.Sc., University of British Columbia, 2001   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Medical Genetics)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   December 2009   © Bibiana Ka Yan Wong, 2009   ii Abstract Nuclear receptor 2E1 (Nr2e1) is expressed in the developing and adult brain and eye, and controls proliferation and differentiation of neural and retinal stem/progenitor cells by regulating genes important in these cellular processes. The Simpson laboratory discovered and characterized a spontaneous deletion of mouse Nr2e1 (the fierce allele, frc) and demonstrated the functional equivalence of human and mouse NR2E1 when the behavioural and neuroanatomical phenotypes of Nr2e1frc/frc mutants were rescued by introducing human NR2E1. NR2E1 has recently been implicated in human psychiatric disorders and variants in NR2E1 were identified in patients with brain and behavioural abnormalities, including bipolar I disorder (BPI). Although NR2E1 had been implicated in BPI, the validity of Nr2e1frc/frc mice to model BPI has not yet been tested. In anticipation of subtle behavioural phenotypes, the hypothesis that dark-phase testing affects the outcome of high-throughput behavioural tests was tested. We demonstrated that dark-phase testing improved discrimination between genetically distinct inbred mouse strains. This result was integrated into the experimental design for evaluating Nr2e1frc/frc mice as a model for BPI by behavioural measures and lithium treatment. Nr2e1frc/frc mice exhibited behavioural traits used to model BPI in rodents, including hyperactivity and learning deficits; however, adult Nr2e1frc/frc mice were resistant to the effects of lithium treatment, and therefore our results did not provide support for Nr2e1frc/frc mice as an appropriate pharmacological model for BPI. Since the nature of patient variants in NR2E1 is likely regulatory, resulting in transcriptional alterations, and the effects of variable levels of Nr2e1 are currently unknown, I tested the hypothesis that variable Nr2e1 levels will affect gene expression and neurological and ocular development. Mice overexpressing Nr2e1 showed alterations in transcription   iii levels of key target genes in both the brain and the eye, significant increase in neural stem/progenitor cell proliferation in the subventricular zone of the adult brain, and severe eye abnormalities. Gene expression changes in Gfap, Gsk3β, Pax6, and Nr2e3 suggest a role for Nr2e1 in genetic pathways important in psychiatric and eye disorders, including BP, Alzheimer Disease, cancer, Aniridia, and enhanced S-cone syndrome. Collectively, these results justify the further investigation of NR2E1 in these human disorders.    iv Table of contents Abstract.................................................................................................................................... ii Table of contents .................................................................................................................... iv List of tables.......................................................................................................................... viii List of figures.......................................................................................................................... ix List of abbreviations .............................................................................................................. xi Acknowledgments ................................................................................................................ xiii Dedication ...............................................................................................................................xv Co-authorship statement ..................................................................................................... xvi Chapter 2............................................................................................................................ xvi Chapter 3............................................................................................................................ xvi Chapter 4............................................................................................................................ xvi Chapter 1: General introduction............................................................................................1 1.1 Nuclear receptor superfamily...........................................................................................1 1.2 The importance of Nr2e1 in neurodevelopment and cell cycle regulation......................2 1.2.1 Structure and interspecies homology of Nr2e1 .......................................................2 1.2.2 Expression pattern of Nr2e1 in the developing and adult brain..............................3 1.2.3 Targeted and spontaneous deletions of Nr2e1 in mice ...........................................4 1.2.4 Role of Nr2e1 in neurodevelopment and neurogenesis...........................................5 1.2.5 Genetic and protein interactions of Nr2e1 in the brain ...........................................6 1.3 The lack of Nr2e1 results in adult neuroanatomical and behavioural abnormalities.......7 1.3.1 Neuroanatomical anomalies ....................................................................................7 1.3.2 Behavioural abnormalities.......................................................................................8 1.4 The importance of Nr2e1 in eye development.................................................................9 1.4.1 Adult eye anomalies in Nr2e1-null mice...............................................................10 1.4.2 Genetic and protein interactions of Nr2e1 in the eye............................................12 1.4.3 The function of Nr2e3, a relative of Nr2e1, in the eye .........................................12 1.5 The emerging role of Nr2e1 in cancer ...........................................................................13 1.6 NR2E1: A candidate gene for bipolar disorder .............................................................13 1.6.1 Genetics of bipolar disoder ...................................................................................13 1.6.2 Genetic support for NR2E1 in brain disorders ......................................................15 1.6.3 Role of neural stem/progenitor cells in brain disorders ........................................17 1.6.4 Different mouse “models” of bipolar disorder ......................................................17 1.7 Thesis objectives............................................................................................................20 1.7.1 General hypothesis and sub-hypotheses................................................................20 1.7.2 Evaluation of Nr2e1frc/frc as a model for bipolar I disorder ...................................21   v 1.7.3 Evaluation of overexpression of Nr2e1 in mice....................................................21 1.8 References......................................................................................................................23 Chapter 2: The dark phase improves genetic discrimination for some high throughput mouse behavioural phenotyping...........................................................................................40 2.1 Introduction....................................................................................................................40 2.2 Methods and materials ...................................................................................................43 2.2.1 Mouse facility........................................................................................................43 2.2.2 Mice.......................................................................................................................44 2.2.3 Testing procedures ................................................................................................44 2.2.4 Home cage activity................................................................................................46 2.2.5 Open-field test .......................................................................................................47 2.2.6 SHIRPA primary screen........................................................................................48 2.2.7 Social interaction test ............................................................................................48 2.2.8 Social recognition test ...........................................................................................49 2.2.9 Rotarod test ...........................................................................................................49 2.2.10 Tail-flick test .......................................................................................................50 2.2.11 Hot-plate test .......................................................................................................50 2.2.12 Statistical analysis ...............................................................................................51 2.3 Results............................................................................................................................52 2.3.1 Home cage activity showed expected diurnal patterns in response to reverse L/D cycle .......................................................................................................................52 2.3.2 Open-field test discriminates better in the dark phase ..........................................53 2.3.3 SHIRPA primary screen discriminates better in the dark phase ...........................56 2.3.4 Social interaction test is not improved by the dark phase .....................................58 2.3.5 Social Recognition test is not improved by the dark phase...................................60 2.3.6 The rotarod test discriminates better in the dark phase.........................................60 2.3.7 The tail-flick test discriminates only in the light phase ........................................62 2.3.8 The hot-plate test does not discriminate better in the dark phase .........................63 2.4 Discussion......................................................................................................................64 2.5 References......................................................................................................................67 Chapter 3: Hyperactivity, startle reactivity and cell-proliferation deficits are lithium resistant in Nr2e1frc/frc mice ...................................................................................................71 3.1 Introduction....................................................................................................................71 3.2 Methods and materials ...................................................................................................73 3.2.1 Mice.......................................................................................................................73 3.2.2 Genotyping ............................................................................................................73 3.2.3 Testing procedure ..................................................................................................74 3.2.4 Pup body weight and milk consumption ...............................................................74 3.2.5 Pup open field activity...........................................................................................75 3.2.6 Home cage activity................................................................................................75 3.2.7 Open field activity and habituation .......................................................................75 3.2.8 Tail suspension......................................................................................................76 3.2.9 Hot plate and tail flick...........................................................................................76 3.2.10 Auditory brainstem response...............................................................................77 3.2.11 Passive avoidance................................................................................................77   vi 3.2.12 Acoustic startle reactivity....................................................................................77 3.2.13 Lithium administration and testing procedure ....................................................78 3.2.14 Serum analysis.....................................................................................................79 3.2.15 Brain harvesting and immunohistochemistry......................................................79 3.2.16 Statistical analysis ...............................................................................................80 3.3 Results............................................................................................................................80 3.3.1 Young Nr2e1frc/frc mice show early hyperactivity .................................................80 3.3.2 Adult Nr2e1frc/frc mice show hyperactivity in three behavioural tests...................83 3.3.3 Nr2e1frc/frc mice showed a deficit in two different learning and memory tasks ....86 3.3.4 Nr2e1frc/frc mice lack startle reactivity ...................................................................90 3.3.5 Nr2e1frc/frc hyperactivity resistant to lithium treatment .........................................92 3.3.6 Nr2e1frc/frc open field habituation deficit is unaffected by lithium treatment........96 3.3.7 Lithium-treated Nr2e1frc/frc mice show no improvement in startle reactivity........96 3.3.8 Cell proliferation in subventricular zone and dentate gyrus is unaffected by lithium treatment....................................................................................................98 3.4 Discussion....................................................................................................................100 3.5 References....................................................................................................................105 Chapter 4: Increased Nr2e1 transcription affects gene regulation, cell proliferation, and brain and eye morphology in mice .....................................................................................115 4.1 Introduction..................................................................................................................115 4.2 Methods and materials .................................................................................................117 4.2.1 Mice.....................................................................................................................117 4.2.2 Genotyping ..........................................................................................................118 4.2.3 Interphase and metaphase FISH..........................................................................119 4.2.4 Quantitative reverse transcriptase PCR...............................................................119 4.2.5 Brain and eye harvesting .....................................................................................120 4.2.6 Immunofluorescence ...........................................................................................121 4.2.7 Statistical analysis ...............................................................................................122 4.3 Results..........................................................................................................................122 4.3.1 High copy integration of B6-pacEMS into mouse genome ................................122 4.3.2 B6-bacEMS4A mice show increased Nr2e1 transcription..................................124 4.3.3 PAC mice show overexpression of human NR2E1 .............................................126 4.3.4 Characterization of gross brain and eye morphology of four transgenic strains.128 4.3.5 B6-bacEMS4A mice show altered transcription level of Gfap and Gsk3β.........131 4.3.6 Cell proliferation in the subventricular zone was altered in B6-bacEMS4A......134 4.3.7 B6-bacEMS4A eyes showed thinning and disorganization of retinal cell layers136 4.3.8 Gene transcription is altered in B6-bacEMS4A eyes..........................................139 4.4 Discussion....................................................................................................................141 4.5 References....................................................................................................................148 Chapter 5: General discussion............................................................................................156 5.1 Overview of major findings.........................................................................................156 5.2 Considerations for modeling behavioural traits of human disease in mice .................158 5.2.1 Dark-phase behavioural testing can improve detection of behavioural differences in genetically distinct mice ..................................................................................158 5.2.2 The power of dissecting complex disorders into endophenotypes......................159   vii 5.3 Nr2e1frc/frc mice – an appropriate model for bipolar disorder? ....................................159 5.3.1 Nr2e1frc/frc mice show phenotypes observed in bipolar disorder.........................160 5.3.2 New direction stemming from inconsistencies in Nr2e1-null behavioural abnormalities........................................................................................................162 5.4 Overexpression of Nr2e1 illuminates important genetic pathways .............................163 5.5 Future directions: NR2E1, bipolar disorder, and eye disorders...................................165 5.5.1 Testing bipolar disorder variants in mice............................................................165 5.5.2 Identifying NR2E1 variants in human eye disorders...........................................166 5.5.3 The use of genetic crosses to identify novel pathways .......................................166 5.6 Conclusion ...................................................................................................................168 5.6 References....................................................................................................................169 Appendix A: Deletion of the nuclear receptor Nr2e1 impairs synaptic plasticity and dendritic structure in the mouse dentate gyrus ................................................................175 Appendix B: Certificate of animal care .............................................................................182    viii List of tables  Table 1.1 Significant association between NR2E1 SNP and bipolar I disorder ......................16 Table 1.2 Mouse behavioural tests used to evaluate phenotypes similar to bipolar disorder symptoms .................................................................................................................18 Table 4.1 Ct values obtained from human-specific NR2E1 TaqMan assay ..........................126 Table 4.2 Gross phenotypic description of the four transgenic strains..................................128 Table 4.3 Gross brain measurements in the four transgenic strains ......................................130 Table 4.4 Fold change of target gene transcript in the four transgenic strains ......................133   ix List of figures Figure 1.1 DNA-binding domain of Nr2e1 contains two distinct differences...........................3 Figure 1.2 Nr2e1frc deletion results in the loss of all Nr2e1 exons............................................5 Figure 1.3 Cellular structure of mature mouse retina ..............................................................11 Figure 1.4 NR2E1 located near a putative bipolar I disorder susceptibility locus...................16 Figure 2.1 One room, two test times........................................................................................45 Figure 2.2 Home cage activity is affected by L/D cycle..........................................................52 Figure 2.3 The open-field test discriminates better in the dark phase .....................................54 Figure 2.4 Discriminant function plots of open-field data show improved strain discrimination in the dark phase...............................................................................55 Figure 2.5 The SHIRPA primary screen discriminates better in the dark phase .....................57 Figure 2.6 Discriminant function plots of SHIRPA data show improved strain discrimination in the dark phase.......................................................................................................58 Figure 2.7 The social interaction test was affected by L/D cycle but discrimination was not clearly better in one phase than the other .................................................................59 Figure 2.8 The rotarod test discriminates better in the dark phase ..........................................61 Figure 2.9 The tail-flick test does not discriminate better in the dark phase ...........................63 Figure 2.10 The hot-plate test does not discriminate better in the dark phase.........................64 Figure 3.1 Reduced body weight of Nr2e1frc/frc pups not explained by milk consumption .....81 Figure 3.2 Nr2e1frc/frc mice showed hyperactivity as early as postnatal day (P)18..................82 Figure 3.3 Nr2e1frc/frc mice showed hyperactivity in the home cage .......................................84 Figure 3.4 Nr2e1frc/frc mice showed hyperactivity and habituation deficiency in the open   field...........................................................................................................................85 Figure 3.5 Nr2e1frc/frc mice struggled more during the tail suspension test .............................86 Figure 3.6 Nr2e1frc/frc mice showed increased pain sensitivity ................................................88 Figure 3.7 Nr2e1frc/frc mice showed normal hearing ................................................................89 Figure 3.8 Nr2e1frc/frc mice showed impaired performance in the passive avoidance test ......90 Figure 3.9 Nr2e1frc/frc mice showed no startle reactivity to auditory stimuli ...........................91 Figure 3.10 Lithium-treated mice showed therapeutic levels of lithium in their serum..........92 Figure 3.11 Nr2e1frc/frc-induced hyperactivity in the home cage was unaffected by lithium treatment...................................................................................................................94 Figure 3.12 Hyperactivity and habituation deficits in Nr2e1frc/frc mice unaffected by lithium treatment...................................................................................................................95 Figure 3.13 Lithium treatment did not significantly improve startle reactivity deficit in Nr2e1frc/frc mice ........................................................................................................97 Figure 3.14 Lithium treatment did not increase cell proliferation in Nr2e1frc/frc mice ............99 Figure 4.1 FISH mapping of pacEMS1 transgenes ...............................................................123 Figure 4.2 B6-bacEMS4A show increased Nr2e1 expression in E12.5 whole head and adult brain........................................................................................................................125 Figure 4.3 B6-pacEMS1B and 1D showed significant increase in level of human NR2E1 ..127 Figure 4.4 B6-bacEMS4A showed significant increase in cell proliferation in the subventricular zone ................................................................................................135 Figure 4.5 Adult B6-bacEMS4A eyes show abnormal cellular staining ...............................137 Figure 4.6 Adult B6-bacEMS4A eyes show thinning of retinal layers .................................138   x Figure 4.7 Adult B6-bacEMS4A eyes showed significant alteration in gene transcription ..140   xi List of abbreviations 129S1/SvImJ 129 Auditory brainstem response ABR Bacterial artificial chromosome BAC Bipolar disorder  BP Bipolar I disorder BPI Bipolar II disorder BPII Bromodeoxyuridine BrdU C57BL/6J B6 Clumped pigmentary retinal degeneration CPRD Cycle threshold Ct Dentate gyrus DG DNA-binding domain DBD Embryonic day E Enhanced S-cone syndrome ESCS Fierce allele – spontaneous deletion of mouse Nr2e1 frc Fluorescence in situ hybridization FISH Ganglion cell layer GCL Goldmann-Favre syndrome GFS Inner nuclear layer INL Inner plexiform layer IPL Ligand-binding domain LBD Light-dark L/D Lithium chloride LiCl Logarithm of odds LOD Long-term potentiation LTP Neural stem/progenitor cell NSC Nuclear receptor subfamily 2 group E member 1 – Human gene NR2E1 Nuclear receptor subfamily 2 group E member 1 – Mouse gene Nr2e1 Nuclear receptor subfamily 2 group E member 1 – Human protein NR2E1 Nuclear receptor subfamily 2 group E member 1 – Mouse protein Nr2e1 Outer nuclear layer ONL Outer plexiform layer OPL Phage artificial chromosome PAC Photoreceptor outer segment OS Polymerase chain reaction PCR Postnatal day P Quantitative reverse-transcriptase PCR qRT-PCR Retinal progenitor cell RPC Retinitis pigmentosa RP Retinoic acid RA Revolutions per minute RPM Room temperature RT Single nucleotide polymorphism SNP   xii Subventricular zone SVZ Transgenic Tg Untranslated region UTR Ventricular zone VZ Wildtype Wt      xiii Acknowledgments I would like to thank my supervisory committee, Drs. Elizabeth Simpson (supervisor), Leigh Field, Blair Leavitt, Anthony Phillips, and Fabio Rossi, for all their support and expert guidance over the years. To the many administrative support staff, Dora Pak, Tracey Weir, Veronica Yakoleff, Rhonda Ellwyn, Heather Baker, Anna Moorhouse, and Tammy Philippo, I am grateful for all your assistance throughout my studies, from scheduling seemingly impossible meetings, to helping with grant applications, manuscript preparation, and thesis proofreading. To the wonderful students I had the honour of mentoring, John Chen, Emilija Todorovic, Jasmen Sze, Jason Cheng, Catherine Tam, Winnie Yang, and Glen Ottmann; thank you for all your help and giving me the opportunity to experience my own little “lab” within a lab. I would also like to thank the numerous Simpson lab members that I have had the pleasure of working with throughout the many years I have been here. There are too many of you to name, but I appreciate all the meaningful discussions, technical and emotional support, and friendships that have been formed. To my fellow Simpson lab Ph.D. students, old and new, Drs. Brett Abrahams and Ravi Kumar, Charles de Leeuw, Jean-François (Jeff) Schmouth, and Ximena Corso-Dìaz, thank you for all your scientific insights and ideas and support especially through rejected manuscripts, failed experiments, and unexpected results. I would especially like to thank Kathy Banks, my lab manager and my bay mate of over 5 years, and a voice of strength and reason through many of my trials and tribulations at and away from the lab. My graduate experience would also not have been the same without many other CMMT staff and students; you have all touched my life in your own unique ways and I am forever grateful for all the wonderful memories.    xiv I would like to thank all my family and friends for their love and steadfast support, without them I would not have made it this far. To all my friends, you know who you are, without you this would not have been possible. To my two amazing grandmothers, all my aunts, uncles, and cousins thank you for your words of wisdom, your delectable food, and your constant encouragement. To the only grandfather I have known, sorry I took so long to graduate that you could not be here to share this with me. To make up for it, I dedicate this work to your memory. To my parents, thank you for supporting me in everything I do and allowing me the freedom to pursue my dreams. I hope that I can live up to all your expectations and be someone you are proud of. I thank God everyday for all the amazing people He has surrounded me with to help me through this stage of my life.    xv Dedication    To the loving memory of my grandfather (1925 – 2009) Thank you for always believing in me.     xvi Co-authorship statement Chapter 2  The project described in this chapter was conceived and initiated by Dr. E.M. Simpson. The project became focused under the direction of Dr. S.M. Hossain. Dr. S.M. Hossain performed all behaviour testing. I analyzed the data, created all tables and figures, edited, and saw the manuscript through to publication.  Chapter 3  The project described in this chapter was conceived and initiated by Dr. E.M. Simpson. The initial characterization of Nr2e1frc/frc mice was performed under the direction of Dr. S.M. Hossain, and students he trained (ET and SB). After Dr. Hossain’s departure from the laboratory, I took over, focused, and directed the project by independently reproducing the original behavioural observations and further characterizing Nr2e1frc/frc mice with and without lithium treatment. I was also in charge of the collaboration with Dr. Q.Y. Zhen. I generated all the data presented in this chapter. A student (GAO), whom I trained, assisted in capturing images for quantification of cell proliferation. I performed all data analysis, wrote the paper, created all the figures, edited and saw the manuscript to submission.  Chapter 4  The project described in this chapter was conceived and initiated by Dr. E.M. Simpson. The project became focused under my direction. Dr. E.R. Linnell generated the four transgenic strains studied in this chapter. Initial screening of the transgenic strains was   xvii performed by Ms. M.L. Berry. Dr. B.S. Abrahams analyzed, using fluorescent in situ hybridization, the genomic integration site, with the help of Dr. M.B. Valentine and Ms. A.C.O. Chong, of the four transgenic strains (resulting in Fig. 4.1). Students (JS, JCYC, CT, WHWY, and GAO), whom I trained, assisted in sample preparation and image collection and tracing. Ms. A.E. Borrie assisted in the harvesting of the eye samples. I generated the results presented, performed all data analysis, wrote the paper, and created all the figures for this manuscript in preparation.    1 Chapter 1: General introduction  1.1 Nuclear receptor superfamily  The nuclear receptor superfamily is made up of numerous transcription factors important in the regulation of gene expression involved in processes including, but not limited to, metabolism, inflammation, cancer, neural and organ development, and cell proliferation and differentiation, metamorphosis, and organ physiology (Lee et al., 2008, Mangelsdorf et al., 1995, Yang et al., 2006). Nuclear receptors bind specific DNA elements via their DNA-binding domain (DBD) and their function is largely controlled by conformational changes through binding of ligands to the receptor’s ligand binding domain (LBD). These receptors include: thyroid hormone receptors (TR), peroxisome proliferator- activated receptors (PPAR), retinoic acid receptors (RAR), and the steroid receptor group containing glucocorticoid receptor (GR), mineralocorticoid receptors (MR), progesterone receptor (PR), and androgen receptors (AR) (Maglich et al., 2001). However, there is a class of nuclear receptors, known as orphan nuclear receptors that have no known ligand; some of these have been shown to function in the absence of a ligand. For example, the function of Nr4e2, also known as Nurr1, is regulated by stable conformational folding of its LBD that resembles a ligand-bound nuclear receptor (Wang et al., 2003). Many members of the orphan nuclear receptor family are involved in the development of the central nervous system (Armentano et al., 2006, Chen et al., 2001, Lutz et al., 1994, Zetterstrom et al., 1997). One of these orphan nuclear receptors, nuclear receptor 2e1 (Nr2e1) and its role in neural and ocular development, cell proliferation, and behaviour, is the focus of this thesis.   2 1.2 The importance of Nr2e1 in neurodevelopment and cell cycle regulation 1.2.1 Structure and interspecies homology of Nr2e1  Nr2e1, previously known as MTll, Tlx, was first identified in Drosophila melanogaster with similarity in its DBD and LBD to steroid hormone receptors. When mutated, Drosophila Nr2e1 affects the embryonic development of the anterior and posterior poles (Pignoni et al., 1990). Vertebrate Nr2e1 was first cloned and investigated in chicken (Yu et al., 1994), then in mouse (Monaghan et al., 1995), and finally in human (Jackson et al., 1998). The DBD of vertebrate Nr2e1 contains two distinct differences when compared to other nuclear receptors. First, the proximal box (P box) sequence contains a serine residue in place of the canonical lysine residue that is found in all other nuclear receptors (Figure 1.1). Secondly, the distal box (D box) sequence encodes for seven amino acids instead of the typical five in other nuclear receptors (Figure 1.1). DNA-binding assays showed that the Nr2e1 DBD binds to a target sequence AAGTCA, either as a monomer to a single half-site or as dimers to a pair of half-sites (Kobayashi et al., 1999, Yu et al., 1994). The human and mouse Nr2e1 proteins are 385 amino acids large in size and show 100% and 99.5% conservation in the DBD and LBD, respectively (Kobayashi et al., 2000). Genomic analysis of Nr2e1 also showed elements of extreme conservation from human to mouse down to F. rubripes (Fugu), indicative of regulatory and functional importance (Abrahams et al., 2002).   3  Figure 1.1 DNA-binding domain of Nr2e1 contains two distinct differences Amino-acid sequences of the DNA-binding domain of chick Nr2e1 and Drosophila Tll show two distinct differences compared to other nuclear receptors. The proximal box (P box) and distal box (D box) are boxed and labelled. The P box sequences differ from the consensus sequence such that both encode aspartic acid (D) instead of glutamic acid (E); in addition, a lysine (K) that is absolutely conserved in all other members is substituted with either serine (S) or alanine (A). The D box encodes 7 amino acids between the 5th and 6th coserved cysteines (C) instead of the usual 5 amino acids. Vertical lines identify sequence homology between chick and Drosophila Nr2e1 within the P and D boxes. (Modified from (Yu et al., 1994))  1.2.2 Expression pattern of Nr2e1 in the developing and adult brain  In mouse, Nr2e1 transcription is first detected at the 5-somite stage (embryonic day (E)8) in a few cells adjacent to the neural epithelium caudal to the anterior limit of developing proscencephalon (Monaghan et al., 1995). By E8.5, expression has spread caudally into the presumptive diencephalon and can be detected in newly formed optic and olfactory evaginations. At E12.5, Nr2e1 transcripts are restricted to a subset of forebrain periventricular zones and presumptive amygdala, except for transcripts remaining in the neural retina and olfactory epithelium. Nr2e1 expression decreases to undetectable levels perinatally, but by adulthood expression is again observable in a subset of cells in the subgranular layer of the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) lining the lateral ventricles of the adult brain (Monaghan et al., 1995, Shi et al., 2004). Furthermore, antibody staining revealed that Nr2e1 protein is localized in the cell nucleus (Li et al., 2008).   4 In humans, detailed information about expression patterns is lacking; however, NR2E1 transcripts are detected in adult tissues including the amygdala, caudate nucleus, cerebral cortex (including frontal lobe, occipital lobe, putamen, temporal lobe), corpus callosum, hippocampus, substantia nigra, subthalamic nucleus, and thalamus (Jackson et al., 1998, Kumar et al., 2008).  1.2.3 Targeted and spontaneous deletions of Nr2e1 in mice  The use of Nr2e1-null mouse mutants has provided significant insight into the function of Nr2e1. Several laboratories have used homologous recombination to generate mice carrying deletions of exons two and three (Monaghan et al., 1997) and exons three, four, and five (Yu et al., 2000) of Nr2e1. Alternatively, the Simpson laboratory has reported on mice homozygous for the Nr2e1frc allele, which is a spontaneous deletion of all nine exons of Nr2e1 as well as its proximal promoter, without disruption of neighbouring genes (Figure 1.2) (Kumar et al., 2004). These various Nr2e1-null mice present similar phenotypes and will be discussed as a whole below. The work in Chapter 3 was done using Nr2e1frc/frc homozygous mice from the Simpson laboratory.   5  Figure 1.2 Nr2e1frc deletion results in the loss of all Nr2e1 exons A schematic representation of Wt Nr2e1 and Nr2e1frc loci illustrating the 44.4 kb deletion of Nr2e1 and the transposition of 188-bp sequence (red box) from Lace1. Grey arrowheads indicate deletion boundaries. Diagonal hatched lines represent discontinuous DNA sequence. Number above each gene indicates distance from the centromere. Horizontal arrows below each gene indicate transcriptional direction. Horizontal bars represent exons of each gene. (Modified from (Kumar et al., 2004))  1.2.4 Role of Nr2e1 in neurodevelopment and neurogenesis   The critical role of Nr2e1 in normal neurodevelopment is obvious when one examines the extreme neuroanatomical phenotypes in Nr2e1-null mice. The earliest neurological phenotypes observed from E9.5 to E14.5 are increased staining of two panneural markers, Tuj1 and Map2, and a marker for Cajal-Retzius cells, Cr, in Nr2e1-null compared to wild- type (Wt) telencephalon; this increase in neuronal differentiation is attributable to shorter cell cycle as demonstrated using bromodeoxyuridine (BrdU) birthdating analysis (Roy et al., 2004). By E12.5, Nr2e1-null mice show reduced populations of dorsal telencephalon progenitor-derived excitatory neurons and, ventrally, medial ganglionic eminence (MGE)- generated inhibitory interneurons (Roy et al., 2004). This reduction in cell proliferation is also revealed by the flattening of the lateral ganglionic eminence (LGE) and MGE in the Nr2e1-null telencephalon by E12.5 (Roy et al., 2004). After E14.5, depletion and slower cell division rate of Nr2e1-null neural progenitor cells result in reduction of the superficial layers   6 of the cortex, namely layers 2 and 3 (Li et al., 2008, Roy et al., 2004, Shi et al., 2004, Sun et al., 2007). Since TUNEL assay showed no difference in apoptosis between Nr2e1-null and Wt mice (Li et al., 2008), the 20% reduction of neocortical thickness is a result of the inability of the Nr2e1-null progenitor cell population to be sustained throughout late prenatal development (Land & Monaghan, 2003). Nr2e1 has been shown to be essential for patterning in the lateral telencephalon, in establishing the pallio-subpallial boundary through its interaction with Pax6 (Stenman et al., 2003a, Stenman et al., 2003b). Recently, in utero electroporation studies also showed that Nr2e1 has a role in regulating cell migration from the VZ into the intermediate zone and cortical plate during embryogenesis (Li et al., 2008). This deficit in the proliferative potential of Nr2e1-null progenitor cells is also evident in the adult mouse forebrain, where Nr2e1-null cells showed reduced proliferation and increased gliogenesis in vitro and in vivo (Shi et al., 2004). Furthermore, these cellular phenotypes can be corrected by viral reintroduction of Nr2e1 in vitro (Shi et al., 2004).  1.2.5 Genetic and protein interactions of Nr2e1 in the brain  The mechanism by which Nr2e1 exerts its control on cell proliferation and differentiation is by directly binding to the AAGTCA consensus sequence in the promoters of Pten, Gfap, S100β, and Aqp4, thereby repressing the expression of these genes (Shi et al., 2004, Yu et al., 2000, Zhang et al., 2008). Nr2e1 has also shown direct binding to histone demethylase, LSD1 and histone deacetylases, HDAC3, 5, and 7, to recruit these protein complexes for transcription repression (Sun et al., 2007, Yokoyama et al., 2008). The level of Nr2e1 is itself regulated by a negative feedback loop by microRNA-9 (miR-9) that binds to the 3′ UTR of the Nr2e1 mRNA. The overexpression of miR-9 results in a decrease of Nr2e1 transcripts leading to reduced proliferation, premature differentiation, and outward   7 migration of neural stem cells (Zhao et al., 2009). Further analysis of the miR-9 locus also identified multiple Nr2e1 binding sites downstream of the mature miR-9 sequence (Zhao et al., 2009), supporting the role of Nr2e1 in repression of miR-9.  1.3 The lack of Nr2e1 results in adult neuroanatomical and behavioural abnormalities 1.3.1 Neuroanatomical anomalies  In the adult, gross neuroanatomical abnormalities can be seen in Nr2e1-null brains including: hypoplasia of the cerebral cortex, olfactory bulb, hippocampus, corpus callosum and amygdala; increased volume of the lateral ventricles; reduced thickness in superficial cortical layers II and III; reduced population of excitatory neurons and inhibitory interneurons; reduced neurogenesis in the dentate gyrus (DG) and subventricular zone (SVZ); reduced dendritic branching of DG granule neurons, and long-term potentiation (LTP) deficit in granule neurons of the DG (Christie et al., 2006, Land & Monaghan, 2003, Monaghan et al., 1997, Roy et al., 2004, Roy et al., 2002, Shi et al., 2004, Stenman et al., 2003a, Stenman et al., 2003b, Young et al., 2002). Nr2e1-null mice also show slower weight gain during development and small stature compared to Wt mice (Young et al., 2002). The characterization of DG granule neuron branching and LTP deficit was work I did in collaboration with Dr. Brian Christie and the subsequent publication is presented in the appendix of this thesis (Appendix A).   8 1.3.2 Behavioural abnormalities  Nr2e1-null mice exhibit various behavioural abnormalities. Most striking of these behaviours is their pathological aggression, with Nr2e1-null males often killing their siblings or intended mates (Young et al., 2002). Depending upon strain background, Nr2e1-null females also showed aggression and poor maternal behaviour (Young et al., 2002). When handled by humans, Nr2e1-null mice exhibit a ‘hard to handle’ phenotype, characterized by vocalization, struggling, jumping, and biting (Young et al., 2002). Furthermore, impaired olfaction and vision were revealed during sensorimotor examination of Nr2e1-null mice (Young et al., 2002). They also show reduced anxiety and memory for fear and hyperresponsiveness (Roy et al., 2002).  More recently, conditional knockouts of Nr2e1 have been analyzed to decipher the developmental versus adult role of Nr2e1 in behaviour. Of particular interest are (1) mice deleted for the floxed Nr2e1 allele using a tamoxifen-induced cre in the adult brain show significant reduction in stem cell proliferation that corresponds to impairments only in spatial learning, but not to contextual fear conditioning, locomotion, or diurnal rhythmic activities (Zhang et al., 2008), and (2) mice deleted for the floxed Nr2e1 allele using CaMKIIα-Cre during brain development but sparing the eye (eye phenotypes are discussed below) show reduced anxiety and aggression, but no impairment in fear conditioning and Morris water- maze compared to Wt mice (Belz et al., 2007). These results suggest that disruptions in contextual fear conditioning, locomotion, or diurnal rhythmic activities are likely the result of developmental abnormalities and that learning and memory paradigms are dependent on reduced vision in Nr2e1-null mice (Belz et al., 2007, Zhang et al., 2008).    9 Interestingly, human NR2E1, with its endogenous promoter and regulatory regions, when reintroduced into Nr2e1-null mice has been shown to rescue the mutant neuroanatomical and behavioural phenotypes (Abrahams et al., 2005). This result suggests that the regulation and function of human NR2E1 is equivalent to that of mouse Nr2e1, which is supported by the high conservation observed at both the genomic and amino acid sequence level.  1.4 The importance of Nr2e1 in eye development  As mentioned previously, Nr2e1 transcripts are detected at E8.5 in the optic evagination and are expressed in the mouse neural retina throughout development and into adulthood (Monaghan et al., 1995). Nr2e1 expression during early eye development has also been demonstrated in chicken (Yu et al., 1994), Xenopus (Hollemann et al., 1998), and Medaka (Oryzias latipes) (Nguyen et al., 1999). Ectopic expression of the Nr2e1-DBD fused with the engrailed repressor domain in Xenopus showed reduced Pax6 expression and inhibition of eye vesicle evagination (Hollemann et al., 1998). In addition to the interaction of Nr2e1 and Pax6 in establishing boundaries in the brain (Stenman et al., 2003a, Stenman et al., 2003b), this is the second piece of evidence for an interaction between Nr2e1 and Pax6, a gene that encodes for a transcription factor essential for normal vertebrate eye development (Grindley et al., 1995, Ramaesh et al., 2005).  Excess and/or deficiency of retinoic acid (RA) can also cause eye malformations (Cvekl & Wang, 2009, Fujieda et al., 2009); and a cis element, named the silencing element relieved by TLX (SET), found in the RA receptor β2 (RARβ2) promoter supports a regulatory role for Nr2e1 in the expression of RARβ2 in the eye (Kobayashi et al., 2000).   10 Furthermore, Pax2, a gene involved in both human and mouse retinal development, contains the Nr2e1 consensus binding site in its promoter and is a direct target of Nr2e1 (Yu et al., 2000).  1.4.1 Adult eye anomalies in Nr2e1-null mice  Given that Nr2e1 is expressed in the developing eye and the above evidence supporting its role in eye development, it is not surprising that Nr2e1-null adult mice have numerous ocular abnormalities. The adult neural retina consists of 5 layers: the outer nuclear layer (ONL), the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), and the ganglion cell layer (GCL) (Figure 1.3). Ocular phenotypes seen in Nr2e1-null mice include: optic nerve hypoplasia; retinal degeneration and dystrophy, especially the INL and the ONL, which are later forming; enhanced S-cone generation; shortened axons and dendrites of rods, cones, and bipolar, horizontal, and ganglion cells as evident by reduced thickness of the IPL, OPL, and photoreceptor outer segment (OS); impaired astrocyte network formation on the inner retinal surface; diminished retinal vascularization; impaired regression of hyaloid vessels; and reduced to flat electroretinogram (Miyawaki et al., 2004, Young et al., 2002, Yu et al., 2000, Zhang et al., 2006).     11  Figure 1.3 Cellular structure of mature mouse retina (A) A cross section of an adult mouse retina. (B) A schematic representation of the layers and its cellular components. Cell bodies of photoreceptors (PhR; rods and cones) make up the outer nuclear layer (ONL). In the outer plexiform layer (OPL), PhR synapse with bipolar (RBC) and horizontal cells (HC). Cell bodies of RBC, HC, and amacrine cells (AC) are located in the inner nuclear layer (INL). In the inner plexiform layer (IPL), ganglion cells (GC) synapses with RBC, HC, and AC. The cell bodies of GC are located in the ganglion cell layer (GCL). (Modified from (Tian, 2004)).    12 1.4.2 Genetic and protein interactions of Nr2e1 in the eye  Mechanistically, Nr2e1 acts in similar cellular processes in the eye and the brain. Nr2e1 is expressed in retinal progenitor cells (RPCs) and regulates cell cycle progression by directly regulating Pten expression, which then dictates the levels of cyclinD1 and p27Kip1 (Miyawaki et al., 2004, Zhang et al., 2006). In the mature retina, Nr2e1 expression is restricted in Müller cells, glial cells of the eye, and is shown to be necessary for their proper development (Miyawaki et al., 2004). Nr2e1 also recruits co-repressor Atrophin1 (Atn1) for coordinating retina-specific proliferation and differentiation (Wang et al., 2006a, Zhang et al., 2006). The expression of Nr2e1 in retinal astrocytes can be regulated by oxygen concentration and is proposed to participate in the formation of proangiogenic scaffolds under hypoxic conditions (Uemura et al., 2006). 1.4.3 The function of Nr2e3, a relative of Nr2e1, in the eye  The role of Nr2e1 in eye development and disorders is further supported by the function of Nr2e3 in eye. Nr2e3, also known as photoreceptor-specific nuclear receptor (PNR) is the closest relative to Nr2e1. Nr2e3 is expressed in the photoreceptor layer of the neural retina during chick embryogenesis (Kobayashi et al., 2008) and when mutated causes enhanced S-cone syndrome, a disorder of retinal cell fate determination (Akhmedov et al., 2000, Corbo & Cepko, 2005, Escher et al., 2009, Haider et al., 2000, Schorderet & Escher, 2009).     13 1.5 The emerging role of Nr2e1 in cancer  Nr2e1 directly controls the expression of Pten, plays a critical role in regulating cell cycle, and has now been implicated in cancers, such as retinoblastomas and neurocytomas (Sim et al., 2006, Yokoyama et al., 2008). Nr2e1 is expressed in Y79 retinoblastoma cells and acts as an inhibitor of Pten (Sun et al., 2007, Yokoyama et al., 2008). Overexpression of Nr2e1 has also been found in neurocytoma (Sim et al., 2006)  1.6 NR2E1: A candidate gene for bipolar disorder 1.6.1 Genetics of bipolar disoder  Bipolar disorder (BP) is mainly characterized by mood disturbances ranging from extreme elation (mania) to extreme depression with a lifetime prevalence of 0.4 to 1.6% (American Psychiatric Association., 2000). A manic episode is defined by at least 1 week (or less if hospitalization is required) of elevated, expansive, or irritable mood. This mood disturbance must be accompanied by at least three to four additional symptoms from a list that includes: inflated self-esteem or grandiosity, decreased need for sleep, pressure of speech, flight of ideas, distractibility, increased involvement in goal-directed activities (e.g. sexual and social behaviours) or psychomotor agitation, and excessive involvement in pleasurable activities with a high potential for painful consequences (American Psychiatric Association., 2000). A major depressive episode lasts a period of at least two weeks during which there is either depressed mood or the loss of interest or pleasure in nearly all activities, and may include persistent feelings of sadness, anxiety, guilty, anger, isolation, or hopelessness; disturbances in sleep and appetite; fatigue; problems concentrating; apathy or indifference; loss of interest in sexual activity; social anxiety; irritability; and morbid suicidal   14 ideation (American Psychiatric Association., 2000). There are two main subtypes of BP, bipolar I disorder (BPI) and bipolar II disorder (BPII). The clinical course for BPI is the occurrence of one or more manic episodes, and often also one or more major depressive episodes. Patients have to have one or more major depressive episodes accompanied by at least one hypomanic episode, a mild to moderate level of mania, for a diagnostic of BPII. The average age of onset for BP is 20 for both men and women, with BPII more common in women. There are lines of evidence supporting a strong genetic influence for BP. Twin studies have shown high heritability for BP (60-85%) (Burmeister et al., 2008) with concordance rates for monozygotic twins ranging from 40-97% and 5-38% for dizygotic twins (Angst et al., 1980, Kieseppa et al., 2004, Mcguffin et al., 2003). Family studies show increased risk in first-degree relatives of individuals with BP to exhibit earlier age of onset and to develop BP and other related psychiatric disorders, including hypomania and schizoaffective disorder (Baron et al., 1982, Gershon et al., 1988, Kendler et al., 1993, Maier et al., 1993, Winokur et al., 1982). Linkage studies of BP and other psychiatric disorders have also identified several reproducible loci of interest, including the 6q region containing NR2E1 (Dick et al., 2003, Hayden & Nurnberger, 2006, Kohn & Lerer, 2005, Mcqueen et al., 2005, Middleton et al., 2004, Pato et al., 2005, Pato et al., 2004, Schulze et al., 2004). Because of the complex inheritance and other external factors underlying BP, causative genes are only beginning to be identified (Craddock & Sklar, 2009, Le-Niculescu et al., 2009, Martinowich et al., 2009, Ogden et al., 2004).    15 1.6.2 Genetic support for NR2E1 in brain disorders  Human NR2E1 is mapped to chromosome location 6q21. In the largest meta-analysis of BP to date, this 108.5 Mb region (6q21-22) showed the highest LOD score (4.19) specifically for bipolar I disorder (BPI), the subtype dominated by mania (Figure 1.4) (Mcqueen et al., 2005). A follow up study by this group identified a significant association between BPI and a single nucleotide polymorphism (SNP) near the solute carrier family 22, member 16 gene (SLC22A16) (Fan et al.). Under this same linkage peak, the Simpson laboratory found a significant association between a SNP in NR2E1 (marker rs217520, for the A allele) and BPI (Table 1.1) (Kumar et al., 2008). Novel regulatory mutations in NR2E1 that were absent in controls were also identified in patients with impulsive-aggressive disorder, schizophrenia, BP, and microcephaly (Kumar et al., 2007, Kumar et al., 2008).    16  Figure 1.4 NR2E1 located near a putative bipolar I disorder susceptibility locus The highest LOD score to date (4.19) from pooled analysis of original genotype data from 11 BP genomewide linkage scans was identified at physical location 108.5 Mb (115 cM), a region close to where NR2E1 maps (108.6 Mb). The LOD scores from the pooled analysis (solid black line) are overlaid with the LOD scores from the data set-specific analysis (solid non-black lines). Genomewide significance threshold (3.03) is indicated by the horizontal dotted line. (Modified from (Mcqueen et al., 2005))  Table 1.1 Significant association between NR2E1 SNP and bipolar I disorder  (Modified from (Kumar et al., 2008))     17 Furthermore, alterations in genes that interact with NR2E1 have also been identifed in brain-behavioural disorders, including: NURR1 (Buervenich et al., 2000, Carmine et al., 2003), PAX6 (Ellison-Wright et al., 2004, Heyman et al., 1999), RARβ2 (Van Neerven et al., 2008), GFAP (Barley et al., 2009, Steffek et al., 2008), and S100β (Schroeter et al., 2009, Steiner et al., 2006).  1.6.3 Role of neural stem/progenitor cells in brain disorders  The role of neural stem/progenitor cells has also been implicated in various neurological and psychiatric disorders, including BP. For example: neuroprotective effects can be triggered by deep brain stimulation treatment that increases proliferation in the dentate gyrus (Toda et al., 2008); lithium treatment for mania in BPI acts through the Gsk3β pathway to induce neurogenesis (Wada et al., 2005); and Parkinson’s and Alzheimer’s animal models have shown motor and cognitive improvement through stem cell implantation (Bjorklund & Lindvall, 2000, Wang et al., 2006b). Since Nr2e1 plays an important role in the regulation of neural stem/progenitor cells and brain development that results in abnormal behavioural phenotypes, NR2E1 is both a strong positional and functional candidate for psychiatric disorders, especially BPI. 1.6.4 Different mouse “models” of bipolar disorder  The heterogeneity and complexity of behavioural traits represented in patients with BP is difficult, if not impossible, to accurately model in animals. Therefore, it is commonly accepted to study facets of this disease in rodent models (Einat, 2006a, Einat, 2006b). Since BP is a disorder diagnosed by abnormal behavioural traits, many current mouse models are supported by the presence of behavioural phenotypes exhibited in patients (Table 1.2). The   18 mania component of BP is often modeled in increased spontaneous and psychostimulant- induced hyperactivity, increased aggressive behaviours, and decreased anxiety-like behaviours (Einat & Manji, 2006). Depressive behaviours are measured in rodents as attempts to escape during tests such as the forced swim, tail suspension, and learned helplessness test. Cognitive impairments are also noted in some patients with BP, and therefore, are examined in rodent models using learning and memory tasks such as the Morris water maze, conditioned and passive avoidance, and fear conditioning. Table 1.2 Mouse behavioural tests used to evaluate phenotypes similar to bipolar disorder symptoms  (Modified from (Einat, 2006b))  1.6.4.1 Genetic mouse models Candidate genes of BP, identified in human linkage and genome-wide association studies, are being tested in genetic mouse models. There are several single-gene knockout and transgenic mice that have been useful in deciphering the involvement of these genes in   19 BP. Neuronal nitric oxide synthase (nNOS) metabolism has been suggested to contribute to pathogenesis and pathophysiology of BP (Bernstein et al., 1998, Lauer et al., 2005, Reif et al., 2006). nNOS knockout mice have now been shown to exhibit hyperlocomotor activity, increased social behaviours, reduced depressive-like behaviours, and impaired spatial memory retention (Tanda et al., 2009). Given the sleep disturbances and dysregulation in patients with BP, circadian rhythm genes have also been indicated in susceptibility of BP (Shi et al., 2008). Clock mutant mice exhibit behaviours similar to those seen in patients with mania including hyperactivity, decreased sleep, reduced depression-like behaviour, lower anxiety, and enhanced behavioural responses to reward (e.g. cocaine, sucrose, and medial forebrain bundle stimulation; these behaviours were also attenuated by chronic administration of lithium (Roybal et al., 2007). Transgenic mice overexpressing glycogen synthase kinase 3β (Gsk3β), a gene downregulated by lithium treatment, showed increased locomotor activity and acoustic startle response, and decreased habituation to the open field and to acoustic startle (Prickaerts et al., 2006). Recently, reduced expression of Disrupted in schizophrenia 1 (Disc1) has been shown to result in premature proliferation and differentiation of neuronal progenitors that can be compensated by treating with Gsk3β inhibitors (Mao et al., 2009). Interestingly, mice expressing truncated Disc1 showed neurological abnormalities such as those seen in Nr2e1-null mutants, including: enlarged ventricles, reduced cerebral cortex, especially thinning of layers II/III, hypoplasia of corpus callosum and hippocampus, and reduced dendritic branching and length (Shen et al., 2008). Behaviours of mice carrying Disc1 mutations range from hyperactivity, increased immobility during depression-related tests, impaired conditioned response, and abnormal sensorimotor gating (Hikida et al., 2007, Shen et al., 2008).    20 1.6.4.2 Drug-induced model of hyperactivity Another common approach to modeling mania in BP is the use of drugs, in particular a mixture of D-amphetamine and chlordiazepoxide, to induce hyperactivity in rodents (Aylmer et al., 1987, Gould et al., 2007). D-amphetamine/chlordiazepoxide-induced hyperactivity in rodents became a widely used model of manic behaviour because of its response to lithium treatment. Lithium, the drug of choice for treating BP, can attenuate this drug-induced hyperactivity without affecting spontaneous activity levels (Gould et al., 2001).  1.7 Thesis objectives 1.7.1 General hypothesis and sub-hypotheses  Work presented in this thesis tested the general hypothesis that variable levels of Nr2e1 in mice, ranging from no expression to overexpression, will result in neurological, ocular, and gene expression phenotypes that reflect traits observed in human psychiatric and eye disorders. The first two chapters of this thesis focused on evaluating Nr2e1frc/frc mice as a model for bipolar I disorder (BPI) and tested two sub-hypotheses. These sub-hypotheses were: (1) that dark-phase testing is more etiologically-correct and, therefore, will improve discrimination between genetically distinct mouse strains (Chapter 2); and (2) that Nr2e1frc/frc mice will exhibit behavioural anomalies similar to those seen in some patients with BPI (Chapter 3). Based on Nr2e1-null brain and eye phenotypes, Chapter 4 evaluated the sub- hypothesis that Nr2e1 overexpression will also result in dysmorphia of neuroanatomical and ocular development, and that target gene transcription levels will inversely correlate with Nr2e1 levels.   21 1.7.2 Evaluation of Nr2e1frc/frc as a model for bipolar I disorder  Since NR2E1 has been implicated in BPI and the Nr2e1-null mice have not yet been studied as a potential mouse model, this is the focus of the first two manuscript-based chapters. We anticipated that there might be subtle behavioural phenotypes in modeling aspects of BPI in mice, and therefore Chapter 2 evaluated the effect of light-phase versus dark-phase testing on detection of behavioural differences in genetically different inbred mouse strains to establish best practice standards to be used in Chapter 3. The focus of Chapter 3 was to use a battery of behavioural tests to examine Nr2e1frc/frc mice for behavioural traits used in other mouse models of BP, including activity levels, cognition, information processing, and cell proliferation in neurogenic regions. I also tested the effect of lithium treatment on these parameters to assess the pharmacological validity of this model.  1.7.3 Evaluation of overexpression of Nr2e1 in mice  Mice lacking Nr2e1 have been studied in detail by the Simpson laboratory and many others; however, the genetic, neurological, and ocular consequence of Nr2e1 overexpression has not been examined. Our laboratory has previously established four random insertion transgenic strains, two carrying a bacterial artificial chromosome (BAC) containing mouse Nr2e1 and two carrying a phage artificial chromosome (PAC) spanning human NR2E1. The strains carrying the PACs, when bred onto the fierce background, rescue the Nr2e1frc/frc phenotype to demonstrate functional conservation of mouse and human NR2E1 (Abrahams et al., 2005). In Chapter 4, I first quantified the expression of Nr2e1 in these four transgenic strains and then further examined changes in transcript levels of genes previously known to be altered by the absence of Nr2e1. Gross neurological measurements and presence of eye phenotypes were also studied. The B6-bacEMS4A strain was chosen, based on Nr2e1   22 expression and showing the most affected gross neurological and eye phenotypes, for quantification of cell proliferation in neurogenic regions. 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Nat Struct Mol Biol, 16, 365-371.    40 Chapter 2: The dark phase improves genetic discrimination for some high throughput mouse behavioural phenotyping1 2.1 Introduction  Behaviour testing in mice is undergoing a rapid evolution as genetically modified and chemically mutated mice are being applied to the field. Now is the time to set the standards for test conditions (Brown et al., 2000, Crabbe et al., 1999, Crawley & Paylor, 1997, Van Der Staay & Steckler, 2002, Würbel, 2002). An important and often overlooked parameter is the effect of light-dark (L/D) cycle (Wahlsten, 2001). Mice are nocturnal animals and thus more active in the dark phase (Whishaw et al., 1999). Ironically, most researchers conduct behaviour testing during the day, when mice are normally sleeping and less active (Marques & Waterhouse, 1994) and in the light, a condition mice normally avoid. Although convenient, this practice is ethologically incorrect. The alternative is to test mice in the dark phase. Some of the inconvenience of dark-phase testing can be minimized through the use of reverse light cycle (lights on 23:00 hr to 11:00), dim red light, and low-light level camera - but is the effort warranted? The importance of dark-phase test conditions may depend on the type of testing being done. We consider mouse behavioural phenotyping to consist of a continuum of situations with three principle nodes. The first is the ‘classical’ testing situation, paralleling many rat studies, in which a strain is fully characterized and at least three tests purported to measure a specific attribute                                                  1 A version of this chapter has been published. Hossain, S.M., Wong, B.K.Y., Simpson, E.M. (2004) The Dark Phase Improves Genetic Discrimination for Some High Throughput Mouse Behavioral Phenotyping. Genes, Brain, and Behavior 3(3): 167-77. [PMID 15140012]   41 are applied before a conclusion regarding the strain’s ability or psychosocial state is drawn. Such studies are often combined with brain analyses that reveal features supportive of the conclusion. Often such classical testing is driven by a specific hypothesis regarding the interaction of brain and behaviour. Examples of classical testing would include the Morris water task to assess learning and memory, or the elevated plus maze to assess anxiety, both performed with pretest conditioning or training and no particular concern for throughput. Although not always done, from an ethological standpoint, it makes sense to conduct these tests in the dark since the purpose is to learn about the abilities and state of the strain. Previous experimental findings demonstrate that dark-phase testing may affect the outcome. For example, behavioural responses such as emotional reactivity, acoustic startle response, memory performance, and locomotor activity were influenced by lighting conditions (Kopp, 2001, Valentinuzzi et al., 2000). During a water tank social interaction test, mice were more willing to wade in search of food in the dark phase than in the light phase (Nejdi et al., 1996).  The second is the ‘mutant versus wild type’ testing situation. In such a situation the test battery is often not sufficiently comprehensive to permit strong conclusions about overall abilities or psychosocial states. In contrast, the panel of tests, some of which may have been adapted for high throughput, is designed to identify a phenotypic difference between two cohorts of mice that differ by only one modified gene. Often there is no a priori hypothesis and the search is focused on finding a test that can discriminate mutant and wild type. A positive result from such a study would be appropriately followed up with a ‘classical’ test situation before a strong conclusion about ability or psychosocial state could be made. Although the effect of dark-phase testing in the mutant versus wild type test situation has not been thoroughly studied, its value has been demonstrated. Discrimination between wild type   42 and mutant mice by wheel running activity was observed only in dark-phase testing (Kriegsfeld et al., 1999). The third is the ‘screening’ test situation. Such a situation has developed out of the application of mouse behaviour to the world of genomics; as exemplified by quantitative trait loci analyses and ENU mutagenesis. These applications require screening thousands of genetically variant mice to identify unknown phenotypes using high throughput phenotyping, with the goal of rapid discrimination of rare outliers. For this work, tests such as the SHIRPA primary screen (Rogers et al., 1997), coupled with non-invasive high throughput assays for major behavioural domains (e.g. open field), are employed to allow rapid assessments and identification of a subset of mice worth further study. Such studies are resource-driven and conclusions about the ability or psychosocial state of the animal cannot be appropriately made. However, with heritability testing and the generation of a cohort of mice, testing situations mutant versus wild type and classical can subsequently be applied. The value of dark-phase testing in the screening situation has not been investigated. Since the importance of ethological correctness and the effect of L/D cycle on high throughput behavioural tests, such as those required of ‘mutant versus wild type’ and ‘screening’ situations, are poorly characterized or unknown, we set out to test two hypotheses: 1) Dark-phase testing affects the outcome of high throughput behavioural tests, and 2) dark-phase testing improves discrimination between genetically distinct mice using high throughput behavioural tests. Our study includes an initial assessment of home cage activity and the following behavioural tests conducted in a high throughput manner: open- field, SHIRPA primary screen, social interaction, social recognition, rotarod, tail-flick, and hot-plate test performed on three strains: C57BL/6J (B6) inbred, 129S1/SvImJ (129) inbred,   43 and B6129F1 (F1) hybrid. The tests were chosen for their value in screening genetically diverse mice for both physical and social behaviours. The strains were chosen for their importance in genomic and genetic studies (Silva et al., 1997). B6 mice were previously selected for genome sequencing and 129 mice are widely used for targeted mutagenesis in ES cells; both strains are recommended for behavioural phenotyping (Paigen & Eppig, 2000, Silva et al., 1997). The mixed B6129 background is the most commonly used background for the generation of transgenic mice; we chose B6129F1 since F1 mice provide both genetic and phenotypic uniformity, as well as hybrid vigor (Dierssen et al., 2002). Importantly, the goal of this report was not to characterize the strains themselves but to explore the interaction of test conditions and discrimination power.   2.2 Methods and materials 2.2.1 Mouse facility  All mice were born, reared, and tested in the pathogen-free behaviour suite under reverse L/D cycle (light 23:00-11:00 h at 320 lux), at the Centre for Molecular Medicine & Therapeutics, Vancouver, Canada. The three-room behaviour suite consists of a breeding room and a dedicated testing room, separated by an anteroom. The lighting in all three rooms was synchronized. Care was taken not to expose the mice to any inappropriate light, even during testing. When light was needed by the investigator during experiments in the dark phase, a dim red light (8 lux) was used. Since limited color vision renders mice insensitive to red light at wavelengths >630 nm (Jacobs et al., 1999), phase was not disturbed (Crawley, 2000). The mice were maintained at 20 ± 2°C with relative humidity of 50 ± 5% and had food and water ad libitum.   44 2.2.2 Mice  Behavioural testing was started at 12 weeks of age on 168 test mice (24 for home cage activity and 72 each for experiment 1 and 2). The 168 mice represented 56 from each strain (C57BL/6J (JAX®00664) (B6), 129S1/SvImJ (JAX®02448) (129), and B6129F1 (F1)). An additional 24 mice, 8 from each strain (12 female and 12 male, 8 months old ± 1 week) were used as target animals in the social interaction test (experiment 1). All test mice and target mice were weaned at 18 days of age and then individually housed in polycarbonate cages (28x17x12 cm). A further 99 prewean pups B6129F2 at 11-17 days old were used as the stimulus “same” (n=31) or “different” (n=68) animal in the social recognition test (experiment 2). Handling of all mice was minimized. 2.2.3 Testing procedures  To test the phase conditions, home cage activity was measured in 24 mice (4 females and 4 males from each strain) for 24 hours. Eight individually housed mice were tested at a time. In experiment 1, 72 mice were divided into two sets: 36 mice always tested in a 3- hour period (6:00-9:00 h) in the light phase (18 each in groups 1 and 3) and 36 mice always tested in a 3-hour period (18:00-21:00 h) in the dark phase (18 each in groups 2 and 4). All four groups were matched for strain and sex. Groups 1 and 2 underwent open-field testing and SHIRPA primary screening on day 1 (Fig. 2.1). Groups 3 and 4 underwent the same testing on day 2. The social interaction test was performed on groups 1, 2 and groups 3, 4 on days 3 and 4, respectively. The rotarod test was performed on day 5 for all four groups. The tail-flick test was conducted on day 7. The testing order for strain and sex was based on a   45 constrained randomized sequence. The order of tests was chosen such that the procedures most likely to be affected by prior handling were conducted first. All mice were handled by the tail. A single investigator conducted all tests.  Figure 2.1 One room, two test times  Protocol for comparing L/D cycle effects under reverse light cycle (lights on at 2300 hr. and off at 1100 hr.). a) The phase test was designed to validate the testing conditions used. b) Experiments 1 and 2 were a similar battery of tests conducted on different sets of mice. Groups were tested during the same relative 3-hour period, 7 hrs after a lighting change and 2 hrs before the next lighting change. The light phase is indicated in white, dark phase in grey; the test periods are hatched.   46 In experiment 2, the experimental design from experiment 1 was repeated with a different set of 72 mice and a modified set of tests. The open-field test was repeated with a modified protocol but the SHIRPA was not repeated. The social interaction test was replaced by the social recognition test, the rotarod test was repeated with a modified protocol, and the tail-flick test was replaced with the hot-plate analgesia test.  Each behaviour-testing session began at 6:00 h (for light-phase mice) and 18:00 h (for dark-phase mice). Thus, the tests were conducted during a 3-hour period beginning 7 h after the onset of a new lighting phase and completed 2 h before the onset of the next phase (Figs. 2.1 and 2.2). At the start of each test session, an assistant transported all required mice from the breeding room to the anteroom at one time on a mobile cart. Mice stayed in their home cages in the anteroom with food and water available at all times until they were tested so as not be exposed to other mice being tested. After testing, each mouse was immediately returned to the anteroom. Before each mouse, all equipment was cleaned with 70% ethanol except for the SHIRPA primary screen, where Clidox (Pharmacal Research Laboratory Inc., Naugatuck, CT) was used. 2.2.4 Home cage activity  Home cage activity was measured using eight identical Cage Rack Systems (San Diego Instruments, San Diego, CA). Each mouse home cage is placed in the center of a metal cage rack frame that generates a uniformly spaced 8 x 4 photobeam grid. The mice were provided with food and water and spontaneous locomotor activity was measured by counting the total number of beam breaks each hour during the 24-hour period (Kopp, 2001).    47 2.2.5 Open-field test  Spontaneous exploratory locomotor activity was measured by the open-field test (Slow et al., 2003) using a digiscan photocell-equipped automated open-field apparatus (Med Associates, St. Albans, VT). In experiment 1, all mice were tested in the same open field apparatus 27.5L x 27.5W x 20H cm with lower and upper beams at 1.5 cm and 5.5 cm from floor, respectively. In experiment 2, half the mice were tested in a second open field apparatus 27.3L x 27.3W x 20H cm with lower and upper beams at 1.5 cm and 3.6 cm from floor, respectively. Computer software was used to define two zones: the center 16 x 16 cm (center zone), and the surrounding periphery (residual zone). Each mouse was placed in the center zone of the novel arena and allowed to explore for 3 min while the software tallied spatially identified beam breaks. The following parameters were derived separately for the central and residual zones: distance traveled, ambulatory counts (consecutive interruption of at least four beams within 0.5 sec), time of ambulation, stereotypic counts (number of beam breaks within a virtual ‘box’ of a 4 x 4 beam), time of stereotypic counts, time of rest, vertical counts, and vertical time. The number of jumps in either zone was also recorded.  Since our aim was to examine spontaneous exploratory locomotor activity, and not to subject the mice to a strongly anxiogenic situation, standard room illumination was used during the open-field test in experiment 1 in the light phase. However, to test the hypothesis that a strong anxiogenic situation such as open field with an illuminated center would increase the strain discrimination in the light phase, we added a 60-w bulb 30 cm above the arena in experiment 2. The lighting conditions in the dark phase were the same in experiments 1 and 2.   48 2.2.6 SHIRPA primary screen  The SHIRPA primary screen is a battery of high throughput tests that provides a behavioural and functional profile based on 40 separate parameters observed for each mouse. The SHIRPA screen was conducted 5 minutes after the open-field test, one mouse at a time. The tests followed the SHIRPA protocol primary screen as described by the mouse mutagenesis consortium partners including observations of defecation and urination, but excluding the measure of body length (Rogers et al., 1997). 2.2.7 Social interaction test  The social interaction test evaluates the ethological response of a test mouse to natural conflicts experienced during an encounter with a target mouse in a neutral arena. All encounters occurred in the arena of the open-field apparatus and were recorded by low-light video camera (Panasonic, model AG-5710). Recordings were scored using video analysis software (Observer Video Pro, Noldus, The Netherlands). A test mouse, and then a target mouse, was placed in the centre zone of the arena. Partners only met at the time of testing. The number of social events towards an introduced target mouse was recorded for 3 min. Each target mouse was used for three consecutive tests and then retired. The behavioural responses to the target mouse were classified as either social or non-social, based on ethological profiles (Calamandrei et al., 2000). Social responses were sniffing (sniffing the anogenital region, head, or snout of the partner), following the partner around the cage, without any quick or sudden movement, push under (pushing the snout or the whole anterior part of the body under the partner’s body, and then resting), and crawl over (crawling over the partner’s back, crossing it transversally from one side to the other). Non-social responses were exploring (moving around, rearing, sniffing the air and the walls), and self-grooming   49 (wiping, licking, combing or scratching any part of own body). The non-social response of immobility (laying flat, sitting, or standing still) was also measured.  2.2.8 Social recognition test  The social recognition test measures the ability of the test mouse to recognize a familiar mouse as measured by a reduction in social investigation time between the first and second encounters. Using a modification of the procedure described by Engelmann et. al (Engelmann et al., 1995), the test consisted of a 5-min learning trial during which a novel pup was introduced into the home cage of the test mouse, and the time spent by the test mouse on investigating the pup was measured. Social investigation was defined as the tip of the nose being within approximately 10 mm of the pup and accompanied by sniffing or anogenital investigation. The number of aggressive behaviours towards the pup (tail rattle, attack, biting) was also recorded. The pups were then isolated in individual small plastic cages with paper towel. After a 30-min interval, the test mouse is exposed to either the same or novel pup for 5 min. Observations were recorded by low-light video camera (Panasonic, model AG-5710, Matsuhita Electric Co. of America, Los Angeles, CA). Recordings were scored using video analysis software (Observer Video Pro, Noldus, The Netherlands).  2.2.9 Rotarod test  Motor coordination and balance, and motor learning were measured with the rotarod test as previously described (Slow et al., 2003). The rotating drum (San Diego Instruments, San Diego, CA) accelerated at a constant rate from 0 to 45 rpm over 2 minutes (experiment 1) or 1 minute (experiment 2). Mice were tested in squads of four. Mice were submitted to stationary training (non-moving rod) for 60 sec to adapt to the environment before receiving   50 four consecutive trials with a 3-minute interval between each trial. After a 1-hour rest, a final test was given. In each trial and test, the latency to fall off the rotarod was recorded. Mice clinging on to the rod and rotating for three consecutive rotations were scored as a fall. Motor coordination and balance was scored as the mean latency of the four trials. Motor learning was defined as the test score minus trial 1 score. 2.2.10 Tail-flick test  Pain sensitivity in mice was measured with the tail-flick test (Crawley et al., 2003) using an automated tail-flick analgesia meter (Columbus Instruments, Columbus, OH). Mice were placed in a clear restraining tube (Model 33033, Columbus Instruments) and the tail was placed freely on a level surface between two photo detector panels. Immediately after a 90-sec habituation, radiant heat from a 20-V beam of light was focused on the ventral surface of the tail and the time for the mouse to flick its tail away from the surface was automatically recorded. A 10 sec cut-off time was employed to prevent tissue damage. The average of two consecutive trials, separated by 1 min interval, was calculated. 2.2.11 Hot-plate test  The thermal nociceptive threshold in mice was assessed using a hot plate apparatus (Columbus Instruments, Columbus, OH). Mice were placed on a hot plate thermostatically set at 55.0 ± 0.5 º C (Crawley et al., 2003). The latency of first licking or kicking of the fore or hind paws was recorded. A cut-off time of 60 sec was employed to avoid tissue damage. The average of two consecutive trials, separated by 1 min interval, was calculated.   51 2.2.12 Statistical analysis  All data were analyzed using SPSS® statistical package (SPSS, Chicago, IL). Data from the home cage activity were analyzed by repeated measures ANOVA. Data from the SHIRPA, open field, and social interaction tests were subjected to discriminant analysis separately for the light and dark phases. Discriminant functions, which are linear composites of the original parameters, were defined by Eigenvalues > 1 and Wilk’s lambda p<0.05 in the classification matrix. These functions were used to discriminate between the strains. The ability of a test to discriminate between strains was evaluated by the spread in the data and the relative position of and the distances between group centroids. Data were validated with the “leave one out” cross validation procedure. The home cage activity, social interaction, social recognition, rotarod, hot-plate, and tail-flick data were analyzed with a multifactorial ANOVA for sex, phase, and strain. For all tests, no effect of sex was found so this factor was dropped from the analyses. Multivariate analyses found no effect of which apparatus was used in open field testing for experiment 2 and no effect of which of the 24 target mice was used in the social interaction test in experiment 1. Discrimination was defined as a significant difference between strains within a phase using multifactorial post hoc analyses.   52 2.3 Results 2.3.1 Home cage activity showed expected diurnal patterns in response to reverse L/D cycle  As expected, the home cage activity of the three strains showed a typical diurnal pattern of increased activity levels during the dark phase (Whishaw et al., 1999) (Fig. 2.2a). The sum of beam breaks during the 3-hour period chosen for behaviour testing showed a significant increase in activity level during the dark phase for all three strains (Fig. 2.2b). Thus, we conclude that the mice were responding appropriately to the reverse L/D cycle.  Figure 2.2 Home cage activity is affected by L/D cycle  a) More beam breaks occur in the dark phase than in the light phase as summed every hour over 24 hours. b) Sum of beam breaks during the 3-hour test period confirms higher activity in the dark phase for all three strains, thus validating the test conditions. *p<0.004. N = 4 per strain/sex.   53 2.3.2 Open-field test discriminates better in the dark phase  Discriminant analysis included all seventeen parameters measured by the open field to define two significant functions for the dark phase data (Function 1 (F1), Eigenvalue 23.72, Wilks’ lambda significance <0.001; F2, Eigenvalue 8.2, Wilks’ lambda significance <0.001), but only one function for the light phase data in experiment 1 (light phase = room lighting) (F1, Eigenvalue 12.53, Wilks’ lambda significance <0.001) (Fig. 2.3, experiment 1). Furthermore, the cross validation of parameters led to 100 % reclassification of original grouped cases in the dark phase, whereas in the light phase the reclassification was 94.4%. Thus, the dark phase provided better discrimination between strains than the light phase, as is demonstrated by the relative position of and distance between centroids on discriminant function plots (Fig. 2.4, experiment 1).   54  Figure 2.3 The open-field test discriminates better in the dark phase Discriminant analysis performed on the 17 parameters measured by the open field defined two significant functions for the dark phase data, but only one significant function for the light phase data in both experiment 1 (light phase = room light) and experiment 2 (light phase = bright light) based on Eigenvalues > 1 and p<0.05 for Wilk’s lambda. Correlation coefficients for parameters used to define function 1 (F1) and function 2 (F2) are shown in bold italic text. The functions are defined by all 17 parameters measured. N = 6 per strain/sex/phase for each experiment.  To test the possibility of improving the strain discrimination in the light phase, the open-field test was repeated in experiment 2 using the stronger anxiogenic situation of brighter illumination during light-phase testing. However, discriminant analysis again defined two significant functions for the dark phase data (F1, Eigenvalue 15.26, Wilks’ lambda significance <0.001; F2, Eigenvalue 2.79, Wilks’ lambda significance 0.007), but only one function for the light phase data (F1, Eigenvalue 28.41, Wilks’ lambda significance <0.001) (Fig. 2.3, experiment 2). Furthermore, the cross validation of parameters led to 100   55 % reclassification of original grouped cases in the dark phase versus 86.1% in the light phase. Thus, increased illumination of the open field did not improve strain discrimination in the light phase as is demonstrated by the relative position of and distance between centroids on discriminant function plots (Fig. 2.4, experiment 2). We conclude that open-field testing discriminates strains better in the dark phase.  Figure 2.4 Discriminant function plots of open-field data show improved strain discrimination in the dark phase Experiment 1: discrimination between strains for function 1 and 2 is better in the dark phase, as demonstrated by the relative position of and distances between group centroids. Experiment 2: The bright lighting conditions used during the light phase of experiment 2 did not significantly improve discrimination in the light phase. The better phase is indicated by a box. N = 6 per strain/sex/phase for each experiment.   56 2.3.3 SHIRPA primary screen discriminates better in the dark phase  Discriminant analysis performed on the 41 SHIRPA parameters successfully defined two significant functions for both the dark-phase (F1, Eigenvalue 87.037, Wilks’ lambda significance <0.001; F2, Eigenvalue 10.954, Wilks’ lambda significance <0.001) and light- phase data (F1, Eigenvalue 25.07, Wilks’ lambda significance <0.001; F2, Eigenvalue 7.895, Wilks’ lambda significance 0.002) (Fig. 2.5). However, the cross validation of parameters led to 91.7 % reclassification of original grouped cases in the dark phase versus only 63.9% in the light phase. In addition, the relative position of and distance between centroids on discriminant function plots showed improved discrimination in the dark phase (Fig. 2.6). Furthermore, the dark phase only required 21 parameters versus 24 for the light phase to define discriminant functions. Thus, we conclude that SHIRPA data discriminate strains better in the dark phase. Interestingly, in either phase, only approximately half the parameters measured were used.    57     Figure 2.5 The SHIRPA primary screen discriminates better in the dark phase  Discriminant analysis performed on the 41 parameters measured by the SHIRPA data defined two significant functions in both the light and dark phases based on Eigenvalues > 1 and p<0.05 for Wilk’s lambda. Correlation coefficients for parameters used to define function 1 (F1) and function 2 (F2) are shown in bold italic text. The functions are defined by 24 parameters in the light phase but only 21 in the dark phase. N = 6 per strain/sex/phase for each experiment.    58  Figure 2.6 Discriminant function plots of SHIRPA data show improved strain discrimination in the dark phase Discrimination between strains for both functions is better in the dark phase, as demonstrated by the relative position of and distances between group centroids. The better phase is indicated by a box. N = 6 per strain/sex/phase.  2.3.4 Social interaction test is not improved by the dark phase  Discriminant analysis of the social interaction data failed to define any significant functions. However, the multifactorial ANOVA indicated significant effects of phase for self grooming (p<0.04), sniffing (p<0.04), and immobility (p<0.001); and effects of strain for self grooming (p<0.006), sniffing (p=0.001), immobility (p<0.03), and exploring (p<0.001). No significant effects on push under were found. Three parameters for B6 mice (self grooming, crawl over, and immobility), and two parameters for 129 mice (sniffing and immobility), gave significantly different results between the light and dark phases; these mice showed more self grooming, more social interactions, and less immobility in the dark phase (Fig. 2.7). F1 mice were not significantly affected by phase. Discrimination between strains did not show a clear benefit to either phase. Three of the parameters (self grooming, crawl over, and exploring) showed better discrimination in the dark phase and three parameters (sniffing,   59 following, and immobility) showed better discrimination in the light phase. Thus, although the social interaction test is affected by L/D cycle, there is no clear advantage to dark-phase testing.   Figure 2.7 The social interaction test was affected by L/D cycle but discrimination was not clearly better in one phase than the other  The six of the seven parameters measured that showed significant effect of phase or strain are shown here. Phase was a significant effect for a, b, d, and f. Three parameters for B6 mice (a,b,f) and two parameters for 129 mice (d,f) gave significantly different results between the light and dark phases; these mice showed more self grooming, more social interactions, and less immobility in the dark phase. The F1 mice were not significantly affected by phase. Discrimination between strains did not show a clear benefit to either phase. Three parameters showed better discrimination in the dark phase (a-c) but three showed better discrimination in the light phase (d-f). The better phase is indicated by a box. *p<0.04. N = 6 per strain/sex/phase.    60 2.3.5 Social Recognition test is not improved by the dark phase  Because the social interaction test failed to show a clear advantage to testing in either phase, we examined the effects of dark-phase testing on a second high throughput test for social behaviour, the social recognition test. This test assesses a mouse’s ability to recognize a familiar pup upon a second encounter as measured by a reduction in time spent investigating it. The multifactorial ANOVA found no effect of phase or strain. However, since the difference in investigation time between the first and second encounter for the ‘same’ pup failed to reach statistical significance for all three strains for both phases (data not shown), we conclude that the test did not work. Therefore, no conclusions can be drawn about the effects of phase. We hypothesize the test failure may be due to the age of the pups used, which may have been too old (11-17 days) and thus perceived by the test mice as intruders. In fact, we did note a significant difference in frequency (p<0.05) and duration (p<0.05) of aggressive behaviours between strains in the dark phase by Kruskal-Wallis ranked sums test and noted the highest score means for the F1 mice. 2.3.6 The rotarod test discriminates better in the dark phase  In experiment 1, no differences in performance (motor coordination and balance), as assessed by the mean latency to fall from the rod during four trials, were observed between the light and dark phases for any of the three strains (Fig. 2.8a). A multifactorial ANOVA found an effect of strain (p<0.001) but not phase. Performance was significantly different between all three strains in both light and dark phases. Motor learning, as assessed by the difference between the final test score minus the first trial, also showed an effect of strain (p<0.009) but not of phase. However, dark phase   61 testing did improve the test’s ability to discriminate between strains; the 129 mice tested in the dark phase showed significantly better learning than both B6 and F1 mice (Fig. 2.8b).    Figure 2.8 The rotarod test discriminates better in the dark phase  a) In experiment 1 (0-45 rpm over 2 min), all three strains showed no difference in average latency to fall from the rotarod between phases. Furthermore, discrimination between the strains worked well in both phases. b) Motor learning was not different between phases but showed improved strain discrimination in the dark phase. c) In experiment 2 (0-45 rpm over 1 min), B6 and 129 mice demonstrated improved performance in the light phase and again, discrimination between the strains worked equally in both phases. d) Motor learning was not affected by phase under these conditions. The better phase is indicated by a box. *p<0.02. Since performance, strain discrimination, and learning were all better in experiment 1 than in experiment 2, we conclude that the slower acceleration time was more appropriate. Only under these conditions did dark phase testing result in improved strain discrimination for motor learning. N = 6 per strain/sex/phase for each experiment.   62  To further optimize the rotarod test by reducing throughput time, we repeated it in experiment 2 using a shorter test time (1 min rather than 2 min) and a faster acceleration. Under these more challenging conditions, B6 and 129 mice showed longer latency to fall in the light phase than in the dark phase (Fig. 2.8c). However, in both phases, the test could not discriminate differences between B6 and F1 mice as had been achieved in experiment 1, but rather, only between 129 and the other two strains. Motor learning in experiment 2 showed no effect of phase or strain (Fig. 2.8d). 2.3.7 The tail-flick test discriminates only in the light phase  The multivariate ANOVA found significant effects of phase (p<0.002) and strain (p<0.001). The 129 mice showed significantly longer latency to flick their tails in the light phase than in the dark phase (Fig. 2.9). This accounted for discrimination between the strains in the light phase that was not possible in the dark phase. B6 and F1 mice were unaffected by phase for this test. Thus, we conclude that, when 129 mice are being tested, the tail-flick test discriminates better in the light phase.  63  Figure 2.9 The tail-flick test does not discriminate better in the dark phase  The 129 mice show a longer latency to remove their tails in the light phase - a response that differentiated them from the other strains in the light phase only. B6 and F1 mice were unaffected by phase. The better phase is indicated by a box. *p<0.001. N = 6 per strain/sex/phase.  2.3.8 The hot-plate test does not discriminate better in the dark phase  As an alternative to the tail-flick test for pain sensitivity, for which phase could have been affected by the light beam used to generate the heat source, we assessed pain response with the hot-plate test. The multivariate ANOVA found significant effects of phase (p<0.001) and strain (p<0.001). The latency to lick or kick a paw was shorter in the dark phase than in the light phase for B6 mice (Fig. 2.10). However, the test could discriminate 129 mice, who had significantly longer latency than the other two strains, equally well in both phases.    64  Figure 2.10 The hot-plate test does not discriminate better in the dark phase The latency to lick or kick a paw was shorter in the dark phase than in the light phase for B6 mice. However, the ability to discriminate 129 mice, which had significantly longer latency than the other two strains, was not different between the phases. The better phase is indicated by a box. *p<0.04. N = 6 per strain/sex/phase.  2.4 Discussion  The present study demonstrates that dark-phase testing affects the outcome of high throughput behavioural phenotyping. Six of the seven tests showed significant phase differences for at least one parameter in at least one strain. Generally, where significant differences existed, activity levels were higher in the dark phase. For example, in the social interaction test, mice tested in the dark phase demonstrated increased self grooming (B6), crawl overs (B6), sniffing (129), and decreased immobility (B6 and 129). Similarly, in each test of pain sensitivity, a shorter latency to move was present in the dark phase: tail flick (129), and hotplate (B6). The only exception to this was in the second rotarod experiment for which the test conditions were too challenging and thus, inappropriate. Importantly, an overall examination of the data does not show the results from a single strain drove the light dark differences, but rather that both B6 and 129 are affected by phase, whereas F1 is generally not.   65 Dark-phase testing provided improved discrimination between strains using the SHIRPA primary screen. Although the discriminant analysis was able to discriminate between the strains in the light phase as others have shown (Rogers et al., 1999), the dark phase was clearly more sensitive. Since discrimination between genetically different mice is the ultimate goal of a behavioural screen such as SHIRPA, we must conclude that in our test conditions, dark-phase testing is superior. Dark-phase testing also provided better strain discrimination using the open-field test. Since the open-field test is an assessment of spontaneous exploratory behaviour, and exploration is naturally a dark-phase behaviour for mice, the improved ability to discriminate strains in the dark supports our hypothesis for ethologically correct testing.  In contrast, we were surprised to find that social interaction did not consistently discriminate strains better in the dark phase, but rather that the significantly different parameters split evenly, in their discriminate ability, between the light and dark phases. A likely explanation is that the test mouse was so focused on the target mouse during the brief interaction time (3 min.), that effects of L/D cycle were less important to the response. However, the significant effects of strain and phase we did find are supported by the work of previous investigators (Lister & Hilakivi, 1988, Paterson & Vickers, 1984, Pieper et al., 1997). Similarly, performance on the rotarod may be so driven by the strong stimulus of the rotating rod, that there is little or no affect of phase. However, despite this strong stimulus, the motor learning ability of the 129 mice in the dark phase was still enhanced sufficiently that strain discrimination was achieved only in this phase.    66 Interestingly, even in the competing presence of a strong pain stimulus, the 129 mice were so affected by phase that strain discrimination occurred in only one phase. Because our goal was to interpret results in terms of strain discrimination, it is the light phase that we score as better. However, a likely explanation for this result is that the 129 mice are ‘frozen’ in the light, a common behaviour in this strain, and so appear differentially more resistant to pain than B6 and F1, whereas in the dark they don’t freeze and thus are indistinguishable from the other strains.  The practicalities of working in the dark phase must also be considered. We found the challenges of reverse light cycle to be easily overcome using the methodology we have described; reverse light cycle, dim red light, and low-light level camera. However, not all tests were easily amenable to the dark-phase. For example, the SHIRPA parameter, skin color, was difficult to assess in the dark. In addition, the researcher may have been less coordinated and had slower movements in the dark. Furthermore, the researcher was less likely to be blinded to sex or strain (as indicated by coat color) in the light phase. Thus, we conclude that these unavoidable differences may have contributed, along with the intended differences in diurnal cycle and illumination, to the effect of phase. Should high throughput behaviour testing be done in the dark phase? Since the preponderance of our data shows dark-phase testing improves, or does not affect strain discrimination, we conclude that for these strains and tests, dark-phase testing provided superior outcomes. If discrimination is not achieved in the dark phase, or if the test is known a priori to have a strong test stimulus, then light phase testing would be undertaken.    67 2.5 References  Brown, R.E., Stanford, L. & Schellinck, H.M. (2000) Developing standardized behavioral tests for knockout and mutant mice. ILAR J, 41, 163-174. Calamandrei, G., Venerosi, A., Branchi, I., Valanzano, A. & Alleva, E. (2000) Prenatal exposure to anti-HIV drugs. long-term neurobehavioral effects of lamivudine (3TC) in CD-1 mice. Neurotoxicol Teratol, 22, 369-379. Crabbe, J.C., Wahlsten, D. & Dudek, B.C. (1999) Genetics of mouse behavior: interactions with laboratory environment. Science, 284, 1670-1672. Crawley, J.N. (2000) What's wrong with my mouse?: behavioural phenotyping of transgenic and knockout mice, A John Wiley and Sons, Inc., New York. Crawley, J.N., Gerfen, C.R., Rogawski, M.A., Sibley, D.A., Skolnick, P. & Wray, S. (2003) Current Protocols in Neuroscience. In Taylor, G.P. (ed), John Wiley & Sons, Inc. Crawley, J.N. & Paylor, R. (1997) A proposed test battery and constellations of specific behavioral paradigms to investigate the behavioral phenotypes of transgenic and knockout mice. Horm Behav, 31, 197-211. Dierssen, M., Fotaki, V., Martinez de Lagran, M., Gratacos, M., Arbones, M., Fillat, C. & Estivill, X. (2002) Neurobehavioral development of two mouse lines commonly used in transgenic studies. Pharmacol Biochem Behav, 73, 19-25. Engelmann, M., Wotjak, C.T. & Landgraf, R. (1995) Social discrimination procedure: an alternative method to investigate juvenile recognition abilities in rats. Physiol Behav, 58, 315-321.   68 Jacobs, G.H., Fenwick, J.C., Calderone, J.B. & Deeb, S.S. (1999) Human cone pigment expressed in transgenic mice yields altered vision. J Neurosci, 19, 3258-3265. Kopp, C. (2001) Locomotor activity rhythm in inbred strains of mice: implications for behavioural studies. Behav Brain Res, 125, 93-96. Kriegsfeld, L.J., Eliasson, M.J., Demas, G.E., Blackshaw, S., Dawson, T.M., Nelson, R.J. & Snyder, S.H. (1999) Nocturnal motor coordination deficits in neuronal nitric oxide synthase knock-out mice. Neuroscience, 89, 311-315. Lister, R.G. & Hilakivi, L.A. (1988) The effects of novelty, isolation, light and ethanol on the social behavior of mice. Psychopharmacology (Berl), 96, 181-187. Marques, M.D. & Waterhouse, J.M. (1994) Masking and the evolution of circadian rhythmicity. Chronobiol Int, 11, 146-155. Nejdi, A., Guastavino, J.M. & Lalonde, R. (1996) Effects of the light--dark cycle on a water tank social interaction test in mice. Physiol Behav, 59, 45-47. Paigen, K. & Eppig, J.T. (2000) A mouse phenome project. Mamm Genome, 11, 715-717. Paterson, A.T. & Vickers, C. (1984) Saline drinking and naloxone: lightcycle dependent effects on social behaviour in male mice. Pharmacol Biochem Behav, 21, 495- 499. Pieper, J.O., Forester, D.C. & Elmer, G.I. (1997) Mice show strain differences in social affiliation. Implications for open field behavior. Ann N Y Acad Sci, 807, 552-555. Rogers, D.C., Fisher, E.M., Brown, S.D., Peters, J., Hunter, A.J. & Martin, J.E. (1997) Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment. Mamm Genome, 8, 711-713.   69 Rogers, D.C., Jones, D.N., Nelson, P.R., Jones, C.M., Quilter, C.A., Robinson, T.L. & Hagan, J.J. (1999) Use of SHIRPA and discriminant analysis to characterise marked differences in the behavioural phenotype of six inbred mouse strains. Behavioural Brain Research, 105, 207-217. Silva, A., Simpson, E.M., Takahashi, J.S., Lipp, H.-P., Nakanishi, S., Wehner, J.M., Giese, K.P., Tully, T., Abel, T., Chapman, P.F., Fox, K., Grant, S., Itohara, S., Lathe, R., Mayford, M., McNamara, J.O., Morris, R.J., Picciotto, M., Roder, J., Shin, H.-S., Slesinger, P.A., Storm, D.R., Stryker, M.P., Tonegawa, S., Wang, Y. & Wolfer, D.P. (1997) Mutant mice and neuroscience: recommendations concerning genetic background. Neuron, 19, 755-759. Slow, E.J., van Raamsdonk, J., Rogers, D., Coleman, S.H., Graham, R.K., Deng, Y., Oh, R., Bissada, N., Hossain, S.M., Yang, Y.Z., Li, X.J., Simpson, E.M., Gutekunst, C.A., Leavitt, B.R. & Hayden, M.R. (2003) Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease. Human Molecular Genetics, 12, 1555-1567. Valentinuzzi, V.S., Buxton, O.M., Chang, A.M., Scarbrough, K., Ferrari, E.A., Takahashi, J.S. & Turek, F.W. (2000) Locomotor response to an open field during C57BL/6J active and inactive phases: differences dependent on conditions of illumination. Physiol Behav, 69, 269-275. van der Staay, F.J. & Steckler, T. (2002) The fallacy of behavioral phenotyping without standardisation. Genes, Brain and Behavior, 1, 9-13. Wahlsten, D. (2001) Standardizing tests of mouse behavior: reasons, recommendations, and reality. Physiol Behav, 73, 695-704.   70 Whishaw, I.Q., Haun, F. & Kolb, B. (1999) Analysis of Behavior in Laboratory Rodents. In Windhorst, U. & Johansson, H. (eds), Modern Techniques in Neuroscience. Springer-Verlag, Berlin, pp. 1243-1275. Würbel, H. (2002) Behavioral phenotyping enhanced - beyond (environmental) standardization. Genes, Brain and Behavior, 1, 3-8.   71 Chapter 3: Hyperactivity, startle reactivity and cell-proliferation deficits are lithium resistant in Nr2e1frc/frc mice2 3.1 Introduction  Although bipolar disorder (BP) is a multifactorial psychiatric disorder that is highly heritable (60-85%) (Burmeister et al., 2008), and the 6q chromosomal region has repeatedly shown evidence for genetic linkage to BP and other neurological disorders (Dick et al., 2003, Hayden & Nurnberger, 2006, Kohn & Lerer, 2005, Mcqueen et al., 2005, Middleton et al., 2004, Pato et al., 2004, Schulze et al., 2004), the causative genes for BP are just beginning to be identified (Craddock & Sklar, 2009, Martinowich et al., 2009, Ogden et al., 2004). The largest meta-analysis of BP to date, found the strongest genome-wide linkage at 6q21-22 (108.5 Mb), with the highest LOD score (4.19) specifically for bipolar I disorder (BPI), the more manic subtype (Mcqueen et al., 2005). One of the genes in the 6q21-22 region is NR2E1. A role for NR2E1 in BP has also been supported by a significant association after correction between NR2E1 and BPI, and candidate mutations in NR2E1 in BP patients (Kumar et al., 2008).  Mice lacking orphan nuclear receptor Nr2e1, the mammalian homolog of the Drosophila Tlx (tailless) gene, have been developed in several laboratories (aka Tlx-/-, Nr2e1frc/frc) and are generally referred to as Nr2e1-null mice. Unlike the Nr2e1 heterozygous mice that have no significant behavioural and neurological phenotypes (Young et al., 2002), the Nr2e1-null mice have a wide range of neurological                                                  2 A version of this chapter has been submitted for publication. Wong, B.K.Y., Hossain, S.M., Trinh, E., Ottmann, G.A., Budaghzadeh, S., Zheng, Q.Y., and Simpson, E.M.. Hyperactivity, startle reactivity and cell-proliferation deficits are lithium resistant in Nr2e1frc/frc mice.    72 abnormalities, of particular interest are those similar to abnormalities seen in some patients with BP, including: increased lateral ventricular volume; reduced volume of the hippocampus, cerebral cortex, corpus callosum, amygdala, and cortical layers II and III; olfactory abnormality and dysfunction; altered cell cycling, cell morphology and plasticity in the hippocampus; reduced neurogenesis; impairment in GABAergic interneurons; and cognitive deficits (Anand & Shekhar, 2003, Brambilla et al., 2003, Christie et al., 2006, Goldberg & Chengappa, 2009, Kruger et al., 2006, Land & Monaghan, 2003, Mccurdy et al., 2006, Monaghan et al., 1997, Roy et al., 2004, Roy et al., 2002, Shi et al., 2004, Stenman et al., 2003, Swayze et al., 1990, Tian et al., 2007, Young et al., 2002, Zhang et al., 2008). These neurological similarities, as well as linkage and association evidences, provide strong support for NR2E1 as a candidate for BP, especially BPI. Despite the support for NR2E1 in BP, Nr2e1-null mice have not been fully characterized for anomalies similar to those seen in some patients with BP. Here, we examine Nr2e1frc/frc mice carrying a spontaneous deletion of Nr2e1 (Kumar et al., 2004) for abnormalities in activity level, cognition, information processing, and cell proliferation in neurogenic regions. To further evaluate the similarity of Nr2e1frc/frc mice and BP, we tested the effect of lithium treatment on these parameters. Lithium is the standard treatment for BP and it has been shown to attenuate psychostimulus-induced hyperactivity in rodent models of mania (O'donnell K & Gould, 2007) and to promote cell proliferation in the dentate gyrus (DG) (Son et al., 2003). Considering that Nr2e1- null neural stem/progenitor cells (NSCs) showed reduced proliferation that could be rescued by reintroducing Nr2e1 in vitro (Shi et al., 2004), we tested whether lithium   73 could allow reengagement of the cell cycle in these quiescent NSCs and whether any behavioural amelioration would accompany.  3.2 Methods and materials 3.2.1 Mice  The B6129F1-Nr2e1 mice used for experimental analysis were all first generation offspring resulting from mating C57BL/6J.129-Nr2e1frc (B6-Nr2e1frc/+) females (backcross generation N17-22) to 129S1/SvImJ.Cg-Nr2e1frc (129-Nr2e1frc/+) males (N15- 20). The Nr2e1frc allele is a 44 kb spontaneous deletion of all 9 exons of Nr2e1 that does not affect transcription of neighboring genes (Kumar et al., 2004). In accordance with Mendelian inheritance, approximately 25% of the offspring were homozygous Nr2e1frc/frc mice and 25% were Nr2e1+/+ (Wt) littermates; the latter were used as controls. All mice were weaned at postnatal day (P)18 - 21, housed with same-sex littermates, and then individually housed by 4 weeks to avoid aggressive incidence with Nr2e1frc/frc mice and to be consistent for all mice. Mice were provided with food and water ad libitum and were provided standard care according to University of British Columbia animal care policies. Handling of all mice was minimized.  3.2.2 Genotyping  All mice were analyzed by two separate polymerase chain reaction (PCR) assays. Wild-type allele of Nr2e1 was amplified using oEMS1859 (5′- CTGGGCCCTGCAGATACTC-3′) and oEMS1860 (5′- GGTGGCATGATGGGTAACTC-3′), and the fierce deletion allele of Nr2e1 was detected using oEMS650 (5′-GGCGGAGGGAGCTTAAATAG-3′) and oEMS1368 (5′-  74 GATTCATCCTATTCCACAAAGTCA-3′). Cycling conditions were as follow: 2 min at 92°C, 30 cycles of 30 s at 94°C, 30 s at 58°C, and 55 s at 72°C; and a final extension of 5 min at 72°C. 3.2.3 Testing procedure  All mice were tested in the pathogen-free behaviour suite under reverse L/D cycle (light 23:00-11:00 h at 320 lux), at the Centre for Molecular Medicine and Therapeutics, Vancouver, Canada, as previously described (Hossain et al., 2004). The multi-room behaviour suite consists of a breeding room and dedicated testing rooms, separated by corridors. The lighting in all areas was synchronized. Care was taken not to expose the mice to any inappropriate light, even during testing. When light was needed by the investigator during experiments in the dark phase, a dim red light (8 lux) was used. All adult mice tested were males between the ages of 2 – 6 months. The testing chambers and equipment were thoroughly cleaned between each test subject, using Clidox (Pharmacal Research Laboratories Inc., Naugatuck, CT) and 70% ethanol. 3.2.4 Pup body weight and milk consumption  The body weights of 15 Wt and 14 Nr2e1frc/frc pups were measured at P0, 7, 14, and 21. Pups were individually placed on a clean plastic weigh boat and body weight was measured on a bench-top balance. The amount of milk consumption was similarly measured in a different cohort of 11 Wt and 12 Nr2e1frc/frc pups. Pups were removed from their mother and weighed, then kept separate from their mother for 2 h after which the pups were returned to their mother and given 15 min for feeding and were weighed again.   75 3.2.5 Pup open field activity  Spontaneous exploratory locomotor activity was measured on 10 Wt and 12 Nr2e1frc/frc pups at P9, 14, and 18 using a digiscan photocell-equipped automated open field apparatus 27.5 cm (L) x 27.5 cm (W) x 20.0 cm (H) with lower and upper beams at 1.5 cm and 5.5 cm from the floor, respectively (Med Associates Inc., St. Albans, VT). Each pup was placed in the center of the novel arena and allowed to explore for 3 min while the software tallied spatially identified beam breaks.  3.2.6 Home cage activity  Home cage activity was measured on a total of 8 Wt and 8 Nr2e1frc/frc mice during a 48-h period using identical Cage Rack Systems (San Diego Instruments, San Diego, CA). Each mouse home cage was placed in the center of a metal cage rack frame that generates a uniformly spaced 8 × 4 photobeam grid. The mice were provided with food and water ad libitum throughout the testing period and spontaneous locomotor activity was measured by counting the total number of beam breaks each hour during the 48-h period (Kopp, 2001). 3.2.7 Open field activity and habituation  Activity and habituation in the open field of 12 Wt and 9 Nr2e1frc/frc mice were measured using the open field apparatus described above (Pup open field activity). Mice were introduced to the open field apparatus for three consecutive days and tested for 10 min each time. The numbers of beam breaks were recorded for all trials.   76 3.2.8 Tail suspension  Struggling during the 3 min tail suspension test was measured on 8 Wt and 4 Nr2e1frc/frc mice using a PHM-300TSS mouse tail suspension system (Med Associates, St. Albans, VT), as previously described (Abrahams et al., 2005). The apparatus was calibrated to normalize for body weight before testing of each animal and the settings for struggle and gain were 15 and 4, respectively. Percent time struggle was calculated as time spent struggling during which force exceeded the struggle threshold (set to 15) divided by the total testing time (3 min).  3.2.9 Hot plate and tail flick  Thermal nociception and pain sensitivity of 8 Wt and 8 Nr2e1frc/frc mice was measured for each mouse using the hot plate and tail flick tests, respectively, as previously described (Hossain et al., 2004). Mice were placed on the hot plate apparatus (Columbus Instruments, Columbus, OH) thermostatically set at 55.0 ± 0.5 °C. The latency of first licking or kicking of the fore or hind paw was recorded. A cut-off time of 60 s was employed to avoid tissue damage.  For the tail flick test, mice were placed in a clear restraining tube (Model 33033, Columbus Instruments, Columbus, OH) and the tail was placed freely on a level surface between two photo detector panels of the automated tail flick analgesia meter (Columbus Instruments, Columbus, OH). Immediately after a 90-s habituation period, radiant heat from a 20-V beam of light was focused on the ventral surface of the tail and the time for the mouse to flick its tail was automatically recorded by the apparatus. A 10-s cut-off time was employed to prevent tissue damage.    77 For both tests, the average of two consecutive trials, separated by a 1-min interval, was calculated for each animal. 3.2.10 Auditory brainstem response  Auditory functions of 5 Wt and 4 Nr2e1frc/frc mice were tested using the auditory brainstem response (ABR) procedure, as previously described (Zheng et al., 1999). Briefly, electrodes were placed under the scalp and recordings taken as different sound intensities were presented to anesthetized mice. 3.2.11 Passive avoidance  Learning and memory of 9 Wt and 6 Nr2e1frc/frc mice was tested in the passive avoidance test using the GEMINITM Avoidance System (San Diego Instruments, San Diego, CA). The equipment has two chambers separated by a sliding door. Mice were introduced to the first chamber in the presence of an auditory stimulus. After 30 s in the first chamber, the door separating the two chambers opened and the mouse was allowed to enter into the second chamber without the auditory stimulus. The time it took for the mouse to enter the second chamber after the door opened was recorded. The maximum time allowed to enter the second chamber was 180 s. Once the mouse entered the second chamber it received a mild electrical shock. The mouse was again tested 24 h later and the latency of entering the second chamber was recorded. 3.2.12 Acoustic startle reactivity  Acoustic startle reactivity was tested using the SR-LAB system (San Diego Instruments, San Diego, CA). Two separate groups of male mice were used: Group 1 (12 Wt, 9 Nr2e1frc/frc) and Group 2 (7 Wt, 7 Nr2e1frc/frc). After a 5-min acclimatization period,   78 each mouse was subjected to 90 acoustic startle stimuli (10 at each of nine intensities ranging from 75 to 125 dB) in a semi-randomized sequence. The startles had a fixed duration of 50 ms and were separated by a variable inter-stimulus interval (ISI) ranging from 20 to 30 s, while the recording window was set at 100 ms. Startle response was measured at each stimulus as well as at 10 no-stimulus trials. 3.2.13 Lithium administration and testing procedure  3 Wt and 5 Nr2e1frc/frc male mice received lithium chloride (LiCl) diets, while 4 Wt and 4 Nr2e1frc/frc male mice received control diets. Mice on the control diet were fed with untreated purified diet with Teklad Vitamin Mix (Harlan Teklad, Madison, WI). Mice on the lithium diet were fed with 1.7 g LiCl/kg added to the untreated purified diet with Teklad Vitamin Mix (Harlan Teklad, Madison, WI) for 4 weeks, and then switched to 2.55 g LiCl/kg added to the untreated purified diet with Teklad Vitamin Mix (Harlan Teklad, Madison, WI) for 2 additional weeks, before behaviour testing. These mice remained on the 2.55 g LiCl/kg of chow diet throughout the testing period. All mice were also given water ad libitum and a water bottle of 450 mM sodium chloride solution. Each mouse was subjected to behaviour tests in the following order: home cage activity, open field activity and habituation, and startle reactivity. The start of each test was performed one week after the end of the previous test. Tests were performed as described in the above sections. At the end of behaviour testing all animals were sacrificed and bled for serum analysis of lithium level, and brains were harvested for immunohistochemical analysis.   79 3.2.14 Serum analysis   Mice from the lithium-treatment experiment were given a lethal injection of 2,2,2- tribromoethanol in tert amyl alcohol (Sigma-Aldrich, St. Louis, MO) (aka avertin) and blood was collected via cardiac puncture using a 25-gauge needle. Blood samples were allowed to separate for 30 min at room temperature (RT). Samples were then centrifuged for 10 min at RT at 3000 RPM for separation of serum. The serum was then isolated and kept at –20°C until lithium levels analyses. The Department of Pathology and Laboratory Medicine at Vancouver General Hospital, blinded to the experimental conditions, analyzed serum lithium level. 0.2 mmol/L was the minimum detection limit of lithium serum assay. 3.2.15 Brain harvesting and immunohistochemistry  Brains of mice from the lithium-treatment experiment were dissected out intact and placed into 4% paraformaldehyde in 1× PBS at 4°C for 48 h, then transferred to a 20% sucrose solution at 4°C until saturated. Brains were then sectioned at 25 μm using the Cryo-Star HM 560 cryostat (MICROM International, Walldorf, Germany) and representative sections (every 24th) starting from the most rostral aspect of the ventricles to the most caudal aspect of the hippocampus were analyzed by immunofluorescence. Sections were blocked with 5% normal goat serum (NGS) + 5% bovine serum albumin (BSA) in 0.1% Triton-X100 in PBS, incubated overnight at RT with rabbit anti- Ki67 polyclonal antibody (1:1000 dil, Cat. #ab15580; Abcam Inc., Cambridge, MA), and further incubated with Alexa Fluor® 594 goat anti-rabbit IgG (H+L) (Cat. #A31631; Invitrogen, Carlsbad, CA). Sections were mounted onto Superfrost® Plus slides (Cat.   80 #12-550-15; Fisher Scientific, Ottawa, ON) and coverslipped using Vectashield Hard Set™ (Cat. #H-1400; Vector Laboratories, Inc., Burlingame, CA). Images were captured on an Olympus BX61 motorized fluorescence microscope (Olympus America Inc., Center Valley, PA) and proliferating cells in the SVZ and DG were analyzed using the ImageJ software. 3.2.16 Statistical analysis  All data were analyzed using STATISTICA© 6 (StatSoft, Inc., Tulsa, OK). Body weight, milk consumption, tail suspension, hot plate, tail flick, auditory brainstem response, and passive avoidance data were analyzed by t-test on genotype. The remaining behavioural data were analyzed using repeated measures ANOVA for genotype and trials. In all repeated measures ANOVAs the Greenhouse-Geisser correction factor (ε) was used to adjust the degrees of freedom (Vasey & Thayer, 1987). Post-hoc tests with Bonferroni correction were performed for repeated between-subject comparisons.  Behavioural data pertaining to the lithium experiment were analyzed using repeated measures ANOVA for interaction between genotype and drug treatment. The same corrections as above were performed for these analyses. Cell proliferation data were analyzed using factorial ANOVA for genotype and drug treatment. All data are reported as mean values ± standard error of the mean (SEM).  3.3 Results  3.3.1 Young Nr2e1frc/frc mice show early hyperactivity  Previously, we showed that Nr2e1frc/frc pups on a C57BL/6J (B6) background failed to gain weight at the rate of their Wt littermates between postnatal weeks 2 and 3   81 (Young et al., 2002). For the current study, we retested this phenotype at postnatal (P) 0, 7, 14, and 21 on the B6129F1 background. We showed that B6129F1-Nr2e1frc/frc mice were also significantly smaller than their Wt siblings at P21 (Fig. 3.1a; Wt = 14.1 ± 0.2 g, Nr2e1frc/frc = 12.7 ± 0.2 g, P < 0.001), but not at P0, 7, or 14. Therefore, small size at wean is a stable phenotype across two genetic backgrounds.  Figure 3.1 Reduced body weight of Nr2e1frc/frc pups not explained by milk consumption (a) Nr2e1frc/frc pups weighed significantly less than Wt pups by postnatal day 21. * P < 0.001. N = 15 Wt and 14 Nr2e1frc/frc pups. (b) Pups were weighed before and after feeding to determine their amount of milk consumption. No significant difference in milk intake was seen between Wt and Nr2e1frc/frc pups (P > 0.1). N = 11 Wt and 12 Nr2e1frc/frc pups.    82 We measured milk consumption in pre-wean pups to test the hypothesis that the failure of Nr2e1frc/frc mice to gain weight normally may depend on a reduction in milk consumption. This hypothesis was not supported by the milk consumption data, where no significant differences were found between the two genotypes (Fig. 3.1b; Wt = 0.059 ± 0.004 g, Nr2e1frc/frc = 0.07 ± 0.01 g, P > 0.1). We then measured activity level in the same group of pre-wean pups at P9, 14, and 18 using the open field apparatus. Activity level was significantly higher in Nr2e1frc/frc mice than Wt controls at P18 (Fig. 3.2; Beam breaks: Wt = 186 ± 15.0, Nr2e1frc/frc = 325 ± 39.9, P < 0.01), but not at P9 and 14 (P > 0.1). Therefore, the post-wean size reduction of Nr2e1frc/frc mice was not apparently the result of a feeding abnormality but may be a secondary effect of hyperactivity.     Figure 3.2 Nr2e1frc/frc mice showed hyperactivity as early as postnatal day (P)18 A 3-min open field test showed that Nr2e1frc/frc mice were significantly more active at P18, but not at younger ages. * P < 0.01. N = 10 Wt and 12 Nr2e1frc/frc pups.    83 3.3.2 Adult Nr2e1frc/frc mice show hyperactivity in three behavioural tests  To fully characterize the extent of the hyperactivity phenotype in Nr2e1frc/frc mice we used the home cage activity monitor, a powerful and ethological test that assesses movement of mice in their home cage. This test showed that Nr2e1frc/frc mice are extremely hyperactive (Fig. 3.3a; genotype effect F(1,11) = 10.6, P < 0.01). The mean number of beam breaks per hour was ~8-fold higher in Nr2e1frc/frc mice than in Wt controls for both light and dark phases (Fig. 3.3b; Beam breaks: Light: Wt = 189 ± 19.0, Nr2e1frc/frc = 1304 ± 118.9, P < 0.001; Dark: Wt = 313 ± 21.6, Nr2e1frc/frc = 2403 ± 148.6, P < 0.001).    84  Figure 3.3 Nr2e1frc/frc mice showed hyperactivity in the home cage  (a) Nr2e1frc/frc mice broke more beams than their Wt littermates over 48 h. (b) Nr2e1frc/frc mice are significantly more active than Wt controls in both light and dark phases. * P < 0.001. N = 8 Wt and 8 Nr2e1frc/frc mice.    Hyperactivity in Nr2e1frc/frc mice was also seen in the open field test. Throughout the three days of open field habituation testing there was a significant effect of genotype on distance traveled (Fig. 3.4; F(1,57) = 80.0, P < 0.001).   85  Figure 3.4 Nr2e1frc/frc mice showed hyperactivity and habituation deficiency in the open field  Distance traveled was measured in the open field on 3 consecutive days for 10 min each day. Nr2e1frc/frc mice were significantly more active than Wt mice on all 3 days. Wt mice showed habituation on day 1 (solid blue). *Wt: P < 0.05. Nr2e1frc/frc mice did not show habituation on day 1 (dotted blue), but showed habituation on days 2 (dotted red) and 3 (dotted green). *Nr2e1frc/frc: P < 0.05. N = 12 Wt and 9 Nr2e1frc/frc mice.   Finally, in the tail suspension test we found that Nr2e1frc/frc mice spent significantly more time struggling than Wt mice (Fig. 3.5; Wt = 8.49 ± 1.60 % time struggle, Nr2e1frc/frc = 33.3 ± 3.30 % time struggle, F(1,10) = 2.13, P < 0.001). This observation is consistent with a similar study testing mice lacking Nr2e1 (Abrahams et al., 2005). Therefore, increased struggle of Nr2e1frc/frc mice in the tail suspension test is a stable phenotype across studies.   86  Figure 3.5 Nr2e1frc/frc mice struggled more during the tail suspension test  The Nr2e1frc/frc mice spent significantly more time struggling compared to their Wt controls. * P < 0.001. N = 8 Wt and 4 Nr2e1frc/frc mice.  3.3.3 Nr2e1frc/frc mice showed a deficit in two different learning and memory tasks  To characterize the behavioural manifestation of hippocampal and cortical hypoplasia, hallmarks of the Nr2e1frc/frc brain, we tested our mice for deficits in learning and memory tasks. Since Nr2e1frc/frc mice have reduced vision and showed deficits in the hidden cookie test, which could result from abnormal olfaction because of hypoplasia of olfactory bulbs (Young et al., 2002), we used two tests that do not rely primarily on visual or olfactory cues.  The ability of mice to habituate in the open field is measured by a decrease in exploratory activity over time. We demonstrate here that although Nr2e1frc/frc mice were able to habituate to the open field arena, they required significantly more time than the Wt controls. Throughout the three days of testing the two genotypes showed different activity patterns depending on the day, as shown by a significant effect of minute, day, and genotype interaction (Fig. 3.4; F(18, 513) = 3.02, P < 0.001, ε = 0.46). More   87 specifically, during day 1 of testing Wt mice already showed habituation by the 4th min of testing (P < 0.05), whereas Nr2e1frc/frc mice did not habituate during the 10 min on day 1 (P > 0.7). Nr2e1frc/frc mice did eventually show habituation on test days 2 and 3, at 10 (P < 0.01) and 7 (P < 0.05) min, respectively. Similarly, Wt mice showed inter-session habituation such that exploratory activity during days 2 and 3 was significantly reduced when compared to day 1 (P < 0.05), whereas Nr2e1frc/frc mice did not show a significant decrease in activity across days (P > 0.05). The passive avoidance test depends on the ability of the mouse to react to pain, and therefore prior to this test, we examined our mice for pain sensitivity using the hot plate and tail flick tests. Nr2e1frc/frc mice began licking their paws in significantly less time compared to Wt mice, indicating increased pain sensitivity in the hot plate test (Fig. 3.6a; Wt = 16.2 ± 1.71 s, Nr2e1frc/frc = 11.3 ± 1.05 s, P < 0.05). In the tail flick test there was no difference in the time required to remove the tail between Nr2e1frc/frc and Wt mice (Fig. 3.6b; Wt = 1.75 ± 0.15 s; Nr2e1frc/frc = 1.90 ± 0.11 s; P > 0.1). Despite the discordance in the results of these two tests we have reason to favor the finding of increased pain sensitivity when Nr2e1frc/frc mice are not restrained (see Discussion). More importantly, both tests showed the ability of Nr2e1frc/frc mice to respond to pain, thus supporting the use of the passive avoidance test.   88  Figure 3.6 Nr2e1frc/frc mice showed increased pain sensitivity (a) The latency to lick paws as a sign of discomfort from heat is measured in the hot plate test. Nr2e1frc/frc mice took significantly less time to lick their paws compared to the Wt controls. * P < 0.05. (b) The tail flick test was also used to test pain sensitivity in these mice; however, there was no significant difference found between the two genotypes (P > 0.1). N = 8 Wt and 8 Nr2e1frc/frc mice for each test.  The standard protocol for passive avoidance testing is to use light as an adverse stimulus to encourage the animal to cross into the second chamber. However, since Nr2e1frc/frc mice have impaired vision, we decided to use sound as the adverse stimulus. We have previously tested 4-month-old Nr2e1frc/frc mice on a B6 background and showed that they have normal hearing as measured by auditory brainstem response (ABR) (Young et al., 2002). However, since our current mice are on a B6129F1 hybrid background, we retested them for ABR. Nr2e1frc/frc mice did not show any significant differences from Wt controls (Fig. 3.7; Click: Wt = 50.0 ± 2.89 dB, Nr2e1frc/frc = 45.0 ± 2.74 dB, P > 0.5, 16 kHz: Wt = 22.5 ± 4.33 dB, Nr2e1frc/frc = 17.0 ± 2.00 dB, P > 0.5). Therefore, normal ABR in Nr2e1frc/frc mice is a stable phenotype across two genetic backgrounds.   89  Figure 3.7 Nr2e1frc/frc mice showed normal hearing Auditory brainstem response was used to assess hearing ability in the mice. No significant differences in click and 16 kHz thresholds were seen between Wt and Nr2e1frc/frc mice (P > 0.1). N = 5 Wt and 4 Nr2e1frc/frc mice.   Since we confirmed that B6129F1-Nr2e1frc/frc mice are able to respond to pain and that their hearing is normal, we used sound to test these mice for passive avoidance. Wt mice demonstrated the expected learning response, showing an average >3-fold increase in latency to re-enter the second chamber upon the second exposure to the condition stimulus (Fig. 3.8; Day 1 = 41.7 ± 3.32 s, Day 2 = 150 ± 12.1 s, P < 0.001). Although Nr2e1frc/frc mice also showed an increase in latency to re-enter, this change was much less than that seen in Wt mice, and did not reach statistical significance (Day 1 = 29.3 ± 7.21 s, Day 2 = 71.0 ± 23.1 s, P > 0.05), demonstrating that they did not perform this learning task as well as Wt mice.   90  Figure 3.8 Nr2e1frc/frc mice showed impaired performance in the passive avoidance test Learning is measured by the increase in latency to enter the chamber where the mouse received a mild shock the day before. Although Nr2e1frc/frc mice did show an increase in latency to enter the 2nd chamber, this was much less than that seen in Wt mice (* P < 0.001), and did not reach statistical significance (P = 0.057). N = 9 Wt and 6 Nr2e1frc/frc mice.  3.3.4 Nr2e1frc/frc mice lack startle reactivity  Hippocampal lesions in rodent models have been well documented to show impairments in prepulse inhibition (PPI), a measure of sensorimotor gating (Kamath et al., 2008, Pouzet et al., 1999). Prior to evaluating PPI, acoustic startle reactivity (ASR) must be tested to establish a startle threshold, as defined as the lowest startle intensity that produces a startle reaction significantly different than at the no-stimulus condition. Nr2e1frc/frc mice showed less acoustic startle reactivity than Wt controls, as shown by a significant main effect of genotype (Fig. 3.9; F(1,19) = 17.5, P < 0.001) and a significant interaction between intensity and genotype (F(9,171) = 29.9, P < 0.001, ε = 0.27). Post- hoc analysis indicated that the startle threshold for Wt mice was at 105 dB (P < 0.001); interestingly, there was no startle threshold for Nr2e1frc/frc mice (P > 0.05). This surprising result was confirmed with a new group of mice (data not shown). Therefore,   91 we conclude that Nr2e1frc/frc mice show a lack of normal startle reaction. When we compared the startle magnitudes of Nr2e1frc/frc and Wt mice at each startle intensity, there were significant differences at no stimulus, 85, 90, 95, 110, 115, and 120 dB (P < 0.005). Significant genotype differences below the Wt startle threshold (105 dB) are indicative of hyperactivity in Nr2e1frc/frc mice. This test becomes the fourth test demonstrating hyperactivity in Nr2e1frc/frc mice. Furthermore, as PPI tests are based on the startle response, PPI results for these mice would be uninformative.    Figure 3.9 Nr2e1frc/frc mice showed no startle reactivity to auditory stimuli Wt controls showed a normal pattern of increasing startle responses as startle stimuli became louder. However, Nr2e1frc/frc mice showed no increase in their startle responses at any decibel level tested. TWt, startle threshold for Wt (P < 0.001). * P < 0.005, between genotype comparison at each individual startle intensity. N = 12 Wt and 9 Nr2e1frc/frc mice.    92 3.3.5 Nr2e1frc/frc hyperactivity resistant to lithium treatment  Lithium chloride is the most effective drug for treatment of mania in patients with BPI, with human therapeutic plasma lithium level between 0.6-1.2 mmol/L (equivalent to mouse plasma lithium level 0.77-1.17 mmol/L), which can attenuate psychostimulus- induced hyperactivity and increase cell proliferation in the dentate gyrus in rodent models (Chen et al., 2000). Using a dietary source of lithium, Wt and Nr2e1frc/frc mice showed therapeutic levels of lithium in their serum (Fig. 3.10; Wt, control diet = below detection limit, Wt, lithium diet = 0.9 ± 0.1 mmol/L, Nr2e1frc/frc, control diet = below detection limit, Nr2e1frc/frc, lithium diet = 0.8 ± 0.1 mmol/L, significant main effect of diet F(1,14) = 78.1, P < 0.001).    Figure 3.10 Lithium-treated mice showed therapeutic levels of lithium in their serum Mice fed with a lithium diet showed significant, and importantly, therapeutic levels of lithium in their serum compared to mice fed control diet (* P < 0.001). There was no significant difference in lithium serum level between genotypes on the same diet (P > 0.1). N = 4 Wt and 4 Nr2e1frc/frc mice on control diet; 3 Wt and 5 Nr2e1frc/frc mice on lithium diet.    93 We showed that lithium treatment was unable to alleviate the hyperactivity seen in Nr2e1frc/frc mice in the 24-h home cage activity test, as demonstrated by the significant effect of genotype (Fig. 3.11a; F(1,12) = 37.7, P < 0.001), but no significant effect of diet (F(1,12) = 0.15, P > 0.5), nor a significant interaction between genotype and diet (F(1,12) = 0.004, P > 0.5). The mean number of beam breaks in both light and dark phases was significantly higher in Nr2e1frc/frc mice compared to Wt controls, regardless of lithium treatment (Fig. 3.11b; Light: Wt, normal diet = 78.9 ± 10.8, Wt, lithium diet = 110.2 ± 21.6, Nr2e1frc/frc normal diet = 285.5 ± 46.0, Nr2e1frc/frc lithium diet = 321.4 ± 55.8; Dark: Wt, normal diet = 158.5 ± 16.7, Wt, lithium diet = 197.2 ± 24.4, Nr2e1frc/frc normal diet = 997.4 ± 65.1, Nr2e1frc/frc lithium diet = 1005.7 ± 79.4; for all comparisons between Wt and Nr2e1frc/frc regardless of diet P < 0.05).   94    Figure 3.11 Nr2e1frc/frc-induced hyperactivity in the home cage was unaffected by lithium treatment (a) Nr2e1frc/frc mice, on control and lithium diet, broke more beams than their Wt littermates over 24 h. (b) Nr2e1frc/frc mice, regardless of diet, were significantly more active than Wt controls in both light and dark phases. * P < 0.05. N = 4 Wt and 4 Nr2e1frc/frc mice on control diet; 3 Wt and 5 Nr2e1frc/frc mice on lithium diet.    95 Nr2e1frc/frc mice hyperactivity in the open field test was similarly unaffected by lithium treatment, where there was a significant effect of genotype on distance traveled (Fig. 3.12; F(1,36) = 44.9, P < 0.001) with no significant effect of diet (F(1,36) = 3.42, P > 0.05), and no significant interaction between genotype and diet (F(1,36) = 0.31, P > 0.5).  Figure 3.12 Hyperactivity and habituation deficits in Nr2e1frc/frc mice unaffected by lithium treatment Nr2e1frc/frc mice, regardless of diet, showed significantly higher activity than Wt mice on all 3 days. Wt mice on a normal diet showed habituation on day 1 (solid black line with diamond; Black *Wt1: P < 0.05), as did lithium-treated Wt mice (solid red line with diamond; Red *Wt1: P < 0.05). Nr2e1frc/frc mice on a normal diet did not show habituation on day 1 (dotted black line with diamond), but showed habituation on days 2 (dotted black line with square; Black *Nr2e1frc/frc2: P < 0.05) and 3 (dotted black line with triangle; Black *Nr2e1frc/frc3: P < 0.05). Lithium-treated Nr2e1frc/frc mice did not show habituation on any of the 3 days. N = 4 Wt and 4 Nr2e1frc/frc mice on control diet; 3 Wt and 5 Nr2e1frc/frc mice on lithium diet.   96 3.3.6 Nr2e1frc/frc open field habituation deficit is unaffected by lithium treatment  To evaluate the effect of lithium treatment on the habituation deficit in Nr2e1frc/frc mice, mice fed control and lithium diets were assayed in the open field habituation test. As before (Fig. 3.4), there was a significant effect of minutes, day, and genotype interaction (Fig. 3.12; F(18,324) = 1.96, P < 0.05, ε = 0.59), indicating that Nr2e1frc/frc mice showed different activity patterns on the different test days compared to Wt controls. Lithium treatment was unable to improve habituation in Nr2e1frc/frc mice, as indicated by the lack of significant interaction between minute, day, genotype, and diet (F(18, 324) = 0.77, P > 0.7, ε = 0.59). 3.3.7 Lithium-treated Nr2e1frc/frc mice show no improvement in startle reactivity  The lack of startle reactivity was one of the most striking phenotypes shown in Nr2e1frc/frc mice. To assess the effect of lithium on this behavioural phenotype, control and lithium-treated Wt and Nr2e1frc/frc mice were assayed in the startle reactivity test. Similar to our previous experiments (Fig. 3.9), the two genotype groups responded differently to the varying acoustic startle stimuli as evidenced by the significant interaction between intensity and genotype (Fig. 3.13; F(9,126) = 9.32, P < 0.001). We showed that lithium treatment did not significantly correct the deficient acoustic startle response in Nr2e1frc/frc mice compared to that shown by Wt mice, as there was no significant effect of diet (F(1,14) = 0.87, P > 0.5), and there were no significant interactions between: genotype and diet (F(1,14) = 0.92, P > 0.5); intensity and diet (F(9,126) = 0.32, P > 0.5); nor genotype, intensity, and diet (F(9,126) = 0.47, P > 0.5). Wt mice showed startle thresholds on control and lithium diets at 110 and 115 dB,   97 respectively (P < 0.05), while control or lithium-treated Nr2e1frc/frc mice lacked a startle threshold at any startle intensity (P > 0.05), paralleling results shown in Fig. 9 and demonstrating the lack of a lithium effect on startle reactivity. As seen previously, when we compared the startle magnitudes of Nr2e1frc/frc and Wt mice on control diet at each startle intensity, there were significant differences at many stimulus levels: no stimulus, 75, 85, 90, 95, and 120 dB (P < 0.05), indicative of Nr2e1frc/frc hyperactivity. Nr2e1frc/frc and Wt mice on a lithium diet also showed significant differences in startle reactivity at 90 and 120 dB (P < 0.05). The reduction in differences was attributable to increase in variability with drug treatment.  Figure 3.13 Lithium treatment did not significantly improve startle reactivity deficit in Nr2e1frc/frc mice Wt controls showed startle thresholds on control (Black TWt: P < 0.05) and lithium (Red TWt: P < 0.05) diets. However, Nr2e1frc/frc mice lacked a startle threshold at any intensity, regardless of diet. Between genotype comparison at each individual startle intensity for mice on control diet (Black * P < 0.05). Between genotype comparison at each individual startle intensity for mice on lithium diet (Red * P < 0.05). N = 4 Wt and 4 Nr2e1frc/frc mice on control diet; 3 Wt and 5 Nr2e1frc/frc mice on lithium diet.   98 3.3.8 Cell proliferation in subventricular zone and dentate gyrus is unaffected by lithium treatment   Reduced neural stem/progenitor cell proliferation has been shown in Nr2e1- knockout mice when compared to their Wt littermates (Shi et al., 2004). Here we show for the first time, using Ki67 staining of proliferating cells, a significant genotype effect (Fig. 3.14a & b; F(2,10) = 44.46, P < 0.001), indicating that mice carrying the Nr2e1frc/frc alleles also show the same reduction in cell proliferation when compared to Wt mice. Since lithium has been shown to act through multiple pathways to increase cell proliferation in vivo (Jope, 1999, Wada et al., 2005), we also analyzed its effect on cell proliferation in Nr2e1frc/frc mice. We showed that lithium treatment was unable to alter cell proliferation, as evident by no significant effect of diet (F(2,10) = 0.13, P > 0.5) and no significant interaction between genotype and diet (F(2,10) = 0.04, P > 0.9). Cell proliferation in normal and lithium-treated Nr2e1frc/frc mice is not significantly different in either the subventricular zone (SVZ) (Fig. 3.14a; Nr2e1frc/frc normal diet = 14.4 ± 3.1 Ki67+ cells/count area, Nr2e1frc/frc lithium diet = 10.6 ± 1.4 Ki67+ cells/count area, P > 0.5) or in the dentate gyrus (DG) (Fig. 3.14b; Nr2e1frc/frc normal diet = 2.3 ± 0.8 Ki67+ cells/count area, Nr2e1frc/frc lithium diet = 1.6 ± 0.5 Ki67+ cells/count area, P > 0.5). Lithium treatment also did not affect cell proliferation of WT mice in the SVZ (Fig. 3.14a; WT normal diet = 129.3 ± 21.8 Ki67+ cells/count area, WT lithium diet = 129.7 ± 2.2 Ki67+ cells/count area, P > 0.5) and in the DG (Fig. 3.14b; WT normal diet = 13.8 ± 2.8 Ki67+ cells/count area, WT lithium diet = 12.5 ± 1.7 Ki67+ cells/count area, P > 0.5).     99     Figure 3.14 Lithium treatment did not increase cell proliferation in Nr2e1frc/frc mice (a) In the subventricular zone (SVZ), there were significantly less Ki67+ cells in Nr2e1frc/frc mice compared to Wt mice, regardless of diet (* P < 0.001). (b) In the dentate gyrus (DG), there were significantly less Ki67+ cells in Nr2e1frc/frc mice compared to Wt mice, regardless of diet (* P < 0.01). N = 4 Wt and 4 Nr2e1frc/frc mice on control diet; 3 Wt and 5 Nr2e1frc/frc mice on lithium diet.     100 3.4 Discussion  This study was the first to characterize a spectrum of phenotypes in Nr2e1frc/frc mice, which have been used in the literature to model aspects of BP (Arban et al., 2005, Cao & Peng, 1993, Decker et al., 2000, Einat, 2006a, Einat, 2006b, Einat et al., 2003, El- Mallakh et al., 2003, Gessa et al., 1995, Ralph-Williams et al., 2003). In addition, it is the first to evaluate the effect of any drug treatment on Nr2e1-null mice. Results from this study showed new important behavioural phenotypes in Nr2e1frc/frc mice including extreme hyperactivity and deficits in habituation and startle reactivity. The presence of reduced cellular proliferation in the SVZ and DG was a novel finding for Nr2e1frc/frc mice and the resistance of these behavioural and proliferative phenotypes to lithium treatment is a novel finding amongst all Nr2e1-null mice. In the present study, the extreme hyperactivity phenotype of the Nr2e1frc/frc animals was documented in four different tests: home cage activity, tail suspension, open field habituation, and startle reactivity. Of these tests, the tail suspension was originally chosen to evaluate depressive behaviour in this study, but because of the overwhelming hyperactivity phenotype, the results were not indicative of depressive behaviour. Currently, the most frequently used model of mania is psychostimulant-induced hyperactivity (Einat, 2006a, Machado-Vieira et al., 2004). Interestingly, hyperactivity seen in Nr2e1frc/frc mice was approximately 8-fold higher than basal activity level in the home cage, while administration of psychostimulant commonly increases activity by 2- to 4-fold over non-induced mice (Arban et al., 2005). As far as we are aware, Nr2e1frc/frc mice show the most extreme hyperactivity phenotype currently documented.    101 Nr2e1-null mice have previously been shown to have hypoplasia of the hippocampus and decreased adult neurogenesis in the granular layer of the DG, regions important for learning and memory (Mainen & Sejnowski, 1996, Shi et al., 2004, Young et al., 2002). Our group also demonstrated that not only is the dendritic branching structure of granule cells in Nr2e1frc/frc mice reminiscent of immature neurons in the DG, they also lack synaptic plasticity, as demonstrated by the absence of long-term potentiation (LTP) in their dentate gyrus (Christie et al., 2006). LTP is thought by some to be an electrophysiological measure of learning and memory (Howland & Wang, 2008, Kinney et al., 2009). Therefore, in an attempt to reveal impairments in cognitive function as seen in some patients with BP (Green, 2006), we showed, using two distinct tests of learning and memory, that Nr2e1frc/frc mice perform poorly on these tasks compared to Wt mice. Since Nr2e1frc/frc mice have reduced vision and may also have abnormal olfaction, many conventional behavioural paradigms of learning and memory were not appropriate. The two different tests used in this study were chosen and designed specifically to assess learning and memory with minimal use of visual or olfactory cues. Both tests provide an internal control for activity level since they consider the change in activity between the same groups of mice on different days, thus normalizing for activity levels. The increased time required to habituate in the open field test and the lack of significant increase in latency to re-enter in the passive avoidance test are suggestive of reduced learning. Yet, we cannot completely exclude the possibility that a slowness to acquire environmental cues due to sensory deficits or an inability to control hyperactivity contributes to their deficits in performance in these tasks. Despite these caveats, we conclude that abnormal habituation and conditioned avoidance, along with the significant neuropathological   102 phenotypes in Nr2e1frc/frc mice (Christie et al., 2006, Young et al., 2002) are all evidence indicative of cognitive deficits in these mice.  This study was also the first to test for acoustic startle reactivity (ASR) in Nr2e1- null mice. Our novel finding of complete lack of startle was unexpected, since previously there has not been a case of hearing mice not showing ASR. ASR was done in preparation for evaluating PPI; however, we are unable to test PPI since PPI requires startle reactivity greater than movements seen at background noise and Nr2e1frc/frc mice showed no startle threshold. This result, along with normal response for the tail flick test, was surprising since our previous results, and those of others (Roy et al., 2002), led us to anticipate a hyper-responsive phenotype. However, we note that the lack of hyperresponsiveness in these instances correlates with the use of restraint, an extreme stressor in mice (Bain et al., 2004). Brain regions shown to contribute to stress-related response include the amygdala and hippocampus (Liberzon & Martis, 2006, Vermetten & Bremner, 2002). Regions suggested to be involved in modulation of ASR, include nucleus accumbens, basolateral amygdala, and prefrontal cortex (Stevenson & Gratton, 2004, Storozheva et al., 2003). All of these regions are structurally abnormal in the Nr2e1frc/frc mice and may underlie the lack of hyperresponsiveness to pain, as well as the lack of ASR. Based on the hot plate test where Nr2e1frc/frc mice were not tested under restraint and showed a significant reduction in time to lick their paws, we concluded that Nr2e1frc/frc mice had increased pain sensitivity. However, in the tail flick test, Nr2e1frc/frc mice were placed in a restrainer and, we concluded that under this stressor, the expected hyper-responsive phenotype of Nr2e1frc/frc mice was masked by the atypical stress response caused by restraint.   103 We chose to evaluate the effect of lithium treatment on Nr2e1frc/frc mice for four reasons: (1) lithium has been shown to attenuate symptoms of mania in patients with BP (Shastry, 2005); (2) lithium reduces amphetamine-induced hyperactivity in rodent models of mania (Gould et al., 2001); (3) lithium has shown neuroprotective effects by inducing neural stem cell proliferation in the mouse DG both in vitro and in vivo assays (Wada et al., 2005); and (4) lithium is thought to act through multiple key neurological pathways (Jope, 1999), thus increasing the probability that lithium would effect Nr2e1frc/frc behavioural phenotypes compared to drugs with restricted modes of action. In this study, we showed that adult lithium treatment was ineffective in attenuating any of the abnormal behavioural phenotypes observed in Nr2e1frc/frc mice including the extreme hyperactivity in the home cage, the habituation deficit in the open field test, and the lack of acoustic startle reactivity. Despite the fact that lithium can induce cell cycle in vitro and in vivo (Wada et al., 2005) and that the introduction of Nr2e1 can “rescue” quiescent stem cells from Nr2e1-null brains in vitro (Shi et al., 2004), here we showed that lithium administration to adult Nr2e1frc/frc mice was unable to trigger an increase in cell proliferation in the SVZ and DG. The lack of significant lithium effect could be interpreted as a result of the low number of mice examined in the lithium experiment. However based on the literature of other genetic and psychostimulant-induced mouse models of mania, lithium treatment, was able to reduced the hyperactivity phenotype by at least half, if not returning activity level to that seen in wild-type controls (Gould et al., 2007, Gould et al., 2001, Roybal et al., 2007). Therefore, since Nr2e1frc/frc mice exhibit ~8-fold increase in locomotor activity compared to Wt controls, the number of mice tested in the lithium experiment had   104 sufficient power to detect lithium effect given the anticipated reduction in locomotor activity. The development of an all-encompassing mouse model for complex diseases, such as mental illness, is challenging for reasons of environmental factors, minor multiple gene effects, and appropriate pharmacological responsiveness. However, many single gene mouse models, such as Gsk3b overexpressing mice, nitric oxide synthase (NOS-III) and nNOS knockout mice, and DISC1 mutant mice (Flint & Shifman, 2008, Kato et al., 2007, Prickaerts et al., 2006, Reif et al., 2006, Tanda et al., 2009) have proven valuable as they exhibit aspects of complex disorders. We have now added Nr2e1frc/frc mice to this group. We have shown here that Nr2e1frc/frc mice, although complicated by sensory defects, demonstrate the behavioural traits of hyperactivity and deficit in habituation and learning tasks, which are commonly used in genetic models of BP. However, since Nr2e1frc/frc mice failed to respond to lithium treatment, they do not meet the criteria of pharmacological validity as a model for BP (Kato et al., 2007). We hypothesize for future consideration that in utero or perinatal administration of lithium might further elucidate the effectiveness of lithium treatment. 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Nr2e1 controls proliferation and differentiation of neural and retinal stem/progenitor cells by regulating gene expression important in these cellular processes (Hollemann et al., 1998, Kobayashi et al., 2000, Li et al., 2008, Liu et al., 2008, Miyawaki et al., 2004, Shi et al., 2004, Sun et al., 2007, Yokoyama et al., 2008, Yu et al., 2000). In particular, Nr2e1 acts as a transcriptional repressor by binding to the promoters of Pten, Gfap, S100b, and Aqp4 (Shi et al., 2004, Yu et al., 2000, Zhang et al., 2006) that further affects downstream molecules important for cell cycle progression, such as CyclinD1 and p27Kip1 (Miyawaki et al., 2004, Zhang et al., 2006). Nr2e1-null mice (also known as Tlx-/-, Nr2e1-/-, Nr2e1frc/frc) display numerous neurological and ocular phenotypes including: hypoplasia of the cerebral cortex and olfactory bulbs; increased exposure of the colliculi; enlarged ventricles; reduced proliferation in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus; hypoplasia of the optic nerve; retinal degeneration especially the inner nuclear layer (INL) and the outer nuclear layer (ONL); enhanced S-cone generation; thinning of the inner plexiform layer (IPL), outer plexiform layer (OPL), and                                                  3 This chapter is in preparation for submission for publication. Wong, B.K.Y., Borrie, A.E., Tam, C., Cheng, J.C.Y., Sze, J., Yang, W.H.W., Ottmann, G.A., Abrahams, B.S., Wallace, V. and Simpson, E.M.. Increased Nr2e1 transcription affects gene regulation, cell proliferation, and brain and eye morphology in mice.   116 the photoreceptor outer segment (OS); and reduced to flat electroretinogram (Christie et al., 2006, Land & Monaghan, 2003, Li et al., 2008, Liu et al., 2008, Miyawaki et al., 2004, Monaghan et al., 1997, Monaghan et al., 1995, Roy et al., 2004, Roy et al., 2002, Shi et al., 2004, Stenman et al., 2003a, Stenman et al., 2003b, Young et al., 2002, Yu et al., 2000, Zhang et al., 2006).  The role of NR2E1 in human neurological diseases is also starting to become evident. The 6q21-22 region, where NR2E1 is located, has been shown by a meta- analysis of original data from 11 genome-wide linkage studies to have the highest LOD score (4.19) for bipolar I disorder (BPI), a psychiatric disorder characterized by mood fluctuations ranging from mania to depression (Mcqueen et al., 2005). Recent work from our laboratory identified novel candidate-regulatory mutations in NR2E1 in patients with either severe cortical malformations or BPI, as well as a significant association between NR2E1 and BPI (Kumar et al., 2007, Kumar et al., 2008).  Although NR2E1 has not yet been studied in human eye disorders, numerous mutations in NR2E3, the closest relative of NR2E1, have been characterized in enhanced S-cone syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP) (Bandah et al., 2009, Pachydaki et al., 2009, Schorderet & Escher, 2009). Mouse mutants carrying patient variants of Nr2e3 have also displayed phenotypes similar to those seen in patients (Haider et al., 2006, Wang et al., 2009). Functional changes caused by patient-specific variants in NR2E3 have not been fully characterized; however, it appears that the mutations do not always result in a loss of inhibitory function (Fradot et al., 2007).    117 Since NR2E1 is essential to normal neural and retinal development, variants identified from patients with BPI, and potential future variants from eye disorders, are highly unlikely to be null mutations. NR2E1 variants, especially those found in regulatory and untranslated regions (Kumar et al., 2007, Kumar et al., 2008), may exude their effects by altering NR2E1 transcript levels or stability, which makes it imperative to study the effects of varying Nr2e1 levels.  The bulk of our knowledge has come from studying the phenotypes of Nr2e1-null mice; however, the effects of Nr2e1 overexpression have not yet been examined. This study aims to evaluate the transcriptional, morphological, and cellular phenotypes resulting from Nr2e1 overexpression in mice. This new set of data will further our understanding of the pathways in which Nr2e1 functions.  4.2 Methods and materials 4.2.1 Mice  Random insertion transgenic mice carrying mouse and human NR2E1 examined here have been previously published (Abrahams et al., 2003, Abrahams et al., 2005). Briefly, founders carrying a BAC clone containing the mouse Nr2e1 genomic locus were generated on a C57BL/6J × 129S1/SvImJ hybrid background (B6129F1) and backcrossed to C57BL/6J to generate C57BL/6J.Cg-Tg(Nr2e1bacEMS4A)5Ems and C57BL/6J.Cg- Tg(Nr2e1bacEMS4B)6Ems strains, abbreviated to B6-bacEMS4A and B6-bacEMS4B respectively (Abrahams et al., 2003). Similarly, two mouse strains carrying a PAC clone spanning human NR2E1 were backcrossed to C57BL/6J to generate C57BL/6J.Cg- Tg(NR2E1pacEMS1B)10Ems and C57BL/6J.Cg-Tg(NR2E1pacEMS1D)11Ems,   118 abbreviated to B6-pacEMS1B and B6-pacEMS1D respectively (Abrahams et al., 2005). All mice were full congenics by backcrossing to C57BL/6J for more than 10 generation before analysis. All mice were weaned at postnatal day (P)18 – 21 and housed with same- sex littermates. Mice were provided with food and water ad libitum and standard care according to University of British Columbia animal care policies. Handling of all mice was minimized. Timed pregnancies were set up for collecting embryos of different developmental timepoints. Mice (2 – 6 months old) were used for adult analysis.  4.2.2 Genotyping  Three PCR assays were used to genotype individuals before and after each experiment. A common assay used for both BAC and PAC mice detects the presence of the endogenous mouse Nr2e1 gene using oEMS1859 (5′- CTGGGCCCTGCAGATACTC-3′) and oEMS1860 (5′- GGTGGCATGATGGGTAACTC-3′) (Abrahams et al., 2005). A BAC-specific assay detects the presence of the pBeloBAC11 vector using oEMS1753 (5′- CTGGCGAAAGGGGGATGT-3′) and oEMS1755 (5′- GCTGGAGGGGAATGGAAAAC-3′) (Abrahams et al., 2003). A PAC-specific assay detects the presence of the human NR2E1 using oEMS800 (5′- CCCAGCAGCTGCGGTTTTGC-3′) and oEMS801 (5′- GCAGCGCTCCAGGCAGGAC-3′) (Abrahams et al., 2005). The PCR conditions were 92°C for 2 min, 30 cycles of 94°C for 30 sec, 58°C for 30 sec, and 72°C for 55 sec, and 72°C for 5 min.    119 4.2.3 Interphase and metaphase FISH  Detailed methods for performing interphase and metaphase fluorescence in situ hybridization (FISH) have previously been published (Abrahams et al., 2003). Briefly, interphase chromosomes were prepared from interphase nuclei obtained from peripheral blood smears. Interphase chromosomes were probed with bEMS4 DNA labeled with biotin-14-dCTP and pEMS1 DNA labeled with biotin-14-dCTP for detection of mouse Nr2e1 and PAC clone spanning the human NR2E1, respectively. Metaphase chromosomes were prepared from lung tissue cultures. Metaphase chromosomes were probed with pEMS1 DNA labeled with dig-14-dUTP for detection of PAC clone spanning human NR2E1 and chromosome-specific probes (Incyte Genomics, St. Louis, MO). 4.2.4 Quantitative reverse transcriptase PCR  RNA from embryonic day (E)12.5 whole brain, adult forebrain, and adult eyes were extracted using Qiagen RNA Mini Plus Kit (Cat#74134; Qiagen Inc., Mississauga, ON). RNA was cleaned with Qiagen DNase kit (Cat#79254; Qiagen Inc., Mississauga, ON) and cDNA generated using SuperScript III Master Mix kit (Cat#11752-050; Invitrogen, Carlsbad, CA). cDNA quantification was performed using ABI TaqMan® assays specifically designed for Aqp4, Ccnd1, Dcx, Gfap, Gsk3β, Nes, Nr2e1, NR2E1, Nr2e3, Nr4a2, Opsin1sw, Pax6, Pten, and S100β. The 7500 Fast real-time PCR system and TaqMan® Fast Universal PCR Master Mix (Cat#4352042; Applied Biosystems Inc., Foster City, CA) was used for all the qRT-PCR runs. The cycle threshold (Ct) value was   120 defined as the number of cycles required for the fluorescent signal to cross a threshold above background signals and is inversely proportional to the amount of target cDNA. 4.2.5 Brain and eye harvesting  4.2.5.1 Tissue for RNA extraction Adult brains were freshly dissected from the skull; a coronal cut was made at the caudal end of the cortical lobes and the anterior portion of the cut brains were used for RNA extraction. Embryos were collected at E12.5 in cold saline and whole heads were removed. Eyes were also freshly extracted from adult mice. All tissues harvested for qRT-PCR were flash frozen in liquid nitrogen.  4.2.5.2 Tissue for immunofluorescence Mice for brain harvesting were injected intraperitoneally (i.p.) with heparin and perfused intracardially with 4% paraformaldehyde, 30 min following the heparin injection. Whole brains were dissected from the skull intact and placed into 4% paraformaldehyde in 1× PBS at 4°C for 48 h, then transferred to a 20% sucrose solution at 4°C until saturated. Prior to sectioning, images of brains were taken on the Leica MZ6 (Leica Microsystems Inc., Bannockburn, IL). These images were traced using Image-Pro Express (Media Cybernetics Inc., Bethesda, MD) for quantification of brain regions. Brains were then sectioned at 25 μm using the Cryo-Star HM 560 cryostat (MICROM International, Walldorf, Germany) and every 24th section starting from the most rostral aspect of the ventricles to the most caudal aspect of the hippocampus was analyzed by immunofluorescence.   121 For eye harvesting, mice were sacrificed by cervical dislocation and eyes were removed. Eyes were placed into 4% paraformaldehyde in 1× PBS at 4°C for 24 h, then transferred to a 20% sucrose solution at 4°C until saturated. Eyes were then sectioned at 14 μm using the Cryo-Star HM 560 cryostat (MICROM International, Walldorf, Germany) and every 30th section was analyzed by immunofluorescence. 4.2.6 Immunofluorescence  Free-floating brain sections and mounted eye sections were blocked with 5% normal goat serum (NGS) + 5% bovine serum albumin (BSA) in 0.1% Triton-X100 in PBS. Brain sections were incubated overnight at room temperature (RT) with rabbit anti- Ki67 polyclonal antibody (1:1000 dil, Cat. #ab15580, Abcam Inc., Cambridge, MA), and further incubated with Alexa Fluor® 594 goat anti-rabbit IgG (H+L) (Cat. #A31631, Invitrogen, Carlsbad, CA). Eye sections were incubated overnight at RT with: mouse anti-CRALBP (1:1000 dil, Cat. #sc-48354, Santa Cruz Biotech., Santa Cruz, CA); mouse anti-GFAP (1:1000 dil, Cat. #VP-G805, Vector Laboratories., Burlingame, CA); rabbit anti-Pax6 (1:1000 dil, Cat. #sc-11357, Santa Cruz Biotech., Santa Cruz, CA); mouse anti- rhodopsin (clone B630, 1:150 dil, gift from Dr. Valarie Wallace); and mouse anti- syntaxin (clone HPC-1, 1:1000 dil, Cat. #S0664, Sigma-Aldrich Ltd., Oakville, ON), and further incubated with either Alexa Fluor® 488 goat anti-mouse IgG (H+L) (Cat. #A11001, Invitrogen, Carlsbad, CA) or Alexa Fluor® 594 goat anti-rabbit IgG (H+L) (Cat. #A31631, Invitrogen, Carlsbad, CA). Hoechst 33342 was used for nuclear staining for all sections. All sections were mounted onto Superfrost® Plus slides (Cat. #12-550-15, Fisher Scientific, Ottawa, ON) and coverslipped using Vectashield Hard Set™ (Cat. #H- 1400, Vector Laboratories, Inc., Burlingame, CA). Images were captured on an Olympus   122 BX61 motorized fluorescence microscope (Olympus America Inc., Center Valley, PA). Proliferating cells in the SVZ and DG were counted using the ImageJ software (Rasband, 1997-2009). 4.2.7 Statistical analysis  All data were analyzed using STATISTICA© 6 (StatSoft, Inc., Tulsa, OK). QRT- PCR and brain morphological data were analyzed by factorial ANOVA for strain and genotype effects. When significant effects were found, post-hoc tests with Tukey correction were performed for multiple comparisons to reveal the underlying differences within the main effects. Data are reported as mean values ± 1 standard error of the mean (SEM).   4.3 Results 4.3.1 High copy integration of B6-pacEMS into mouse genome  The generation of B6-bacEMS4 and B6-pacEMS1 mice and the mapping of the BAC inserts have been described in detail in Abrahams et al. (2003, 2005); however, the mapping of the 141-kb PAC in the two strains of B6-pacEMS1 mice has not been shown. Interphase fluorescence in situ hybridization (FISH) showed an intense signal from the human specific probe at Chromosome 6 (band A2) and Chromosome 4 (band A2) in B6- pacEMS1B and B6-pacEMS1D mice, respectively (Fig. 4.1). The intensity of the signals compared to the endogenous locus probed with mouse specific bEMS4 DNA suggested high copy of PAC integration.  123      Figure 4.1 FISH mapping of pacEMS1 transgenes (a & b) Interphase and (c & d) metaphase FISH using probes specific for mouse and human NR2E1 of pacEMS1B and pacEMS1D, respectively. Both pacEMS1B and 1D show two single copy endogenous signals of mouse Nr2e1 (arrowheads) and a more intense signal indicative of transgene (arrow). (e & f) Metaphase FISH using mapping probes and probe specific for human NR2E1 of pacEMS1B and pacEMS1D, respectively. Both pacEMS1B and 1D show two signals from chromosome 6 and 4 mapping probes (arrowheads) respectively, and an intense signal indicative of the transgene (arrow). Further banding localized the human transgene to chromosome 6A2 and 4A2 for pacEMS1B and pacEMS1D respectively.   124 4.3.2 B6-bacEMS4A mice show increased Nr2e1 transcription  Transcription of mouse Nr2e1 and human NR2E1 from the high copy inserts of BAC and PAC, respectively, were examined. Mouse Nr2e1 transcript levels from whole head of embryonic day (E)12.5 embryos of B6-bacEMS4A, B6-bacEMS4B, B6- pacEMS1B, and B6-pacEMS1D were examined using a mouse-specific Nr2e1 TaqMan assay. B6-bacEMS4A transgenic E12.5 heads showed a significant increase in the level of Nr2e1 transcripts (Fig. 4.2a; Wt = 1.00 ± 0.15-fold change, Tg = 2.07 ± 0.26-fold change, P < 0.05), while there were no significant differences in embryonic heads of B6- bacEMS4B (Fig. 4.2a; Wt = 1.00 ± 0.18-fold change, Tg = 1.01 ± 0.19-fold change, P > 0.05), B6-pacEMS1B (Fig. 4.2a; Wt = 1.00 ± 0.26-fold change, Tg = 0.73 ± 0.14-fold change, P > 0.05), and B6-pacEMS1D (Fig. 4.2a; Wt = 1.00 ± 0.21-fold change, Tg = 0.88 ± 0.19-fold change, P > 0.05) transgenics compared to their Wt counterparts.  Similarly, the anterior portion of the adult brain transcript level of Nr2e1 was only significantly higher in B6-bacEMS4A Tg (Fig. 4.2b; Wt = 1.00 ± 0.19-fold change, Tg = 4.79 ± 0.76-fold change, P < 0.05), and not in B6-bacEMS4B (Fig. 4.2b; Wt = 1.00 ± 0.20-fold change, Tg = 1.02 ± 0.19-fold change, P > 0.05), B6-pacEMS1B (Fig. 4.2b; Wt = 1.00 ± 0.19-fold change, Tg = 0.73 ± 0.18-fold change, P > 0.05), and B6-pacEMS1D (Fig. 4.2b; Wt = 1.00 ± 0.19-fold change, Tg = 1.02 ± 0.19-fold change, P > 0.05) when compared to Wt. These results indicate that high copy number of BAC inserts in B6- bacEMS4A results in increased transcription of mouse Nr2e1.   125  Figure 4.2 B6-bacEMS4A show increased Nr2e1 expression in E12.5 whole head and adult brain B6-bacEMS4A was the only strain that showed significant fold increase of Nr2e1 expression in (a) E12.5 whole head and (b) anterior portion of adult brain. * P < 0.05. N = 5 per strain/genotype/age.    126 4.3.3 PAC mice show overexpression of human NR2E1  A human-specific NR2E1 TaqMan assay only detected transcripts in human whole brain, B6-pacEMS1B, B6-pacEMS1D, and rescue transgenic mice and not in Wt B6 mice (Table 4.1), corresponding to qRT-PCR results previously demonstrated in Abrahams et al. (2003).  Table 4.1 Ct values obtained from human-specific NR2E1 TaqMan assay    Expression level of human NR2E1 in both pacEMS1 transgenic strains was compared to the endogenous levels of mouse Nr2e1 in Wt controls. Human NR2E1 levels were significantly higher than endogenous level of mouse Nr2e1 in E12.5 B6-pacEMS1B (Fig. 4.3a; Wt mouse Nr2e1 = 1.00 ± 0.34-fold change, Tg human NR2E1 = 3.91 ± 0.98- fold change, P < 0.05), E12.5 B6-pacEMS1D (Fig. 4.3a; Wt mouse Nr2e1 = 1.00 ± 0.21- fold change, Tg human NR2E1 = 30.7 ± 6.13-fold change, P < 0.05), adult B6- pacEMS1B (Fig. 4.3b; Wt mouse Nr2e1 = 1.00 ± 0.19-fold change, Tg human NR2E1 = 4.97 ± 1.14-fold change, P < 0.05), and adult B6-pacEMS1D (Fig. 4.3b; Wt mouse Nr2e1 = 1.00 ± 0.19-fold change, Tg human NR2E1 = 7.36 ± 1.48-fold change, P < 0.05) indicating that high copy number of PAC inserts results in increased transcription of the   127 human NR2E1 gene, similar to increased transcription of the mouse Nr2e1 gene in B6- bacEMS4A mice.   Figure 4.3 B6-pacEMS1B and 1D showed significant increase in level of human NR2E1 Level of human NR2E1 transcript from B6-pacEMS1B and 1D transgenic (a) E12.5 whole head and (b) anterior portion of adult brain showed significant fold increase when compared to the level of mouse Nr2e1 from equivalent Wt regions. * P < 0.05. N = 5 per strain/genotype/age.    128 4.3.4 Characterization of gross brain and eye morphology of four transgenic strains  Hypoplasia of the olfactory bulbs and frontal lobes and gross neuroanatomical differences are well documented in mice lacking Nr2e1, therefore we obtained detailed measurements of various brain regions to document effects of increased Nr2e1 transcription on brain morphology. B6-bacEMS4A transgenic mice showed significantly reduced brain weight when compared to Wt mice (Table 4.2). However, this weight reduction was not observed in the other strains. All four transgenic strains of mice were also examined for eye phenotypes including: corneal opacity, microphthalmia, and anophthalmia. B6-bacEMS4A Tg mice showed increased frequency of eye phenotypes compared to Wt mice, while the other three Tg strains did not (Table 4.2). B6- bacEMS4A also showed bilateral and unilateral optic nerve hypoplasia (data not shown). These eye abnormalities are grossly reminiscent of those seen in Pax6+/Sey mice (Hill et al., 1991, Ramaesh et al., 2003). Table 4.2 Gross phenotypic description of the four transgenic strains     129 A multifactorial ANOVA was performed on all brain measurements of all four transgenic strains. From this analysis, significant main effects of strain (F(3,39) = 10.4, P < 0.001) and genotype (F(1,13) = 3.49, P < 0.01), and a significant interaction between strain and genotype (F(3,39) = 2.72, P < 0.001) were observed. These results indicate that there were inter-strain differences in brain morphology that was genotype-dependent and that the four strains are significantly different from each other. However, post-hoc analysis did not identify any significant genotype effect in any brain measurements in the four strains (Table 4.3). Since post-hoc analysis did not reveal the underlying source of the significant strain and genotype effects, we analyzed the four strains separately. In these analyses, only B6-bacEMS4A showed a significant effect of genotype (F(1,8) = 66501, P < 0.005). These results collectively suggest that only B6-bacEMS4A transgenic mice show an effect of over-transcription of Nr2e1 on brain morphology.    130  Table 4.3 Gross brain measurements in the four transgenic strains      131 4.3.5 B6-bacEMS4A mice show altered transcription level of Gfap and Gsk3β  The transcription levels of Aqp4, Ccnd1, Dcx, Gfap, Gsk3β, Nes, Nr4a2, Pten, and S100β were examined in the anterior portion of adult brains in all four transgenic strains. With the exception of Gfap that is not transcribed at E12.5, the rest of the gene set was examined in E12.5 whole heads. These genes were selected for analysis based on literature showing an interaction with Nr2e1 or their involvement in cell cycle regulation. Since we had no a priori hypothesis that gene transcription had to be concordant throughout time, we analyzed the two time points separately. A multifactorial ANOVA was first performed on transcription data of all target genes from adult brains of the four transgenic strains. From this analysis, significant effects of strain (F(3,27) = 50.3, P < 0.001) and genotype (F(1,9) = 271.5, P < 0.001), and a significant interaction between strain and genotype (F(3,27) = 51.8, P < 0.001) were observed. Post-hoc analysis revealed that genotype differences were only observed in the B6-bacEMS4A strains. B6- bacEMS4A transgenic adult brain showed significantly increased Gsk3β and a trend for reduced Gfap transcription compared to Wt brains (Table 4.4a). These results are consistent with the brain morphological data indicating that the B6-bacEMS4A strain is the only significantly affected transgenic strain. Analysis of the transcription data from E12.5 whole brain were treated identically to that of adult brain, except for Gfap that was removed from the gene set because it is not transcribed in E12.5 whole head. The multifactorial ANOVA revealed significant main effects of strain (F(3,24) = 5.99, P < 0.001) and genotype (F(1,8) = 23.4, P < 0.001), and a significant interaction between strain and genotype (F(3,24) = 6.77, P < 0.001). When we performed post-hoc analysis, we did not identify any significant   132 genotype effect in any particular gene in the four strains (Table 4.4b). Therefore, at E12.5 the presence of increased Nr2e1 transcripts results in minor transcriptional differences that only collectively contribute to the main effects detected.    133 Table 4.4 Fold change of target gene transcript in the four transgenic strains     134 4.3.6 Cell proliferation in the subventricular zone was altered in B6-bacEMS4A  Cell proliferation in neurogenic regions was quantified in the subventricular zone (SVZ) and dentate gyrus (DG) using Ki67 labeling. B6-bacEMS4A showed a significant increase in cell proliferation in the SVZ (Fig. 4.4a; Wt = 232.0 ± 31.7 Ki67+ cells/count area, Tg = 319.7 ± 46.6 Ki67+ cells/count area, P = 0.05), but not in the DG (Fig. 4.4b; Wt = 31.4 ± 3.1 Ki67+ cells/count area, Tg = 29.1 ± 4.3 Ki67+ cells/count area, P > 0.1) compared to Wt. However, B6-bacEMS4B showed no significant differences in cell proliferation in the SVZ (Fig. 4.4a; Wt = 117.1 ± 9.6 Ki67+ cells/count area, Tg = 122.5 ± 18.3 Ki67+ cells/count area, P > 0.1) or the DG (Fig. 4.4b; Wt = 17.3 ± 5.3 Ki67+ cells/count area, Tg = 19.1 ± 2.5 Ki67+ cells/count area, P > 0.1).   135  Figure 4.4 B6-bacEMS4A showed significant increase in cell proliferation in the subventricular zone (a) In the subventricular zone (SVZ), there were significantly more Ki67+ cells in B6- bacEMS4A, but not B6-bacEMS4B mice compared to Wt mice. * P < 0.05. (b) In the dentate gyrus (DG), there were no significant differences in Ki67+ cells in either B6- bacEMS4 strains compared to Wt. N = 3 per strain/genotype.    136 4.3.7 B6-bacEMS4A eyes showed thinning and disorganization of retinal cell layers  Since B6-bacEMS4A was the only strain to show overt eye phenotypes, further effects of Nr2e1 overexpression in the eye were examined only in B6-bacEMS4A. The adult neural retina consists of 5 layers: the outer nuclear layer (ONL), the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), and the ganglion cell layer (GCL) (labeled in Fig. 4.6a and b). We performed immunofluorescence with cell type specific markers and nuclear staining to compare retinal organization of the B6-bacEMS4A transgenic and Wt mice. In Wt retina, Gfap staining of Müller glia was observed in the GCL, however in B6-bacEMS4A transgenic mice intraretinal Gfap staining indicated that there was gliosis, likely from Muller glia (Fig. 4.5a and b). Rhodopsin staining of rods was reduced and sparse in B6-bacEMS4A transgenic retina compared to Wt retina, consistent with thinning of the ONL indicative of fewer photoreceptors (Fig. 4.5c and d). Syntaxin staining was observed in the IPL of Wt and B6-bacEMS4A transgenic retina (Fig. 4.5e and f); however, Hoechst staining showed cells protruding into the IPL from the normally tightly packed GCL indicating disorganization of the GCL in the B6-bacEMS4A transgenic retina (Fig. 4.6b). Staining for a nuclear marker showed that the ONL, INL, and IPL layers of the B6-bacEMS4A retina were significantly thinner than Wt retina (Fig. 4.6). A multifactorial ANOVA identified a significant main effect of genotype (F(1,145) = 52.3, P < 0.001) and layers (F(4,145) = 76.6, P < 0.001), and a significant interaction between genotype and layers (F(4,145) = 13.1, P < 0.001). Post-hoc analysis identified significant genotype differences in the ONL, INL, and IPL (Fig. 4.6c-g; P < 0.01).   137  Figure 4.5 Adult B6-bacEMS4A eyes show abnormal cellular staining (a & b) Gfap staining (green) of Müller glia in (a) Wt and (b) B6-bacEMS4A eyes, respectively, showed differences in staining between transgenic and Wt eyes. (c & d) Rhodopsin staining (green) of rod photoreceptors in (c) Wt and (d) B6-bacEMS4A eyes, respectively, showed reduced staining in transgenic eyes. (e & f) Syntaxin staining (green) of amacrine cells in (e) Wt and (f) B6-bacEMS4A eyes, respectively, showed abnormal staining in transgenic eyes. All nuclei were counterstained using Hoechst 33342 (blue). Outer nuclear layer (ONL); inner nuclear layer (INL); ganglion cell layer (GCL). White scale bar = 20 μm. N = 3 per genotype.   138   Figure 4.6 Adult B6-bacEMS4A eyes show thinning of retinal layers  Representative pictures of (a) Wt and (b) B6-bacEMS4A retina showed thinning of retinal layers. White arrowheads indicate cells seen in the IPL, which are not present in Wt retina. White scale bar = 20 μm. Five retinal layers: (c) outer nuclear layer (ONL); (d) outer plexiform layer (OPL); (e) inner nuclear layer (INL); (f) inner plexiform layer (IPL); and (g) ganglion cell layer (GCL), were measured for thickness in Wt and transgenic eyes. Significant thinning of the (c) ONL, (e) INL, and (f) IPL were observed in transgenic versus Wt retina. * P < 0.001. N = 3 per genotype.   139 4.3.8 Gene transcription is altered in B6-bacEMS4A eyes  Transcription of Nr2e1 and other important developmental and retinal cell marker genes (Gfap, Nr2e3, Opsin1sw, and Pax6) were examined using quantitative reverse transcriptase PCR (qRT-PCR) in the B6-bacEMS4A adult eye. B6-bacEMS4A transgenic adult eye showed a significant increase in Nr2e1 transcript level compared to Wt eyes (Fig. 4.7a; Wt = 1.00 ± 0.48-fold change, Tg = 3.34 ± 1.23-fold change, P < 0.05).  A significant effect of genotype (F(1,4) = 23469, P < 0.005) was observed for gene transcription of target genes in the adult eye. Similar to gene expression results from the brain, the level of Gfap transcript was also significantly decreased in B6-bacEMS4A transgenic adult eyes compared to Wt eyes (Fig. 4.7b; Wt = 1.00 ± 0.10-fold change, Tg = 0.67 ± 0.10-fold change, P < 0.05). Interestingly, transcript level of Nr2e3, a gene known to be involved in eye disease and the closest relative to Nr2e1, was significantly reduced in B6-bacEMS4A adult eyes (Fig. 4.7c; Wt = 1.00 ± 0.16-fold change, Tg = 0.28 ± 0.05-fold change, P < 0.01). Opsin1sw, expressed specifically in cones, was not significantly different in B6-bacEMS4A transgenic eyes (Fig. 4.7d; Wt = 1.00 ± 0.14-fold change, Tg = 0.74 ± 0.12-fold change, P = 0.07) compared to Wt eyes. And given the similarities observed between the B6-bacEMS4A transgenic and Pax6+/Sey adult eyes, we found, as expected, significant reduction of Pax6 transcripts in B6-bacEMS4A transgenic eyes compared to Wt eyes (Fig. 4.7e; Wt = 1.00 ± 0.17-fold change, Tg = 0.56 ± 0.10- fold change, P < 0.05).    140  Figure 4.7 Adult B6-bacEMS4A eyes showed significant alteration in gene transcription  Adult B6-bacEMS4A eyes were examined for fold differences in (a) Nr2e1, (b) Gfap, (c) Nr2e3, (d) Opsin1sw, and (e) Pax6 expression. * P < 0.05. N = 5 per genotype.   141 4.4 Discussion  This is the first study to characterize the neurological, proliferative, and ocular effects of increased Nr2e1 transcription in mice. Although these mouse strains carry high copies of inserts, quantification of Nr2e1 and NR2E1 transcript levels was necessary to show that transgene transcription was indeed increased. Genes studied for transcriptional changes in these mice were chosen based on (1) previous literature demonstrating differences in Wt versus Nr2e1-null mice (Li et al., 2008, Liu et al., 2008, Miyawaki et al., 2004, Shi et al., 2004, Sun et al., 2007) and (2) genes that are shown to be involved in cell cycle regulation (Eom & Jope, 2009, Ke et al., 2004, Li et al., 2006). The two bacEMS4 strains were analyzed for Nr2e1 transcript levels, where B6-bacEMS4B showed no significant increase and B6-bacEMS4A showed a four-fold increase in the transcriptional level of Nr2e1 in both the brain and eyes of adult mice. Consistent with the over transcription of Nr2e1 in the bacEMS4 strains, only B6-bacEMS4A showed significant transcriptional changes of its target genes. In both pacEMS1 strains, although increased transcription of human NR2E1 was observed, no significant difference in transcription of target genes were detected. Furthermore, increased Nr2e1 transcription was observed in both embryonic and adult time points but significant target gene transcription differences were only observed in adults. These results all point towards a complex effect of Nr2e1 level and its role in transcriptional regulation. Since Nr2e1 is generally known to act as a transcriptional repressor, we had anticipated that Nr2e1 overexpression might result in increased repression of its direct genetic targets (i.e. Aqp4, Gfap, Pten, S100β). However, as evidenced by our qRT-PCR data, this is not necessarily the case. In fact, of the four direct target genes only Gfap   142 expression showed a statistical trend for reduction in B6-bacEMS4A transgenic mice, while others showed no significant differences. Several possibilities may explain these results. First, Nr2e1 is known to recruit histone demethylases (LSD1) and deacetaylases (HDAC3, 5, and 7) for transcription repression (Sun et al., 2007, Yokoyama et al., 2008). The lack of gene expression differences in the presence of Nr2e1 overexpression may be due to the availability of these corepressor proteins. If these corepressors were not proportionally increased with Nr2e1, then transcriptional repression would reach a plateau. Secondly, although our null hypothesis was to predict a linear relationship between transcription level of target genes and Nr2e1 transcript levels, this simplistic model was unlikely to hold up based on the dynamic expression pattern of Nr2e1 (Land & Monaghan, 2003, Stenman et al., 2003b), indicating that Nr2e1 levels requires strict regulation for normal development. And finally, although we have shown increased transcription of mouse and human NR2E1 in the transgenic strains, we have been unable to demonstrate that this transcriptional increase results in increased translation of Nr2e1 protein. Nr2e1 protein levels have to be quantified in these strains before we can accurately correlate the transcriptional data presented here with phenotypes observed. Our work has been impeded by the performance failure of the commercial antibodies for Nr2e1 currently available. In B6-bacEMS4A transgenic brains, aside from the reduction in Gfap transcription, the other transcriptionally-affected gene was Gsk3β. This result strengthens the connection between NR2E1 and bipolar I disorder (BPI). A relationship between NR2E1 and BPI has now been demonstrated by different lines of evidence (Kumar et al., 2008, Mcqueen et al., 2005) and lithium is the standard treatment for mania in patients   143 with BPI (Shastry, 2005). Lithium has been shown to inhibit Gsk3β leading to an increase of CyclinD1 (Ccnd1) that initiates cell cycle re-entry (Chen et al., 2005, Williams & Harwood, 2000), as well as reducing Aqp4 transcription (Mcquillin et al., 2007), all genes shown to be regulated by Nr2e1 (Miyawaki et al., 2004, Shi et al., 2004). B6-bacEMS4A brains showed that increased levels of Nr2e1 transcripts resulted in increased Gsk3β transcription, therefore implying a functional role of Nr2e1 in pathways mediated by drugs effective in treating BPI. Since B6-bacEMS4A and the two B6-pacEMS1 strains showed an increase in mouse and human NR2E1 levels, respectively, and assuming that the over transcription of the human transgene results in increased NR2E1 protein, then the lack of transcriptional changes in the pacEMS1 strains might be the result of subtle functional variation between human and mouse NR2E1. Previously, human NR2E1 has successfully rescued the Nr2e1-null mouse brain and behavioural phenotypes, indicating a functional conservation between mouse and human NR2E1 (Abrahams et al., 2005). Although brain morphology of PAC transgenic mice was deemed to be normal, detailed measurements of various brain regions were not performed. It was also noted that the presence of the human NR2E1 was unable to entirely correct mutant eye phenotypes (Abrahams et al., 2005), which was either the result of the eyes being sensitive to Nr2e1 dose or slight differences in functional efficiency between mouse and human NR2E1. This study shows that there were no significant neuroanatomical differences in pacEMS1 transgenic strains when compared to Wt brains. B6-bacEMS4A was the only strain to show a significant genotype effect, when all brain measurements were analyzed together. The most noticeable difference in the B6-bacEMS4A brains was the significant reduction in brain   144 weight, which was not observed in any other transgenic strains studied here. However, this reduction in brain weight was not accompanied by any significant decrease in size of various brain regions.  The proliferative deficit of neural stem/progenitor cells in Nr2e1-null mice is characterized by a significant decrease in BrdU incorporation in the neurogenic areas of the adult brain: the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus (Shi et al., 2004). We examined whether increased Nr2e1 levels will alter neural stem/progenitor cell proliferation in these two areas. Based on the transcriptional and morphological data, we had a strong hypothesis that B6-bacEMS4A, our most affected strain, would show altered proliferation in the SVZ and DG. Although the pacEMS1 strains showed increased transcription of NR2E1, data from qRT-PCR of target genes and gross brain and eye morphology does not provide support for increased NR2E1 protein. Since B6-bacEMS4B mice showed no significant increase in Nr2e1 transcript level, proliferating cells in the SVZ and DG of these brains were counted as a control experiment. Proliferation was not significantly different between B6-bacEMS4B transgenic brains and Wt brains in either the SVZ or DG, as expected. Cell proliferation in B6-bacEMS4A was significantly increased only in the SVZ but not in the DG. Neural stem/progenitor cells found throughout the brain have been demonstrated to be characteristically different (Lagace et al., 2007, Merkle et al., 2007) and can respond differently to regional regulatory signals (Palmer et al., 1995). Therefore, the disparity in the proliferative effect of Nr2e1 overexpression in the two areas might reflect varying roles of Nr2e1 that are region- and cell type-specific. This ability of Nr2e1 overexpression to increase cell proliferation and reduce Gfap expression is of great   145 interest for understanding and potential treatment of numerous neurological disorders, including Alzheimer’s disease that shows increased Gfap expression (Jesse et al., 2009) and treatments for depression and mania that attenuate symptoms by increasing proliferation in the brain (David et al., 2009). The eye phenotypes observed in B6-bacEMS4A, and not in other strains, were reminiscent of those seen in Pax6 mutants (Hill et al., 1991, Ramaesh et al., 2003). Pax6+/Sey mice show reduced Pax6 expression resulting in decreased retinal ganglion cell genesis and enhanced cone photoreceptor and amacrine interneuron production (Hsieh & Yang, 2009). Interestingly, the closest relative of Nr2e1, Nr2e3 controls photoreceptors fate by repressing cone-specific gene transcription and when mutated shows increased expression of cone-specific genes and enhanced cone generation (Webber et al., 2008). A previous study in Xenopus showed that a fusion protein consisting of Nr2e1 DNA- binding domain (DBD) and the Engrailed repressor ligand-binding domain (LBD) that increases Nr2e1-specific gene repression resulted in a significant decrease in Pax6 expression (Hollemann et al., 1998). This study is the first to demonstrate that overexpression of Nr2e1 in mice results in significant reduction of both Pax6 and Nr2e3 expression. These results place Nr2e1 in these important genetic pathways of eye development.  Given that Nr2e1 and Nr2e3 are expressed in different cell types (Müller glia and photoreceptor layer, respectively (Kobayashi et al., 2008, Miyawaki et al., 2004)), the reduction of Nr2e3 is likely the result of a decrease in cone photoreceptors in these transgenic eyes, as supported by the trend for reduced expression of Opsin1sw, a cone- specific gene. Similarly, rod photoreceptors appear to be reduced in B6-bacEMS4A   146 retina. The significant thinning of the ONL provides further support for the reduction of photoreceptors. Müller cells, the astrocytic cells of the retina, are shown to be reduced and have defective ultrastructure in Nr2e1-null mice (Miyawaki et al., 2004). Gfap immunofluorescent analysis of Müller cells in B6-bacEMS4A retina showed increased and dispersed GFAP staining. In addition to the staining abnormalities seen in the B6- bacEMS4A adult retina, there was significant thinning of the different retinal layers, especially the ONL, INL, and IPL. Similarly, thinning of retinal layers had also been previously reported for mice lacking Nr2e1 (Young et al., 2002). Therefore, proper development and/or maintenance of the retinal layers are not only regulated by presence and absence of Nr2e1, but also by its levels. Since gross eye abnormalities are observed in neonatal B6-bacEMS4A mice, ocular phenotypes are likely developmental instead of degenerative. However, since B6-bacEMS4A mice showed increased cell proliferation in the SVZ, cell proliferation in the retina will also need to be analyzed. If increased cell proliferation was also observed in the retina, then the thinning of the layers may be a result of increased apoptosis. Interestingly, mice with variable levels of Pax6, ranging from underexpressors to overexpressors, also exhibit thinning of the retinal and cortical layers (Sansom et al., 2009, Schedl et al., 1996). The levels of Pax6 have been shown to regulate the balance between self-renewal and differentiation. In the developing cortex, the loss of Pax6 leads to a failure to self-renew resulting in increased early differentiation, where increase in Pax6 enhances division of stem cells and promotes basal progenitor fate that leads to overproduction of early-born cortical neurons; in both cases there is a depletion of cortical stem cell pool (Sansom et al., 2009). Therefore, future experiments are necessary for examining the effect of Nr2e1 overexpression on   147 cell cycle and cell fate determination that underlie the cortical and retinal abnormalities in the B6-bacEMS4A transgenic mice. Collectively, these results suggest that overexpression of Nr2e1 may have a detrimental effect on the development of the mouse retina by significantly reducing Pax6 and Nr2e3 transcript levels, while generally sparing brain development. 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Genes Dev, 20, 1308- 1320.     156 Chapter 5: General discussion The work presented in this thesis examined the effects of varying Nr2e1 levels on brain and eye phenotypes, with particular focus on (1) the validity of using Nr2e1frc/frc mice as a model of bipolar I disorder (BPI) and (2) to identify novel target genes to place Nr2e1 in genetic pathways important in human disease. In my discussion, I will comment on the findings of the three manuscript chapters as a whole, make suggestions on behavioural modeling of human psychiatric disorders, and propose future experiments, in both in vivo (mouse and human) and in vitro systems that will further identify roles of NR2E1 in disease and as a potential therapeutic molecule.  5.1 Overview of major findings  Prior to evaluating the validity of Nr2e1frc/frc mice as a model of BPI, the discriminatory power of dark- versus light-phase testing was examined. Results presented in Chapter 2 supported our hypothesis that dark-phase testing will affect and improve discrimination between genetically distinct mouse strains using high-throughput behavioural tests. The increased ability of dark-phase testing to distinguish behavioural difference in genetically distinct mouse strains influenced the experimental design of Chapter 3 which assessed Nr2e1frc/frc mice for behavioural anomalies similar to those seen in some patients with BPI. Nr2e1frc/frc mice showed several behavioural characteristics that are also observed in other rodent models of mania, including hyperactivity and learning deficits. We also showed reduced cell proliferation in the subventricular zone (SVZ) and dentate gyrus (DG) of Nr2e1frc/frc mice, a trait that is seen in some patients with BPI. To further evaluate the pharmacological validity of Nr2e1frc/frc mice as a model   157 for BPI, the effect of adult lithium administration on behavioural phenotypes and cell proliferation was examined. Chapter 3 represents the first report of drug assessment on mice lacking Nr2e1. Our results indicated that adult administration of lithium was unable to ameliorate behavioural and proliferation deficits in Nr2e1frc/frc mice. Future work employing various drugs with different treatment regimes will be required to fully test the efficacy of drug treatments in Nr2e1frc/frc mice. We began by examining Nr2e1frc/frc mice as a model for BPI because (1) the heterozygous Nr2e1 mice show no observable behavioural phenotypes and only mild cellular and transcriptional alterations (Liu et al., 2008, Roy et al., 2004) and (2) a null mutation would increase our ability to observe potentially subtle behavioural abnormalities; however, the role of NR2E1 in human diseases is unlikely to be a result of null mutations in this highly conserved and functionally important gene. Variants in NR2E1 identified in patients have been located in conserved regions that are thought to be important in transcriptional regulation (Kumar et al., 2007, Kumar et al., 2008), and therefore it was necessary to examine the effects of variable Nr2e1 levels. In Chapter 4, we examined four transgenic mouse strains carrying exogenous copies of either human or mouse Nr2e1 to test the hypothesis that Nr2e1 overexpression will result in dysmorphia of neuroanatomical and ocular development, and that target gene transcription levels will inversely correlate with Nr2e1 levels. We identified significant neuroanatomical, ocular, and gene expression differences in one of the four transgenic strains, B6-bacEMS4A, when compared to Wt mice. The significance and future directions stemming from these findings are discussed in greater detail below.    158 5.2 Considerations for modeling behavioural traits of human disease in mice 5.2.1 Dark-phase behavioural testing can improve detection of behavioural differences in genetically distinct mice  Despite the wide use of mouse models to study behavioural phenotypes, there has been a lack of continuity in the testing protocol throughout the field resulting in inter- laboratory variability (Crabbe et al., 1999, Wahlsten, 2001, Wahlsten et al., 2003). The sensitivity of behaviour to changes in environment and testing conditions has been well documented and differences in testing conditions can result in unnecessary complications when deciphering genetic effects of behavioural outcomes. With the explosion of genome-wide association studies of human psychiatric disorders throughout the last decade, more and more candidate genes will arise and require examination in a model system. Therefore, the evaluation and optimization of current high-throughput behavioural testing conditions was essential.  In Chapter 2, we examined the effects of light- and dark-phase testing on the ability to discriminate behavioural phenotypes in three genetically distinct strains (C57BL/6J, 129S1/SvImJ, and B6129F1). We acknowledged the inconvenience of dark- phase testing for the experimenters if lights were on between 0600-1800 h; therefore, we raised our mice on a reversed light cycle (lights on 23:00-11:00 h), thereby allowing researchers to test mice in their dark-phase during normal work hours. We demonstrated that dark-phase testing was not only more ethologically correct, but also improved discriminatory power of high throughput tests, including SHIRPA primary screen, open- field test, and motor learning on the rotarod. From these results we would recommend that behavioural examination of mouse mutants carrying candidate mutations of human psychiatric disorders be performed in the dark phase. By using a reversed light cycle,   159 researchers could easily benefit from the increased discriminatory power of dark-phase high-throughput behavioural testing, and thus allow for the detection of potentially subtle behavioural phenotypes. 5.2.2 The power of dissecting complex disorders into endophenotypes  Human psychiatric disorders are essentially impossible to accurately model in rodents (Einat, 2006). Although genetic and drug models of BP (presented in Chapter 1.6.4) demonstrate a subset of behavioural and/or neuropathological traits, the variability of symptoms in patients given the same diagnosis indicates the underlying genetic heterogeneity of the group. By evaluating aspects of these disorders separately, instead of the disorder as a whole, we can improve our ability to identify genetic influences of specific traits. This concept of “endophenotypes” stems from dissecting a complex disease into more basic phenotypes that have a clear genetic connection (Gottesman & Gould, 2003, Gottesman & Shields, 1973). Endophenotypes of BP may include behavioural symptoms (e.g. cognitive deficits, olfactory deficits, hyperactivity, sleep disturbances) (Goldberg & Chengappa, 2009, Kruger et al., 2006, Mccurdy et al., 2006) and neuroanatomical differences (e.g. increased ventricular volume, reduced hippocampal and cerebral cortical volume, reduced neurogenesis) (Swayze et al., 1990). By studying these endophenotypes, we can better detect genetic defects that ultimately contribute to the disease.   5.3 Nr2e1frc/frc mice – an appropriate model for bipolar disorder?  Linkage analysis and significant association have suggested a relationship between NR2E1 and bipolar disorder (BP), especially bipolar I disorder (BPI) (Kumar et   160 al., 2008, Mcqueen et al., 2005) (as outlined in Chapter 1.6.2). These findings laid the groundwork for experiments described in Chapter 3. Furthermore, the considerations discussed above (Chapter 5.1) were implemented in the experimental design of Chapter 3. 5.3.1 Nr2e1frc/frc mice show phenotypes observed in bipolar disorder  The neurological abnormalities are well characterized in Nr2e1-null mice and similarities can be drawn between patients with BPI (outlined in Chapter 3.1). This is the first study to evaluate the validity of Nr2e1frc/frc mice as a model for BPI. Behaviourally, Nr2e1frc/frc mice showed extreme hyperactivity and deficits in learning tasks, behavioural traits that are seen in patients with BPI and rodent models of mania. The neurological abnormalities in Nr2e1frc/frc mice, such as hypoplasia of the hippocampus and olfactory bulbs, could underlie both hyperactivity and learning deficit, as lesion models show similar behavioural outcomes (Chaillan et al., 2005, Deacon et al., 2002, Pullela et al., 2006). Developmental abnormalities in Nr2e1frc/frc mice have also resulted in impairment of the GABAergic interneurons and changes to this neurotransmitter system have been shown to be important in the regulation of activity level (Viggiano, 2008). Given that the structural and behavioural phenotypes observed in Nr2e1frc/frc are consistent with the current understanding of brain regions and behavioural control, our findings suggest a neurodevelopmental role of NR2E1 in BPI.  Deficits in neural stem/progenitor cell proliferation are also observed in some patients with BPI, and since Nr2e1 has a role in adult neurogenesis, where the lack of Nr2e1 reduces neural stem/progenitor cell proliferation, NR2E1 variants may also alter the ability of neural stem/progenitor cells to proliferate. Furthermore, a potential   161 mechanism by which lithium acts to attenuate symptoms in patients with BPI is to promote proliferation of neural stem/progenitor cell. Although we show in Chapter 3 that adult lithium treatment was unable to correct for the proliferative deficit, we hypothesize that adult Nr2e1frc/frc cells may be too severely altered to respond to lithium treatment, and therefore for future pharmacological analysis of Nr2e1frc/frc mice, we suggest testing at prenatal time points. To fully characterize the molecular changes occurring in cells lacking Nr2e1, drug effects should be examined at early (E8-12), mid (E14-15), and late (E16-18) embryoic time points, when we know there are dynamic changes in cell cycle rates (Roy et al., 2004).  The in vivo approach is invaluable in studying the genetic and neurological consequences of behavioural phenotypes of psychiatric diseases. However, because the brain is so severely affected by the loss of Nr2e1, it is conceivable that other pathways and/or mechanisms have taken on a compensatory role, a phenomenon shown in mouse models of stroke and mania (Liu et al., 2007, Prickaerts et al., 2006). Therefore, in an in vivo system these compensatory mechanisms might mask drug responses to more basic phenotypes, such as cell proliferation. From our Chapter 3 results, we would also recommend future in vitro studies of adult- and embryo-derived Nr2e1frc/frc neural stem/progenitor cells (e.g. neurosphere assays, neural differentiation assays), that will allow cell cycle regulation to be evaluated in a controlled environment resulting in improved detection of drug effects. The high degree of sequence conservation at the genomic and amino acid level (Abrahams et al., 2002, Yu et al., 1994), as well as the ability of human NR2E1 to rescue the mouse phenotype (Abrahams et al., 2005) demonstrates the functional importance of   162 this gene. Taking into consideration the severity of the Nr2e1-null brain abnormalities and the location of the NR2E1 variants found in patients with BPI (i.e. conserved regulatory regions) (Kumar et al., 2004), it is highly unlikely that these variants result in the complete loss of function or transcription of the NR2E1 gene. Therefore, NR2E1 variants are likely to result in altered NR2E1 function (i.e. hypomorph or hypermorph) leading to abnormal development of forebrain regions and systems resulting in behavioural traits seen in BPI. This hypothesis drove the work presented in Chapter 4 of this thesis that examined the effects of Nr2e1 overexpression. 5.3.2 New direction stemming from inconsistencies in Nr2e1-null behavioural abnormalities  For the most part, the behavioural phenotypes observed in Nr2e1frc/frc mice were consistent with those seen in other Nr2e1-null strains (Monaghan et al., 1997, Roy et al., 2002) and models of mania (Prickaerts et al., 2006). In our findings (Chapter 3), the lack of startle reactivity and the lack of increased pain sensitivity in the tail flick test were surprising and unexpected, as these behaviours were inconsistent with what was previously known about behaviour of Nr2e1-null mice. Since these two unexpected results were observed from tests requiring physical constraint, we hypothesized that Nr2e1frc/frc mice demonstrated abnormal stress response when subjected to a stressor. Although Nr2e1frc/frc mice have been previously tested for corticosterone levels, which were statistically similar to levels seen in Wt controls, the levels observed in Nr2e1frc/frc mice were consistently higher in both sexes on the C57BL/6J and B6129F1 background (Young et al., 2002). It is conceivable that the statistically insignificant increase in corticosterone level in Nr2e1frc/frc mice can easily become significant when these mice are   163 under restraint. The key structures controlling stress response is the hypothalamic- pituitary-adrenal axis (HPA axis) with connections from regions such as the amygdala and hippocampus that facilitates activation of the HPA axis (Herman et al., 1996). Since several of these regions are affected in Nr2e1frc/frc mice, the role of Nr2e1 in altering stress response is an interesting new direction that warrants examination.  5.4 Overexpression of Nr2e1 illuminates important genetic pathways  The pathways in which Nr2e1 exerts its function are beginning to be elucidated. Transcriptional changes resulting from the lack of Nr2e1 has placed Nr2e1 into genetic pathways involving Pten, Gfap, and S100β, just to name a few (Shi et al., 2004). Analysis of Nr2e1-null developing brains had shown an interaction between Nr2e1 and Pax6 in boundary establishment (Stenman et al., 2003). Our data further supports this interaction, by demonstrating that Nr2e1 overexpression decreases Pax6 expression in the mammalian eye. Based on our transcript data of brain and eye from Nr2e1 overexpressing mice (presented in Chapter 4), we can now add Gsk3β and Nr2e3 to this growing gene list.  The expression change in Gsk3β in Nr2e1 overexpressing brains is of particular interest to the field of psychiatric and cancer genetics. Gsk3β plays a major role in regulating cell cycle progression by acting through the Wnt/β-catenin signaling pathway that controls expression of numerous cell cycle regulatory genes, in particular cyclinD1, which is also phosphorylated by Gsk3β, (Ryves & Harwood, 2003, Takahashi-Yanaga & Sasaguri, 2009). Undoubtedly, cell cycle dysregulation is one of the underlying mechanisms in cancer biology, and the interaction between Nr2e1 and Gsk3β not only   164 supports the role of Nr2e1 in cancer, but also places it in the Wnt/β-catenin signaling pathway. In respect to psychiatric genetics, alteration in Gsk3β levels had been observed in BP, schizophrenia, and Alzheimer’s disease, and the regulation of Gsk3β may also be involved in the therapeutic effects of drugs used in psychiatry (Jope & Roh, 2006), as demonstrated with lithium, a standard treatment for BP and a known Gsk3β inhibitor. Given the support for the role of Gsk3β in BP and our new evidence of an interaction between Nr2e1 and Gsk3β, these findings collectively imply a functional role of NR2E1 in the pathology of BP. The overt eye phenotypes and their similarities to Pax6+/Sey eyes suggested that Nr2e1-overexpressing eyes would likely show reduction in Pax6 expression, which is supported by our transcript data. Until now, Nr2e3 expression had not been examined in its relationship to Pax6 and Nr2e1. Since both NR2E3 and PAX6 mutations result in eye disorders, enhanced S-cone syndrome (ESCS) and Aniridia, respectively, NR2E1 would be a good candidate gene for human eye disorders (future direction discussed below). The quantitative reverse transcriptase PCR examination of known interactors and genes of interest in the Nr2e1-overexpressors was a good starting point to evaluate what effects Nr2e1 overexpression would have on gene regulation on a smaller scale. I would recommend that high-throughput analysis be performed using serial analysis of gene expression (SAGE) or cDNA microarray for expression differences on Nr2e1-null, Nr2e1-overexpressing, and wild-type brains and eyes to identify new interactors that are affected by varying levels of Nr2e1.     165 5.5 Future directions: NR2E1, bipolar disorder, and eye disorders 5.5.1 Testing bipolar disorder variants in mice  Variants found in NR2E1 are located in evolutionarily conserved non-coding sequences that reside in the proximal promoter and untranslated regions, suggesting functional importance for transcriptional regulation of NR2E1 (Abrahams et al., 2002, Kumar et al., 2007, Kumar et al., 2008). Sixty-three percent of the novel NR2E1 variants were also predicted to alter transcription factor binding site (Kumar et al., 2007, Kumar et al., 2008), and therefore, each variant should be examined for NR2E1 expression level as well as difference in binding using in vitro techniques such as electrophoretic mobility shift assay. As demonstrated in Chapter 4, variations in Nr2e1 expression results in transcriptional differences of target genes; expression analysis of these genes would identify the functional changes resulting from these NR2E1 variants. The absence of behavioural phenotypes in Nr2e1 heterozygous mice warrants the study of these variants on a genetic background that does not contain endogenous Nr2e1. This strategy is termed the “rescue paradigm” utilized by Abrahams et al. (2005), where human NR2E1 successfully rescued the Nr2e1frc/frc behavioural and neuroanatomical phenotypes. Therefore, even though results originating from Chapter 3 of this thesis do not provide support for Nr2e1frc/frc mice to be an adequate model for BPI, as it fails to show behavioural improvement with adult lithium treatment, it did characterize the baseline for behavioural phenotypes relevant for modeling BPI on which the suspect disease NR2E1 variants will be studied. Drug studies using this in vivo rescue system, as well as in vitro assays (discussed above in 5.2.1) can provide significant insight into the role of each variant in disease susceptibility.   166 5.5.2 Identifying NR2E1 variants in human eye disorders  The role of NR2E1 in human eye disorders has been significantly strengthened by the transcriptional changes observed in Pax6 and Nr2e3 in Nr2e1-overexpressing eyes (Chapter 4). Mouse mutants carrying mutations in both Pax6 and Nr2e3 have been used in modeling human Aniridia and enhanced S-cone syndrome (ESCS), respectively. Given the resemblance between Nr2e1-overexpressing and Pax6+/Sey eyes in mice, patients who have Aniridia not caused by mutations in PAX6 would be suitable for genetic analysis for NR2E1 mutations. Similarly, patients with ESCS, but no known mutation in NR2E3 would also be an appropriate sample population. Based on the transcriptional data from Chapter 4 of this thesis, we would anticipate that at least a subset of patient-specific variants in NR2E1 identified from human sequencing studies might result in increased expression of NR2E1, leading to reduced PAX6 and NR2E3 expression. Similar to patient variants found in psychiatric disorders, variants found in eye disorders could be tested in mouse models to determine the molecular pathways in which they disrupt. 5.5.3 The use of genetic crosses to identify novel pathways  By crossing Nr2e1-null mice with Pax6+/Sey mutants, the genetic interaction between these two genes was discovered to be important for setting boundaries during brain development (Stenman et al., 2003). This technique allows for two distinct genotypes to interact and either enhance or ameliorate specific phenotypes, thereby illuminating the nature of the genetic interaction. For example, if crossing the Nr2e1 overexpressors with Pax6+/Sey mice results in an eye phenotype more severe than the two single mutants, then the two genes are working in additive genetic pathways. However, if Nr2e1 acts upstream to Pax6, then the phenotype might be unchanged because the Nr2e1   167 overexpression phenotype hides the phenotypic effects of Pax6+/Sey. A combination of genetic crosses can be performed, using Nr2e1frc/frc, B6-bacEMS4A, Pax6+/Sey, and Nr2e3-/- mice, to reveal genetic interactions important in brain and eye development. The reduction of Gfap expression in brains of Nr2e1 overexpressors is an exciting first step towards the long-term goal of using Nr2e1 in therapy. Of particular interest is NR2E1 in the treatment of Alzheimer’s disease (AD), the most common cause of dementia, which shows severe neuropathology in the hippocampus consisting of β- amyloid plaques and neurofibrillary tangles (Gotz & Ittner, 2008). Current stem cell therapies in Alzheimer models have shown improved cognitive functions in transgenic mice; however, increased amyloid precursor protein (APP) in these animals reduces neurogenesis and increases glial differentiation of the implanted neural stem cells (Sugaya et al., 2007), and therefore hinders the effectiveness of the therapeutic potential of these stem cells. We hypothesize that Nr2e1-overexpressing neural stem cells would be less susceptible to the anti-neurogenic and pro-gliosis effects of increased APP because of their increased proliferative potential and their reduced levels of endogenous Gfap. This reduced sensitivity to high APP levels could allow for later onset of symptoms resulting from inflammation and subsequent neuronal loss. Although we only showed a significant increase in neural stem/progenitor cell proliferation in the subventricular zone and not the dentate gyrus of the hippocampus, we predict that a diseased hippocampus would provide an environment in which Nr2e1-overexpressing neural stem/progenitor cells would be challenged and so demonstrate their increased potential to proliferate. Nr2e1 overexpressing mice can be subjected to exercise or intracranial lipopolysaccharide (LPS) injections to test the effect of Nr2e1 overexpression in a   168 sensitized system. Similarly, Nr2e1 overexpressing mice can be crossed to mouse models of AD and examined for amelioration of disease phenotypes, such as neuropathological hallmarks (e.g. increased GFAP staining, neuronal loss in dentate gyrus) and cognitive deficits (e.g. impairment in Morris water maze).  5.6 Conclusion  The work presented in this thesis was the first to evaluate the validity of Nr2e1frc/frc mice as a model for BP by behavioural measures and drug treatment, as well as the first to examine the transcriptional and morphological effects of Nr2e1 overexpression in the mouse brain and eye. 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