Effect of Gtf2i Gene on Anxiety

Transcription

1 Effect of Gtf2i Gene on Anxiety by Joana Dida A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto Copyright by Joana Dida (2013)

2 Effect of Gtf2i Gene on Anxiety Joana Dida Master of Science Institute of Medical Science University of Toronto 2013 Abstract Duplication and deletion of a common interval spanning 26 genes on chromosome 7q11.23 cause Dup7q11.23 Syndrome and Williams-Beuren Syndrome, neurodevelopmental disorders with contrasting anxiety phenotypes. The General Transcription Factor 2 I (GTF2I) gene has been implicated in separation anxiety, common in people with Dup7q11.23, and we studied the effects of commonly used anxiolytics on maternal separation-induced USV in mouse models with copy number changes in Gtf2i. Subcutaneous injection of saline affected both USV production and plasma corticosterone levels in a Gtf2i gene-dosage dependent manner. Drugs acting on the glutamate receptors were most effective at attenuating USVs in all genotypes, compared to GABAergic and serotonergic modulators. Brain c-fos expression after separation was reduced by a GABA A agonist, but not a glutamate antagonist. Collectively, these results suggest a potential difference in pain sensitivity based on Gtf2i copy number and implicate the glutamatergic and GABAergic systems in anxiety phenotypes in these two disorders. ii

3 Acknowledgments I would like to extend my deepest gratitude to my supervisor, Dr. Lucy Osborne, for her continuous guidance. Lucy has been a rock to rely on since the first time I collaborated with her on an undergraduate project, and her influence has continued through choosing a supervisor for graduate school and applying for the next stage in my education. I will always remember your support during some of the most critical points in my career. Your contagious laughter in the lab made it that much more enjoyable to come to work every day. I hope you know that any of my future success can be traced back to you. I would also like to express my thanks to the members of my program advisory committee, Dr. John Yeomans and Dr. Paul Arnold. Dr. Yeomans expertise in mouse work assisted me not only through graduate school but as a new researcher when I joined his lab in undergrad. My discussions with him always extended beyond the research I was conducting at the time, covering anything from career paths to politics. Dr. Arnold, whose expertise with clinical work helped me look at the big picture of my research project, was an inspirational example of the clinicianscientist I strive to become. To members of the Osborne lab, thank you for being such an amazing team and sharing these past two years with me. The friendships I have built through champagne celebrations, sangria and cupcake birthday parties, practice presentations, and calming talks are bonds made for life: Emma Strong, Robyn Pereira, Elli Brimble, Elaine Tam, Amy Oh, and Emily Lam. I owe my gratitude to Emily for being a mentor and a friend to me well before I joined the lab and even after she left. Every graduate student needs that special counsel from a past member of the lab, and you were that and more. To old and new friends from high-school, undergraduate and graduate years, thank you for being there. If I could, I would write a book for each and every one of you. I would like to especially thank Cailin, Laura, and Toni for their continuous support during my graduate school specifically. The friendships I have built over the tears of sadness and joy shared with you will forever be cherished. Without your help, I wouldn t be where I am today. Lastly, but most importantly, I would like to thank my family. There was never a moment when I did not feel your endless support. Mom and Dad, you are the greatest role models a daughter could ever ask for. Words can t express my appreciation for your sacrifices and your continuous love and encouragement through all of my amazing and not so amazing life choices. Because of you I never felt alone, and I couldn t ask for more. To my two brothers, Eri and Eraldo, I wouldn t be the strong woman I am today without you. To Eri, thank you for being the best older brother a sister could ever want. You are someone I look up to and can share my biggest problems with, even without having to explain. To Eraldo, thank you for bringing out the child in me and for annoying me like no one else when I m having a bad day, just to make me smile. iii

4 Table of Contents Table of Contents List of Abbreviations List of Tables List of Figures iv ix xiv xv Chapter 1: Introduction Williams-Beuren Syndrome History Clinical Profile Cognitive Profile Behavioural Profile q11.23 Duplication Syndrome History Clinical and Cognitive Profile Behavioral Profile Triplication of Dup7q The Genetics of 7q11.23 Rearrangements Genetic Basis of Williams-Beuren Syndrome Mutational Mechanism in WBS and Dup7q Inheritance Incidence Genotype-Phenotype Correlation Mouse Models Previously Studied Mouse Models Contiguous Gene Deletion Fkbp6 to Gtf2i Gtf2ird1- Knockout Mouse GTF2I Family of Transcription Factors GTF2I (TFII-I) TFII-I in the Nucleus TFII-I in c-fos Promoter Activity TFII-I in the Cytoplasm TFII-I Expression in the Brain Anxiety Introduction to Anxiety Disorders Anxiety Neurocircuitry Pharmacology and Neurotransmitter Hypothesis of Anxiety Current Pharmacological Treatment of Anxiety Neurotransmitter Hypothesis of Anxiety Serotonin Hypothesis of Anxiety GABA and Glutamate Hypothesis of Anxiety Prevelance of Anxiety in WBS and Dup7q Current Treatment of Anxiety in WBS and Dup7q Animal Models of Anxiety 40 iv

5 Maternal Separation-Induced USVs USVs, Serotonin, and anxiety USVs, GABA, and anxiety USVs, Glutamate, and anxiety Role of Maternal Care on Mouse Pups Behavior Anxiety, HPA axis, and Neuronal Activation Anxiety and Rare Disorders Conclusions 49 Chapter II: Behavioral Analysis of Mouse Models with Altered Gtf2i Copy Number Effects of Injection Stress and Gtf2i Pup Genotype on Maternal Separation-Induced USVs Introduction Research Aims Maternal Separation-Induced USVs Duplication of Gtf2i Results in Separation Anxiety in Mice and Humans Hypothesis Materials and Methods Generation of Mice with Altered Gtf2i Copy Number Animal Housing Apparatus and Measurements Maternal Separation-Induced USVs Procedure Statistical Analysis Genotyping and Sexing of PND8 Mice DNA Extraction from Tails Genotyping of Gtf2i +/- Litters Genotyping of Gtf2i +/dup Litters Sexing of PND8 Mice Results Subcutaneous Injection Alters USV Production in Mice with Altered Gtf2i Gene Copy Number Injection Stress, Plasma Corticosterone, and Altered Gtf2i Gene Copy Number Introduction Research Aims Hypothesis Materials and Methods Animals Plasma Collection Corticosterone Assay Statistical Analysis Results Maternal Separation and Subcutaneous Injection Induced Changes in Plasma Corticosterone Levels in a Gtf2i Gene-Dosage Dependent Manner Maternal Separation-Induced USVs Predict Plasma Corticosterone Concentrations Effect of Gtf2i Maternal Genotype on Maternal Separation-Induced USVs Introduction Research Aims 71 v

6 Hypothesis Materials and Methods Animals Apparatus and Measurements Maternal Separation-Induced USVs Procedure Statistical Analysis Results Maternal Genotype Effect on Offspring s Maternal Separation-Induced USVs Discussion and Conclusions Injection Stress Stimulated Changes in Maternal Separation-Induced USVs in a Gtf2i Gene-Dosage Dependent Manner Maternal separation and Subcutaneous Injection Elevate Plasma Corticosterone Levels in a Gtf2i Gene-Dosage Dependent Manner Maternal Separation-Induced USVs Predict Plasma Corticosterone Concentrations Hypothesized Changes in Pain Sensitivity in the Gtf2i Mouse Models Potential Confounding Variables on the Sensitivity of Plasma Corticosterone Changes Role of Maternal Gtf2i Genotype and Parental Genotype Interaction on Offspring Anxiety and Maternal Care 83 Chapter III: Characterizing the Separation Anxiety Phenotype in Mouse Models of Varying Gtf2i Copy Number Effects of Injection Stress and Gtf2i Pup Genotype on Maternal Separation-Induced USVs Introduction Research Aims Hypothesis Maternal Separation-Induced USVs Pharmacological Compounds with Anxiolytic Properties Serotonergic Targeting Drugs GABAergic Targeting Drugs Glutamatergic Targeting Drug Materials and Methods Animals/Housing Apparatus and Measurements Maternal Separation-Induced USVs Procedure Drugs Statistical Analysis Genotyping and Sexing of PND8 Mice Results Screening is not Applicable No Within-Cage Order of Testing Effects on USVs Anxiolytic Effects on Maternal Separation-Induced USVs Based on the Neurotransmitter System Targeted and Gtf2i Gene Copy Number Gtf2i Gene-Dosage Effect of Serotonergic Targeting Drugs on USVs 95 vi

7 Anxiolytic Effects of GABAergic Targeting Drugs on USVs Anxiolytic Effects of Glutamatergic Targeting Drugs on USVs Stress, Immediate Early Gene Expression, and Altered Gtf2i Gene Copy Number Introduction Research Aims Hypothesis Materials and Methods Animals Dissection of Mouse Brain Tissues and RNA Isolation c-fos Expression Analysis Using Quantitative Real-Time PCR Statistical Analysis Results Injection Stress Induces c-fos Expression in a Gtf2i Gene-Dosage Dependent Manner Effective Inhibition of c-fos Expression by Allopregnanolone but not MK Discussion and Conclusions Screening of Mice regarding USVs Potency of Serotonergic, GABAergic and Glutamatergic Drugs on USVs Strong Anxiolytic Properties of Glutamatergic- and GABAergic Targeting Drugs Inhibitory Role of Gtf2i on Serotonergic Transmission Effects of Anxiolytics in Wild-Type PND8 Mice A Shared Route for Allopregnanolone and MK Environmental and Other Factors Influencing USVs Stress- and Pain- Induced Elevations in Brain c-fos Expression with a Change in Gtf2i Gene Copy Number Anxiolytics Induced Changes in Brain c-fos Expression with a Change in Gtf2i Gene Copy Number TFII-I Activation of the c-fos Promoter versus Anxiolytic Mediated Reduction of c-fos Expression 112 Chapter IV: Conclusions and Future Directions Summary Overview Stronger Anxiolytic Effects of Drugs Targeting the Glutamate and GABA Systems than Serotonin System Hypothesized Changes in Pain Sensitivity with Change in Gtf2i Gene Copy Number Stress- and Pain- Induced Elevations of Brain c-fos Expression with a Change in Gtf2i Gene Copy Number Anxiolytic- Induced Suppression of Brain c-fos Expression versus TFII-I Mediated Activation of the c-fos Promoter with a Change in Gtf2i Gene Copy Number Future Directions Assessment of Pain Sensitivity in WBS and Dup7q Additional Measures During and After Maternal Separation-Induced USVs Test 118 vii

8 4.2.3 Assessment of c-fos Changes in Specific Brain Regions Conclusion 120 viii

9 Abbreviations 5-HT 5-HT1A Serotonin Serotonin Receptor 1A 8-OH-DPAT 8-Hydroxy-N,N-dipropyl-2-Aminotetralin AchE ACTH ADHD ADIS-P ANOVA ASD AVP BLA BNST Bp Btk BZ CA1 CA2 CBCL CeM Acetylcholinesterase Adrenocorticotropin Hormone Attention Deficit Hyperactivity Disorder Anxiety Disorders Interview Schedule for DSM-IV-Parent Interview Analysis of Variance Autism Spectrum Disorder Arginine Vasopressin Peptide Basolateral Amygdala Bed Nucleus of the Stria Terminalis Base Pairs Bruton s Tyrosine Kinase Benzodiazepine Cortical Area 1 of the Hippocampus Cortical Area 2 of the Hippocampus Child Behavior Checklist Central Medial Nuclei of Amygdala ix

10 CNV CORT CRE CREB CRF1 CSF D2R DD DNS Copy Number Variation Corticosterone Calcium Cycle AMP Response Element Cyclic AMP Response Element Binding Protein Corticotropin Releasing Factor I Gene Cerebral Spinal Fluid D2 Dopamine Receptor Distal Deletion Down syndrome D/P Deletion of WBS Syntenic Region on Mouse Chromosome 5 DSM-IV Dup7q11.23 EGF ELN ERK ES FPS FISH FOXP2 GABA Diagnostic and Statistical Manual of Mental Disorders 4 th edition Duplication 7q11.23 Syndrome Epidermal Growth Factor Elastin Gene Extracellular Signal-Related Kinase Embryonic Stem Faces Pain Scale Fluorescence In-Situ Hybridization Forkhead Box Protein P2 Gamma Aminobutyric Acid x

11 GAD GPCR GRIN2B GTF2I Generalized Anxiety Disorder G Protein Coupled Receptor Glutamate NMDA Receptor Subtype 2B Gene General Transcription Factor 2I GTF2IRD1 General Transcription Factor 2I Repeat Domain 1 GTF2IRD2 General Transcription Factor 2I Repeat Domain 2 GWAS HLH HMBS HPA ID IIH Inr LCR LHPA LI LoxP LZ Mb MCI Genome Wide Association Study Helix-Loop-Helix Structure Hydroxymethylbilane Synthase Hypothalamic-Pituitary-Adrenal Axis Intellectual Disability Idiopathic Infantile Hypercalcemia Transcription Start Site Initiator Element Low Copy Repeats Limbic-Hypothalamo-Pituitary-Adrenal Axis Language Impairment Loxus of X-Over P1 Leucine Zipper Megabase (Million Base Pair) Minimal Critical Interval xi

12 mglur 2/3 Metabotropic Glutamate Receptors Type 2 and 3 mpfc mrna NAHR NCCPC NMDA OSBD OCD ODD PCR PD PDGF PLC PND PTSD RNA RTK RT-PCR SAD SDHA Medial Prefrontal Cortex Messenger RNA Non Allelic Homologous Recombination Non-Communicating Children s Pain Checklist N-Methyl-D-Aspartate Observational Scale of Behavioral Distress Obsessive Compulsive Disorder Oppositional Defiant Disorder Polymerase Chain Reaction Proximal Deletion Platelet Derived Growth Factor Phospholipase C Post Natal Day Post Traumatic Stress Disorder Ribonucleic Acid Receptor Tyrosine Kinase Reverse-Transcription Polymerase Chain Reaction Separation Anxiety Disorder Succinate Dehydrogenase xii

13 SDS-PAGE SERT Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Serotonin Transporter SH2 Src Homology 2 SINE SRE SRF SSRI SVAS TFII-I TRPC3 US WBS WT Short Interspersed Nuclear Elements Serum Response Element Serum Response Factor Selective Serotonin Reuptake Inhibitor Supravalvular Aortic Stenosis Transcription Factor 2I Transient Receptor Potential C3 Unconditioned Stimulus Williams-Beuren Syndrome Wild Type xiii

14 List of Tables Table 1.1. Clinical features of individuals with WBS Table 1.2. Behavioural profile of individuals with WBS Table 1.3. Behavioral profile of individuals with Dup7q11.23 syndrome Table 1.4. Prevalence of DSM-IV disorders in individuals with WBS and Dup7q11.23 compare to the general populations and individuals with developmental delays Table 2.1. Incidence of Separation Anxiety Disorder (SAD) in children with WBS and Dup7q11.23 compare to the general population Table 2.2. Separation difficulties in children with WBS and Dup7q11.23 compare to the general population Table 2.3. List of Primers Table 3.1. Summary of the doses used for subcutaneous injections of the drugs targetting either the serotonergic, GABAergic, or glutamatergic system Table 3.2. Primers used for quantitative real-time PCR amplification from cdna xiv

15 List of Figures Figure 1.1. Unique facial profile of individuals with WBS Figure 1.2. Visuospatial construction difficulties in individuals with WBS compare to IQ- and age- matched DNS individuals assessed by free drawings Figure 1.3. Distinctive facial characteristics of individuals with 7q11.23 Duplication syndrome Figure 1.4. Schematic representation of the three regions of low-copy repeats (LCRs) Figure 1.5. Schematic representation of 7q11.23 genomic rearrangements Figure 1.6. Schematic representation of possible NAHR Figure 1.7. Individuals with atypical deletions of 7q11.23 have implicated GTF2I and GTF2IRD1 in the behavioral and cognitive profile of WBS Figure 1.8. Schematic representation of mouse chromosome 5G with genes included in the 7q11.23 WBS interval Figure 1.9. Schematic representation of TFII-I family members and their respective amino acid lengths Figure Schematic representation of the structure of the four TFII-I isoforms and their respective amino acids length Figure A schematic representation of an extended emotional network hypothesized to be involved in anxiety responses Figure Schematic representation of a serotonergic synaptic terminal Figure Schematic representation of a GABA synaptic terminal Figure Schematic representation of a glutamate synaptic terminal Figure 2.1. Test of maternal separation anxiety in mice with altered Gtf2i genomic copy number xv

16 Figure 2.2. Generation of mice with decreased or increased Gtf2i dosage Figure 2.3.Schematic diagram depicting the parental crosses set up to generate F1 offspring used for testing the effects of anxiolytics on maternal separation-induced USVs Figure 2.4. Effects of subcutanous injection in maternal separation-induced USVs Figure 2.5. Plasma corticosterone concentrations measured using Corticosterone EIA Assay Figure 2.6. Maternal separation-induced USVs correlate with plasma corticosterone concentrations Figure 2.7. Schematic diagram depicting the parental crosses set up to generate F1 offspring used for testing the effect of maternal Gtf2i genotype on maternal separation-induced vocalizations in post-natal day 8 pups Figure 2.8. Maternal separation-induced USVs in PND8 mouse pups generated from different breeding pups where the Gtf2i duplication is inherited from either the dam or the sire Figure 3.1. Schematic representation of a serotonergic synaptic terminal Figure 3.2. Schematic representation of pentameric GABA-A receptors with five protein subunits that comprise the chloride ion channel Figure 3.3. Schematic representation of a glutamatergic synaptic terminal Figure 3.4. A scatter plot representing correlation between number of USVs emitted during the 30 sec screening and total number of USVs emitted during the 4 min trial for all pups receiving a saline injection Figure 3.5. Number of pups excluded from analysis after removing pups that produced 6 or less USVs during screening Figure 3.6. Gtf2i gene-dosage effect of serotonergic targeting drugs on USVs Figure 3.7. Anxiolytic effects of GABAergic targeting drugs on USVs Figure 3.8. Anxiolytic effects of glutamatergic targeting drugs on USVs xvi

17 Figure 3.9. Expression of immediate early gene c-fos using RT-PCR xvii

18 1 Chapter 1 Introduction The focus of this thesis was on the neurodevelopmental disorders WBS and Dup7q11.23, caused by reciprocal deletion and duplication of genes on chromosome 7, and their respective anxiety phenotypes. These disorders present with unique phenotypic spectra that include anxiety phenotypes common in the general population, such as separation anxiety, general anxiety and phobias. Due to the small number of genes with copy number changes in these two rare disorders, WBS and Dup7q11.23 provide a starting point for the identification of genetic variants and molecular pathways underpinning anxiety. GTF2I, one of the 26 genes commonly deleted/duplication in WBS/Dup7q11.23, has been linked to separation anxiety in a Gtf2i genedosage dependent way through studies of mouse models with altered Gtf2i gene copy number. These single gene mouse models provide the opportunity to better understand the neurochemical and physiological bases underlying separation anxiety disorder. 1.1 Williams-Beuren Syndrome History Williams-Beuren syndrome (WBS)(OMIM: ) is a neurodevelopmental disorder caused by the hemizygous deletion of 26 genes on chromosome 7q The incidence of WBS is estimated to be 1/7500 (Stromme et al., 2002), and the disorder is associated with specific clinical, cognitive, and behavioral phenotypes. Individuals with WBS have a distinctive set of facial features which include a flat nasal bridge, full cheeks, short upturned nose, wide mouth, periorbital fullness, long philtrum and dental malocclusions (Morris et al., 1988). Their cognitive profile is characterized by relative strengths in concrete language and verbal short term memory, but severe weakness in math and visuospatial construction. Cardiovascular problems are also common, including supravalvular aortic stenosis (SVAS) and connective tissue abnormalities. WBS is associated with failure to thrive, growth deficiency and overall developmental delay (Morris et al., 2006). Some people with WBS also have recurrent otitis media, sensory modulation problems and neurological problems (Mervis and Velleman, 2001, Meyer- Lindenberg et al., 2006). Individuals are described as hypersocial, highly approachable, and

19 2 empathetic while paradoxically being diagnosed with non-social anxiety, phobias and ADHD (Klein-Tasman, 2003 and Woodruff-Borden et al., 2010). The first reported cases of the then unknown WBS occurred in the 1950s in both Great Britain and Switzerland ( Lightwood, 1952, Fanconi et al., 1952). These reports described infants with hypercalcemia, constipation, reduced feeding, and growth failure over time. An epidemic of idiopathic infantile hypercalcemia (IIH) was present in Britain and Europe at the time (Jones, 1990) and most investigators suggested that this epidemic was due to excessive dietary vitamin D from supplemented formulas and cereals provided by the government following World War II. Recommendations to adjust supplemental vitamin D in food resulted in the disappearance of most IIH cases, however, IIH persisted in a subgroup of infants (Stapleton, 1957). Two forms of IIH were then recognized: a mild form that disappeared shortly after dietary restrictions, and a more severe form that persisted in individuals with developmental delays, failure to thrive, peculiar facial features and heart murmurs (Fraser at al., 1966). The next decade saw reports describing a condition that included SVAS, as well as dysmorphic facial features and mental disabilities. The first was a report in 1961 of four older children in New Zealand (Williams et al., 1961). Then in the following year, four other individuals were reported to have SVAS and facial features resembling those observed by Williams et al. (1961) in the following year (Beuren et al., 1962). The first report of a patient diagnosed with both SVAS and the severe kind of IIH also described the infant as having the dysmorphic facial features previously seen (Garcia et al., 1964). As an increasing number of similar cases were reported, some lacking a definite IIH diagnosis, a distinct behavioral phenotype emerged. In a subsequent cohort of 11 individuals with SVAS, dental malformations were also reported together with mental disabilities and a unique facial profile (Beuren et al., 1964). These patients were described as overfriendly and very social, which soon became a prominent feature of the WBS diagnosis. The combination of SVAS, characteristic physical features, intellectual disabilities, and unique behavioral profile constituted a previously unidentified syndrome, which is now known as Willams-Beuren syndrome (Beuren et al., 1964).

20 Clinical Profile Individuals with WBS display a characteristic pattern of clinical symptoms including cardiovascular problems (most commonly a narrowing of the ascending aorta SVAS, and connective tissue weaknesses), a distinctive facial profile, and mild to moderate intellectual disability (Williams et al., 1961; Beuren et al., 1962; and Morris, 2010). Although the multisystemic symptoms of WBS are highly variable between individuals, some common clinical presentations of the disorder are detailed in Table 1.1. Unique craniofacial features often make these individuals stand out. Young children typically have a broad forehead, periorbital fullness, stellate iris pattern, strabismus, flat nasal bridge, short upturned nose, full cheeks, wide mouth, dental malocclusion, and prominent ear lobes (Morris, 2010). Older individuals tend to have a more coarse profile with full lips, and a wide smile (Pober, 2010) (Figure 1.1).

21 Table 1.1. Clinical features of individuals with WBS (Adapted from Eronen et al., 2002, Leyfer et al., 2006, Morris, 2010, and Pober, 2010). 4

22 5 Figure 1.1. Unique facial profile of individuals with WBS (Mervis and Velleman, 2012). SVAS is the most common cardiovascular abnormality. SVAS is a stand-alone medical condition that is not necessarily accompanied by the WBS neurocognitive profile, however, it was through the diagnosis of patients with SVAS that WBS was initially recognized. Pulmonary, coronary and renal artery stenoses as well as cardiovascular lesions are also common in individuals with WBS (Pober, 2010). Eronen et al. (2002) showed that heart or vascular disease occurred in 53% of a cohort of 75 WBS patients with an age range of newborn to 76 years. Furthermore, cardiovascular-associated complications are the major cause of mortality in WBS (Wessel et al., 2004). Hypertension is also problematic in WBS, beginning in childhood and developing in approximately 50% of these individuals (Wessel et al., 1997, Broder et al., 1999, and Eronen et al., 2002). Endocrine-related problems include hypercalcemia, which has been documented in anywhere from 5% to 50% of individuals with WBS having one or more episodes of hypercalcemia. The episode can be either asymptomatic or associated with non-specific symptoms such as vomiting, loss of appetite, and constipation (Sforzini et al., 2002). Impaired glucose tolerance, diabetes, and subclinical hypothyroidism have also been noted (Pober et al., 2010).

23 6 Developmental and growth delay are typically observed in all infants with WBS, which is usually accompanied by a premature and shortened pubertal growth spurt (Partsch et al., 1999). Neurological problems present include hypotonia, tongue thrust, strabismus, intention tremor, dysmetria and ataxia (Morris et al., 1988 and Galgiardi et al., 2007). Individuals with WBS also have difficulties with sensory modulation. Hypersensitivity to specific sounds, such as thunderstorms, fireworks and vacuum cleaners, is common and may affect up to 90% of patients (Gothelf et al., 2006). Hypersensitivity to certain sounds and poor muscle control were reported to be the most problematic by parents (John and Mervis, 2010). Imaging studies indicate unique patterns of changes in specific brain regions. The overall volume of the cerebrum is reduced. Cerebellar size and volume, however, remain unaffected (Reiss et al., 2000, and reviewed in Mervis et al., 2000). Decreases in grey matter, evaluated through magnetic resonance imaging studies, have been noted in the intraparietal sulcus, in both children and adults, as well as in the superior parietal lobule in adult women with WBS (Milhorat et al., 2007 and Kippenhan et al., 2005; and Eckert et al., 2005) Cognitive Profile The WBS cognitive profile is characterized by peaks and valleys of ability. Individuals with WBS show mild to moderate intellectual disability, accompanied by relative strengths in language capabilities and verbal short-term memory (auditory route), but extreme weakness in visuospatial construction (Mervis et al., 2000). Mild to moderate intellectual disabilities are noted in these individuals with an average IQ of 55 to 60 (Martens et al., 2008). Most studies report a higher average of verbal IQ in comparison to performance IQ in individuals with WBS (Pober, 2008). Language acquisition, including onset of vocabulary and grammar acquisition as well as production and comprehension of gestures, is almost invariably delayed in children with WBS (Mervis and Klein-Tasman, 2000). Although language acquisition is delayed, expressive language capabilities are not affected. Toddlers with WBS have difficulty producing and/or comprehending referential gestures, such as pointing. These toddlers are also less likely to engage in triadic joint attention episodes, wherein simultaneous attention to the speaker and object is required (Laing et al., 2002 and Osborne and Mervis, 2007). In older children, language

24 7 is fluent and grammatically correct. However, these children often have difficulties with the pragmatics of language, such as maintaining the topic of a conversation (Mervis, 2006). Communicative aspects of language are also impaired. In terms of their vocabulary, individuals with WBS show strengths in concrete vocabulary, but weaknesses in relational vocabulary, which includes words used for spatial, dimensional, and temporal concepts. This weakness in relational vocabulary is thought to stem from their weakness in visuospatial construction (Osborne and Mervis, 2007 and Mervis et al., 2010). Visuospatial construction abilities are extremely weak in individuals with WBS. They have difficulties constructing a pattern as a whole and, instead, focus on the individual components of the pattern. In a study comparing age- and IQ- matched WBS children with Down syndrome (DNS) children, subjects were asked to draw an object such as a bicycle (Bellugi et al., 1990). The drawings by children with WBS contained many parts of the object but the parts were not organized coherently. In contrast, the drawings by individuals with DNS had the correct overall configuration of the object but lacked the individual details (Figure 1.2) (Bellugi et al., 2000). WBS individuals seemed to attend to details at the expense of the whole. However, despite their emphasis on local rather than global processing in drawings, individuals with WBS show strengths in facial recognition, discrimination and memory (Pober, 2010).

25 8 Figure 1.2. Visuospatial construction difficulties in individuals with WBS compare to IQand age- matched Down syndrome (DNS) individuals assessed by free drawings (Bellugi et al., 2000). Focus on the details is seen in the drawings of individuals with WBS at the expense of the overall configuration whereas the opposite is true for individuals with DNS Behavioral Profile The behavioural characteristics of individuals with WBS include overfriendliness, attention deficit-hyperactivity disorder (ADHD) and anxiety (Mervis et al., 2000). Social and interpersonal skills are strengths for these individuals, although comprehension in some social settings might be abnormal. Their personality is often referred to as the cocktail party type of personality, depicting a highly social, friendly, and empathetic individual (Tager-Flusberg et al., 2000 and Pober, 2010). They are often described as socially disinhibited and as seekers of interactions, even with strangers (Frigerio et al., 2006 and Mervis et al., 2000). Even as infants, WBS children show more positive and less negative behaviors in social settings. Furthermore, they are recognized as being dramatic storytellers (Bellugi et al., 2000). Although hypersocial and friendly, individuals with WBS have difficulty making friends and perceiving social cues (reviewed in Morris, 2010). This impaired social judgment is hypothesized to be due to impaired function of the orbitofrontal cortex, which is important for regulation of the amygdala, an area critical for social propriety (Meyer-Lindenberg et al., 2006).

26 9 Elevated rates of psychiatric disorders occur in WBS, with approximately 50% to 90% of these individuals meeting the diagnostic criteria for one or more anxiety disorders, phobias and/or ADHD. Leyfer et al. (2006) studied 119 children with WBS aged 4-16 years old and found that 65% of them met the criteria for ADHD and 57% met DSM-IV criteria for at least one anxiety disorder. Despite their highly social personality, individuals with WBS have excessive worries and fears. Anticipatory anxiety about upcoming events and non-social anxiety are common, whereas social anxiety about meeting strangers is absent (Dykens, 2003). A longitudinal study of WBS patients over time found that 82% of patients had an anxiety diagnosis at some point in their lives (Woodruff-Borden et al., 2010) (Table 1.2). A clear difference needs to be established between the presence of symptoms and meeting the diagnostic criteria for an anxiety disorder since the latter requires not only the presence of symptoms, but also distress, interference with everyday life, and the potential need for intervention (Woodruff-Borden et al., 2010). Although anxiety is a common feature of WBS, only a small number of symptomatic children are treated for it (Morris, 2010). Table 1.2. Behavioral profile of individuals with WBS (Adapted from Leyfer et al., 2009, 2012, and Mervis et al., 2012) q11.23 Duplication Syndrome History The first case of an individual with a duplication of the 7q11.23 region was only recently identified (Somerville et al., 2005). In theory, duplications of the region should occur at a similar frequency as deletions due to the mechanism through which deletions and duplications arise (see 1.3.2), however, such duplications had not been previously described. This was hypothesized to

27 10 be due to either a lethality associated with the duplication, a lack of observable phenotypes, or a completely different clinical presentation of the 7q11.23 duplication compared to the WBS deletion (Osborne and Mervis, 2007). The latter turned out to be the case. Fluorescence in-situ hybridization (FISH) was used to identify the first individual with duplication of 7q11.23 (Dup7q11.23). Results revealed a tandem duplication localized to the region commonly deleted in WBS, and microsatellite marker analysis confirmed the duplication by showing three distinct alleles in the proband for markers within the WBS region. One allele was inherited from the father whereas the other two alleles were inherited independently from each maternal grandparent. This suggested that meiotic interchromosomal recombination led to the duplication of the region and took place in the maternal germ cells (Somerville et al., 2005). The clinical presentation of this eight year old boy was characterized by a unique set of phenotypes, including severe delay in expressive language and intellectual strengths, which were in direct contrast to findings in individuals with WBS. The boy weighed 2.52kg at birth (at the fifth percentile) and with a length of 44.5cm (at the fifth percentile). At 13 months old, he was evaluated for failure to thrive and hypotonia. At 2 years old, he was diagnosed with moderate to severe language delay and, one year later, was diagnosed with a severe delay in receptive and expressive language. Diagnosis of ADHD, unspecified sleep disorder and delays in overall development as well as in speech, language and fine motor skills followed at 4 years and 2 months old. In terms of physical characteristics, growth retardation and mild dysmorphism were observed. Facial features included a high and narrow forehead, long eyelashes, high and broad nose, a short philtrum and an asymmetric face (Somerville et al., 2005). Gene expression analyses revealed increased expression in 5 out of the 6 genes that were examined within the duplicated region. Expression of GTF2I, LIMK1, EIF4H, RFC2, and BAZ1B genes was increased in the individual with Dup7q11.23 and reduced in people with WBS (Somerville, 2005). The characteristics of this first duplication case were soon recognized as another unique syndrome that comprised the following features: a subtle but recognizable facial phenotype, including a high and broad nose, high-arched palate, thin lips, short philtrum, and posteriorly rotated ears, together with a delay in expressive language. This disorder, associated with a

28 11 duplication of the 7q11.23 chromosomal region, is now known as Dup7q11.23 syndrome (OMIM: ) Clinical and Cognitive Profile Although the profile of Dup7q11.23 has not yet been fully established due to the small number of cases (approximately 80) currently identified, almost all of those identified so far exhibit speech delay, a characteristic facies, developmental delay, hypotonia, and behavioural problems such as anxiety or oppositional defiant disorder (Somerville et al., 2005, Berg et al., 2007, Osborne and Mervis, 2007, Van der Aa et al., 2009, Velleman and Mervis, 2012). In contrast to individuals with WBS who show a relative strength in language capabilities and weakness in visuospatial construction, children with Dup7q11.23 show developmental delay in speech (Somerville et al., 2005, Torniero et al., 2007). Speech developmental delay has been observed in all individuals identified so far with Dup7q11.23 (Sommerville et al., 2005, Osborne and Mervis, 2007, Van der Aa et al., 2009, Velleman and Mervis, 2012). Intellectual abilities were evaluated by Velleman and Mervis (2012) in two different age groups. Intellectual abilities in 13 children aged 1-4 years were assessed using the Mullen Scales of Early Learning and found to be in the moderate to average range of developmental delay compared to the general population. A second group of 25 children aged 4-15 years were assessed using the Differential Ability Scales-II and were found to have an IQ in the low average range (Velleman and Mervis, 2012). Therefore, intellectual disabilities in individuals with Dup7q11.23 are often minor with IQ hovering between mild intellectual disability and average IQ. The profile of Dup7q11.23 appears to be milder, more variable, and not as well defined as that of WBS. A facial phenotype of mild dysmorphisms has been associated with this duplication. Dysmorphisms described in Dup7q11.23 patients include a high and broad nose, straight eyebrows, short philtrum, prominent forehead, deep set eyes, thin upper lip, and high palate (Figure 1.3) (Somerville et al., 2005, Berg et al., 2007, and Van der Aa et al., 2009 Velleman and Mervis, 2012). Various congenital anomalies, such as patent ductus arteriosus and cryptorchidism, have also been reported in some cases (Van der Aa, 2009). Although the phenotype of Dup7q11.23 appears to be more variable than seen in WBS, unique characteristics are present that characterize this neurodevelopmental disorder. This suggests that factors other than gene-dosage

29 12 effects, such as genetic and/or environmental interactions, may play a role in determining the phenotypic outcome of patients with a duplication of 7q11.23 (reviewed in Schubert et al., 2009). Figure 1.3. Distinctive facial characteristics of individuals with 7q11.23 Duplication syndrome (Velleman and Mervis, 2012) Behavioral Profile In the approximately 80 individuals with Dup7q11.23 syndrome assessed so far, a difference in social behaviour is striking when compared to WBS individuals. Individuals with Dup7q11.23 appear more socially withdrawn and shy, unlike WBS individuals, who are described as being hypersocial. Anxiety, ADHD, and autism-spectrum disorder (ASD) have been reported in a large number patients (Berg et al., 2007, Depienne et al., 2007, Torniero et al., 2008 and Van der Aa et al., 2009 and Velleman and Mervis, 2012) (Table 1.3).

30 13 Table 1.3. Behavioral profile of individuals with Dup7q11.23 syndrome (Adapted from Van der Aa et al., 2009, Velleman and Mervis, 2012, and Mervis et al., 2012). Studies have also looked for an association between autism spectrum disorder (ASD) and Dup7q11.23 due to the overlap of phenotypes. In a recent study of 206 patients with ASD, one male patient was identified with a de novo duplication of the WBS critical region and presented with a much more severe phenotype than previously identified Dup7q11.23 individuals. The child had severe language delay, mental disabilities, ADHD, sudden outbursts of aggression, hyperphagia, and mild dysmorphic features (Depienne et al., 2007). Due to the pattern of phenotypes that this child presented in addition to the lack of some of these phenotypes in other Dup7q11.23 individuals, it was suggested that the child might also have an additional genetic disorder (Osborne and Mervis, 2007). A second genome-wide analysis study (GWAS) of rare copy-number variation (CNV) in 1124 autism-spectrum disorder (ASD) families found four cases of de novo duplication of the 7q11.23 interval, thus implicating autism in the clinical profile of Dup7q11.23 syndrome (Sander et al., 2011) Triplication of Dup7q11.23 Although rare, triplication of the 7q11.23 region has also been reported. A case report of an individual with 7q11.23 triplication described similar but more severe phenotypic features as compared to Dup7q11.23 syndrome (Beunders et al., 2010). These features included mental disability, severe expressive language delay, behavioral problems, and facial dysmorphisms. Triplications are very rare chromosomal rearrangements. Nonetheless, studies of triplications of the Xq22, Xq28, and 3q chromosomal regions have shown that the resulting phenotype is

31 14 similar to, but more severe than, phenotypes observed in those with a duplication of the same region (Reddy et al., 2000 and Beunders et al., 2010). This suggests that gene-dosage effects are important considerations when examining genotype-phenotype correlations. 1.3 The Genetics of 7q11.23 Rearrangements Genetic Basis of Williams-Beuren Syndrome Evidence for the genetic basis of WBS came through studies of patients with SVAS, who showed an autosomal dominant inheritance pattern (Curran et al., 1993). Firstly, it was found that SVAS was associated with chromosome 7 through linkage studies in two families with autosomal dominant SVAS (Curran et al., 1993). Next, a disruption via translocation within the elastin gene that was mapped to chromosome region 7q11, was found to co-segregate with SVAS (Curran et al., 1993). These two findings confirmed that disruption of the elastin gene can cause SVAS (Curran et al., 1993). Genetic analysis in four familial and five sporadic cases of WBS uncovered a hemizygous deletion of the ELN gene in these WBS individuals who were also diagnosed with SVAS (Ewart et al., 1993). As such, SVAS can also be inherited as a feature of WBS, and hemizygous deletion of the elastin gene is implicated in the vascular and connective tissue abnormalities present in WBS (Ewart et al., 1993; Curran et al., 1993). Fluorescence in-situ hybridisation (FISH) analysis with probes for the elastin locus emerged as the standard method for WBS diagnosis. Although elastin disruption or deletion was present in autosomal dominant SVAS and caused comparable connective tissue abnormalities, none of the neurobehavioral features of WBS had been documented in the autosomal form of SVAS. This suggested that ELN hemizygosity alone could not account for the full spectrum of phenotypes observed in WBS. Subsequent studies focused on mapping the deletion in individuals with WBS to pinpoint the exact chromosome region and genes implicated. Further genetic mapping revealed that WBS is a genomic disorder caused by a hemizygous contiguous gene deletion on chromosome 7q11.23 (Ewart, 1993). This deletion encompasses between 1.5 to 1.8 mega base pairs (Mb) with a resultant loss of between 26 and 28 genes, depending on the breakpoint (Ewart, 1993). The role that these genes play in the phenotype of WBS was unclear and thus, the focus of many subsequent studies attempting to establish genotype-phenotype correlations.

32 Mutational Mechanism in WBS and Dup7q11.23 WBS is caused by the hemizygous deletion of genes on chromosome 7q The single copy region of WBS is flanked by repetitive sequences known as low copy repeats (LCR). These LCRs are arranged in three LCR blocks, namely A, B and C, and these blocks occur centromeric, medial and telomeric to the WBS locus (Figure 1.4). Alu repeats, short interspersed nuclear elements (SINE) of less than 500 bp in size, comprise the boundaries of these LCRs, and are postulated to have played a role in the evolution of these complex LCRs (Batzer, 2002, Antonell 2005). Figure 1.4. Schematic representation of the three regions of low-copy repeats (LCRs). The centromeric (c ), middle (m), and telomeric (t) LCRs each contain three blocks of repetitive sequences (A, B, and C) (Merla et al., 2010). Non-allelic homologous recombination (NAHR) between the LCRs during meiosis is responsible for the WBS deletion, the reciprocal Dup7q11.23 duplication, triplication, and inversions of the same region (Figure 1.5). NAHR can occur between homologous chromosomes (interchromosomal), homologous chromatids (interchromatidal) or within a chromatid (intrachromatidal). The WBS deletion is caused by intrachromosomal, interchromosomal or intrachromatidal NAHR exchange during meiosis, whereas the duplication is caused by interchromosomal and intrachromosomal NAHR (Figure 1.6A and 1.6B) (reviewed in Merla et al., 2010). Two-thirds of the deletions arise from interchromosomal exchange between chromosome 7 homologs, whereas the rest of the deletion cases occur via intrachromatidal rearrangements (Dutly 1996; reviewed in Schubert, 2009). Interchromosomal recombination also accounts for most duplication cases (Cusco et al., 2008). Inversions of chromosome 7q11.23, on

33 16 the other hand, are generated by meiotic or mitotic intrachromatidal misalignment between the inverted homologous LCR blocks (Bayes et al., 2003) (Figure 1.6C). Figure 1.5. Schematic representation of 7q11.23 genomic rearrangements. Deletion, duplication, triplication, and inversion are depicted as generated by non-allelic homologous recombination (NAHR) during meiosis between the LCRs (Merla et al., 2010).

34 17

35 18 Figure 1.6. Schematic representation of (A) interchromosomal and interchromatid non-allelic homologous recombination (NAHR) between low-copy repeats (LCRs) resulting in deletions and duplications of the intervening region. (B) Intrachromatidal NAHR between LCRs resulting in deletions of the intervening region and formation of a reciprocal acentric chromosome with high risk for segregation loss. (C) Intrachromatid misalignment of inverted LCRs leading to an inversion of the intervening region with breakpoints within that block (Schubert, 2009). Both common and atypical deletions have been reported in WBS individuals. Common deletions span a genomic region of around ~1.5 Mb with breakpoints in the centromeric and medial LCR block B. Atypical deletions on the other hand, can be larger or smaller in size. A deletion of ~1.8Mb present in about 5% of WBS cases, is caused by recombination between the centromeric and medial LCR block A (Figure 1.5). Smaller deletions have also been reported with breakpoints within the WBS region (Bayes et al., 2003). NAHR between LCRs is possible because of a high sequence homology (average 99.6%) between the centromeric and medial block B and between the centromeric and medial block A (average 98.2%). The higher sequence homology between the B blocks and the shorter interval size (~1.5Mb) generated from NAHR between the B blocks is thought to account for the higher frequency of this deletion in WBS (Bayes, 2003) Inheritance Incidence Although WBS usually occurs sporadically, a few cases of autosomal dominant inheritance have been reported. The disease-transmitting parent often presented with a milder WBS phenotype (Morris et al., 1993 and Metcalfe et al., 2005). Morris et al., (1993) reported three families in which parent-to-child transmission of WBS had occurred, with one male-to-male transmission.

36 19 An autosomal dominant pattern of inheritance was concluded as the most likely mode of inheritance. None of the parents, however, had evidence of SVAS, and they were only diagnosed with WBS after their child s diagnosis (Morris et al., 1993). Although most reported cases of WBS occur sporadically, the lack of familial cases can be due to either a true lack of inherited cases of WBS or a lack of reported cases of familial inheritance because of external factors. Such external factors may include lower reproductive fitness as a result of decreased opportunities for reproduction due to intellectual disabilities. Furthermore, a lack of diagnosis of WBS in individuals with WBS phenotypes but without SVAS is also possible since cardiovascular problems are often the reason these individuals are diagnosed in the first place (Morris et al., 1993). In contrast, Dup7q11.23 is inherited from one parent in approximately one third of the cases and the chromosome-transmitting parent in many families displayed duplication symptoms (personal communication, Carolyn Mervis). Inversion of the WBS locus has breakpoints external to the WBS region, does not disrupt actively expressed genes in the WBS region, and has no clinical symptoms associated with it (Tam et al. 2008). This inversion is present in around 6% of the population but in 25-33% of transmitting parents of children with WBS (Osborne et al., 2001, Bayes 2003, Hobart 2010). This suggests that its presence predisposes the chromosome to mispairing during meiosis, and increases the risk of deletion or duplication of the region in the gametes (Hobart, 2010). 1.4 Genotype-Phenotype Correlation Since the discovery of the exact region disrupted in WBS, studies have focused on determining the potential function(s) of the deleted genes. Such studies are complicated, however, because of the presence of a large number of genes that may act individually or in combination. Furthermore, variability in the clinical phenotypes observed in individuals with the same chromosomal disruptions makes it harder to pinpoint precise gene contributions. The discovery of individuals with atypical deletions has proven invaluable in providing a unique opportunity to investigate the contribution of specific genes within the 7q11.23 region to the complex WBS phenotype. In atypical cases, one or both of the breakpoints differ from those seen in individuals with classic deletions of the WBS region. However, these individuals are rare, with the majority of WBS cases having a deletion spanning the entire region. To date, about 30

37 20 individuals with atypical deletions have been identified and assessed to determine genotypephenotype correlations (Osborne et al., 2010). Unfortunately, clear correlation has been limited by the identification of only a small number of individuals with atypical deletions. Furthermore, most cases present with different deletions and the roughly mapped breakpoints make it difficult to compare results. In addition, the possible effects on the expression of neighbouring, nondeleted genes have not been examined in most cases. Another issue with this population is that they have been evaluated by different physicians and are often subject to a different battery of tests to assess clinical, cognitive, and psychological function. Lastly, there is a significant ascertainment bias towards individuals with deletions including ELN due to the distinctive cardiovascular phenotype that results from the deletion of this gene. This makes it easier to distinguish those with a deletion of the entire WBS region but harder to identify individuals with smaller deletions that do not encompass ELN (Osborne et al., 2010). ELN was the first gene mapped to the WBS region and linked to a specific phenotype. Disruption of this gene whether via mutation, complete or partial deletion, causes the cardiovascular and connective tissue abnormalities seen in individuals with WBS and also contributes to the hypertension and hoarse voice phenotypes (Curran et al., 1993, Collins, 2010). SVAS, a phenotype observed in approximately 73% of individuals with WBS, is due to mutations of ELN gene (Tassabehji, 1997, Li, 1997). Studies of individuals with atypical deletions have pointed to a sub-region within the known 7q11.23 interval that is sufficient on its own to cause the core phenotypes typically seen in individuals with WBS; this region is referred to as the minimal critical interval (MCI) (Figure 1.7). This MCI spans the region from ELN to the common distal breakpoint encompassing just nine genes, including the two members of the General Transcription Factor 2I family, GTF2I and GTF2IRD1 (Morris et al., 2003, and Tassabehji et al., 2003). Further atypical patients have been reported to have deletions that spare one or more of the genes in the MCI. Through the comparison of phenotypes presented by individuals with typical and atypical deletions, the GTF2I gene family has been implicated in the behavioral and cognitive aspects of WBS (Antonell et al., 2010). These genes are located at the telomeric end of the common WBS deletion and, when left intact, a lack of the unique facial features, cognitive disabilities, and behavioral symptoms is observed (Antonell et al., 2010 and Morris, 2003). The GTF2I gene

38 21 family encodes transcription factors, and as such, their deletion may alter the expression of downstream genes and molecular pathways that are affected in WBS. Figure 1.7. Individuals with atypical deletions of 7q11.23 have implicated GTF2I and GTF2IRD1 in the behavioral and cognitive profile of WBS. A minimal critical interval (MCI) has also been recognized within the 7q11.23 region that is sufficient on its own to cause the core phenotypes typically seen in individuals with WBS. 1.5 Mouse Models Mouse models provide a unique opportunity to study the functions of genes while circumventing some of the problems encountered with studying human populations. An alteration in the genome can be genetically induced in mice and allow investigators to hypothesize and/or draw conclusions about the effects of that specific manipulation by studying many mice with the same genetic background (Osborne, 2010).

39 22 When it comes to neurodevelopmental disorders such as WBS, it is difficult, if not rare, to examine the disorder at the prenatal stages in the patient population. However, the use of mouse models allows investigation into this disorder at both the pre-natal and post-natal periods. The scope of potential analyses is also far wider when studying animal models; this can range from the whole animal level to individual tissues and molecules. Although clinical resources are not replaceable when studying human disease, animal models provide a unique window into the study of the mechanism of disease. In particular, mouse models have been widely used and validated as animal models to study the underlying pathogenic mechanisms of various disorders (Osborne, 2010). The mouse genome sequence reveals very similar gene content to the human sequence, and as such, mice exhibit many of the clinical symptoms observed in human disorders, which can then be assessed with the help of phenotyping tools (Waterson, 2002, and Rossant, 2001). High conservation of gene content and order of the WBS region on the syntenic, although inverted, mouse chromosome 5G has allowed for the creation of different mouse models and dissection of the contributions of individual genes (Valero et al., and Osborne, 2010). Once a genetic manipulation has been induced, whether it involves disruption of a single gene, multiple genes, at the whole genome level or tissue-specific level, its effects on both phenotype and gene function can be studied. This is made possible due to the unlimited access to tissues and embryonic time points in the mouse that are not possible in humans. Such mouse models, which display clinical symptoms similar to the population of patients affected by the particular disorder in question, then provide an experimental model for developing and testing therapeutic interventions. Behavioural and physiological analyses of mouse models have revealed that many of the phenotypes in WBS patients can be mirrored in the mouse. To date, several knock-out mouse models of a single gene or a combination of the genes implicated in WBS have been generated and characterized. Semi-dominant phenotypes in these mouse models suggest that several genes may be haploinsufficient in WBS (Hoogenraad et al., 2002 and Osborne, 2007). However, some aspects of WBS, such as language deficits, are complex neurobehavioral traits that are difficult to study in mice (Osborne, 2010). One of the main advantages of using mouse models is the ability to develop and test therapeutic interventions. From the mouse models depicting the

40 23 cardiovascular problems in WBS, the study of the Eln-null mice has already proven beneficial. In Eln-null mice, which have a deletion of the ELN gene, the cardiovascular abnormalities were ameliorated following introduction of a human ELN gene (Hirano, 2007). This mouse model may therefore be critical in the pre-clinical testing of therapies targeting the cardiovascular deficits in people with WBS Previously Studied Mouse Models Mouse models with single gene deletions currently exist for 12 of the 26 genes commonly deleted in WBS (Figure 1.8). Single-gene deletion mouse models are critical for establishing genotype-phenotype correlations. In contrast, contiguous gene deletion mouse models allow for evaluating the combinatorial effects of genes and are likely to be more representative of WBS, which is defined as a contiguous gene disorder (Osborne, 2010). A mouse model that spans the entire WBS region currently exists (Li et al., 2009). Although many single gene mouse models have been generated, of the several Gtf2ird1 -/- mouse models, only one reveals an anxiety phenotype (reviewed in Osborne, 2010, Durkin et al., 2001, Tassabehji et al., 2005, Palmer et al., 2007, Howard et al., 2011, Young et al., 2008, Proulx et al., 2010, and Enkhmandakh et al., 2009).The following are characterizations of the mouse model that spans the entire WBS region and Gtf2ird1 -/- single gene mouse model with an anxiety phenotype. Figure 1.8. Schematic representation of mouse chromosome 5G with genes included in the 7q11.23 WBS interval. A mouse model with contiguous gene deletion Fkbp6 to Gtf2i has been generated. Single gene mouse models also exist for all the genes highlighted in green.

41 Contiguous Gene Deletion Fkbp6 to Gtf2i The only mouse model that spans the entire WBS region encompasses all the genes between and including Fkbp6 and Gtf2i (Li et al., 2009). Using Cre-loxP recombination, a mouse line was first generated spanning the proximal part of the commonly deleted WBS region (PD), which encompassed a deletion of the region from Limk1 to Gtf2i and was equivalent to deletion of the human MCI, except for Eln. A second mouse line was generated with a distal deletion (DD) spanning the region from Limk1 to Fkbp6. Crossing of the two lines generated P/D mice with deletions that encompassed the entire WBS common deletion region. P/D mice were heterozygous for all of the genes in the interval except for Limk1, for which they were homozygous null due to the presence of loxp sites used to generate the deletion. P/D mice showed growth delay accompanied by hernias and rectal prolapse. Neuroanatomically, an increase in neuronal density was observed in the somatosensory cortex coupled with a decrease in lateral ventricle volume. Sociability was increased in P/D mice as indicated by heightened social interest and interaction and accompanied with an increase in anxiety measured using the open field test. Increased sensitivity to sound was reported with the baseline startle response. P/D mice also exhibited attenuated pre-pulse inhibition. Lastly, performance in the rotarod test was reduced, which indicated impairment of motor skills (Li et al., 2009) Gtf2ird1- Knockout Mouse Many Gtf2ird1 -/- mouse models have been previously created and characterized (Durkin et al., 2001, Tassabehji et al., 2005, Palmer et al., 2007, Howard et al., 2011, Young et al., 2008, Proulx et al., 2010, and Enkhmandakh et al., 2009). One such models was generated by a targeted knockout of Gtf2ird1 exons 2 to 5 and it is the only one that exhibited an anxiety-related phenotype (Young et al., 2008). Behavioral characterization of these mice showed decreased aggression and increased social interaction measured via the resident-intruder test. Mice also exhibited reduced anxiety measured via the elevated plus maze and open field test of anxiety (Young et al., 2008). In the amygdala-dependent cued fear conditioning test, both Gtf2ird1 heterozygous and homozygous mice exhibited decreased fear. These altered behaviors were accompanied by increased levels of the serotonin (5-HT) metabolite, 5-hydroxyindoleacetic acid, in the amygdala as well as frontal and parietal cortices (Young et al., 2008). Furthermore,

42 25 enhanced inhibitory 5-HT currents were recorded in layer V pyramidal neurons of the prefrontal cortex further suggesting altered serotonergic transmission in these mice (Proulx et al., 2010). 1.6 GTF2I Family of Transcription Factors Genotype-phenotype correlations in WBS individuals with atypical deletions point to a MCI for WBS that, when deleted, gives rise to the core phenotypes associated with WBS. This interval includes two of the three GTF2I family members (Morris et al., 2003 and Tassabehji et al., 2003). GTF2I, together with GTF2I Repeat Domain containing protein 1 (GTF2IRD1) and GTF2IRD2, are genes that code for members of the transcription factor 2I (TFII-I) family, and cluster at the telomeric end of the typical WBS deletion. GTF2I and GTF2IRD1 lie within the commonly deleted region and the MCI, whilst GTF2IRD2 is variably deleted depending on the point of non-allelic recombination. The GTF2I proteins are characterized by the presence of DNA-binding I-repeat domains that are 90 amino acids in length, a helix-loop-helix-like structure (HLH), a putative leucine zipper (LZ) essential for homodimerization, and a nuclear localization signal important for entry into the nucleus (Hinsley et al., 2004). (Figure 1.9). Figure 1.9. Schematic representation of TFII-I family members and their respective amino acid lengths. All three genes contain DNA-binding I-repeat domains (R#) that are 90 amino acids in length, a putative leucine zipper (LZ) essential for homodimerization, and a nuclear localization signal important for entry into the nucleus.

43 GTF2I (TFII-I) GTF2I, which encodes the TFII-I protein, was identified as the first member of the family, and as such, it is the one that is best understood (Cheriyath and Roy, 2001). Like the other members of its family, it contains a leucine zipper (LZ) important for homomeric dimerization, a nuclear localization signal, as well as six I-repeat domains with HLH motifs that enable protein-protein interactions, and DNA binding through a basic region just before I-repeat 2. Four alternatively spliced variants of TFII-I exist, all of which contain the six I-repeats as well as a nuclear localization signal (Figure 1.10) (Cheriyath and Roy, 2001). The four spliced isoforms, α, ß, Δ, and γ, have been characterized and found to interact with one another in both homomeric and heteromeric manner. The different combinations of these isoforms might lead to differential gene expression through their activity on promoters. The γ isoform is the predominant form found in the brain (Cheriyath and Roy, 2000). Figure Schematic representation of the structure of the four TFII-I isoforms and their respective amino acids length. All four isoforms contain a leucine zipper (LZ), nuclear localization signal (NLS), basic region/dna binding domain (BR), DNA-binding I-repeat domains (R#), exon a (a, 21 amino acids) and exon b (b, 22 amino acids) encoded regions (Roy et al., 2007). a, b: exons a (21 aa) and b (22 aa) encoded regions TFII-I in the Nucleus Unlike other transcription factors, TFII-I acts as both a basal factor and an activator. It acts as a basal factor by binding the transcription start site initiator element (Inr), whereas it serves the role of an activator by binding to Inr or E-box elements at enhancers (Roy et al., 1997). The

44 27 activity of TFII-I is regulated by phosphorylation on its many serine and tyrosine residues. Bruton s tyrosine kinase (Btk) is a cytoplasmic kinase important during B-cell development (Tsukada et al., 2001). In B-cells, Btk associates with TFII-I, phosphorylates it, and regulates TFII-I transcriptional activity (Yang and Desiderio, 1997; and Kim et al., 1998). After phosphorylation of TFII-I by Btk, TFII-I dissociates from Btk and translocates into the nucleus where it regulates transcription (Novina et al., 1999) TFII-I in c-fos Promoter Activity TFII-I also associates with serum response factor (SRF) and Phox1 protein, both of which are involved in the regulation of the c-fos promoter (Grueneberg et al., 1997). C-fos is an immediate early gene that is responsive to many different extracellular signals through its promoter. The c- fos promoter contains a TATA-box and several upstream elements. Such elements include a calcium cycle AMP response element (CRE), a serum response element (SRE), and a cis platelet derived growth factor (PDGF)-inducible factor element (SIE). The SRE binds to the SRF factor and other transcription factors to form complexes responsive to MAP kinase (reviewed in Kim et al., 1998). SRE can also be activated by small proteins such as RhoA, a GTP-binding protein (G proteins) and ultimately stimulate the c-fos promoter. TFII-I can bind to three different upstream sites of the c-fos promoter, including the SIE and SRE, and enhance the transcriptional activity of the c-fos promoter. In addition, TFII-I can form complexes with critical promoter binding proteins that are involved in the regulation of the c-fos promoter, such as SRF, and enhance c-fos promoter activity. TFII-I enhancement of c-fos promoter activity requires a functioning ras pathway, and this role of TFII-I is enhanced by epidermal growth factor stimulation (EGF) causing a significant increase in phosphorylation of TFII-I tyrosine residues and ultimately increasing TFII-I activity. Therefore, tyrosine phosphorylation of TFII-I may regulate transcriptional activity of TFII-I and subsequently TFII-I enhancement of c-fos promoter activity (Kim et al., 1998). C-fos promoter is also regulated by nitric oxide, cgmp and a cgmpdependent protein kinase (G kinase). cgmp association with G-kinase causes the translocation of G-kinase into the nucleus. Interestingly, G-kinase can phosphorylate TFII-I on its serine phosphorylation site in the nucleus after TFII-I binding to the G kinase via TFII-I s N-terminal LZ. TFII-I phosphyration by G kinase activates TFII-I function in the nucleus and ultimately its

45 28 activation of the c-fos promoter (Casteel et al., 2002). Therefore, TFII-I is an important player in the activation and regulation of the c-fos promoter activity (Kim et al., 1998). The activity of TFII-I is regulated by phosphorylation on its many serine and tyrosine residues. MAP kinase phosphorylation of TFII-I can activate TFII-I transcriptional activity in vitro. TFII-I is also regulated by kinases belonging to the Src family (reviewed in Roy., 2007). TFII-I contains a region homologous to the D-box of Elk-1 (a c-fos transcription factor) and extracellular signalrelated kinase (ERK). The D-box is a MAP kinase interaction domain, and similar domains are present in the MAP kinase phosphorylation sites on TFII-I (reviewed in Kim et al., 2000). This suggests that TFII-I may interact with, and be phosphorylated by the MAP pathway in a similar manner as Elk-1 through its consensus D-box. Specifically, it`s the interaction with ERK of the MAP pathway, via the D-box, that is required for TFII-I activity on the c-fos promoter. Phosphorylation sites serines 627 and 633 were critical for ERK phosphorylation of TFII-I and consequent activation of TFII-I enhancing activity on the c-fos promoter. Therefore, TFII-I function is dependent on a functionally intact MAP kinase pathway that includes ERK. TFII-I function was not dependent, however, on Src kinases. The interaction between TFII-I and ERK is also regulated by other pathways. The Ras and Rho pathways regulate the activity of TFII-I on the c-fos promoter. However, it`s only the Ras pathway that specifically regulates the activity of TFII-I through its modulation of the MAP kinase interaction (interaction between TFII-I and ERK). TFII-I is therefore functionally dependent on the Ras/ERK pathway to activate the c-fos promoter (Kim et al., 2000) TFII-I in the Cytoplasm Receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCR) initiate intracellular calcium signalling by activating phospholipase C (PLC) and ultimately leading to an increase in Ca 2+ influx (reviewed in Caraveo et al., 2006). PLC- γ activation increases Ca 2+ influx through both the triggering of calcium release from intracellular stores, as well increase in cell surface expression of transient receptor potential C3 (TRPC3) Ca 2+ channels (Caraveo et al., 2006). When specific cell surface receptors are activated by agonists, PLC-γ binds to the PH-like `half domain` of TRPC3 Ca 2+ channel subunits via its C-terminal half of the PH domain. The binding of PLC-γ to TRPC3 causes insertion of the subunits into the plasma membrane and ultimately increases Ca 2+ influx (Rossum et al., 2005). PLC-γ contains multiple domains, including the PH

46 29 domains and Src homology 2 (SH2) domain. The SH2 domain of PLC-γ is bound by TFII-I upon phosphorylation of TFII-I by Btk. The C-terminal of the PH domain of PLC-γ important for binding to TRPC3 channels is also important for interaction with TFII-I. More specifically, investigators have suggested that TFII-I is a negative regulator of the agonist-controlled calcium entry through TRPC3 function due to the increase in Ca 2+ influx observed after reduction of TFII-I levels. Similarly, TFII-I overexpression reduced the Ca 2+ influx, further suggesting the role of TFII-I as a negative regulator of the agonist-controlled calcium entry (Rossum et al., 2005). The next question was to investigate whether this change in Ca 2+ influx due to changes in TFII-I expression was due to changes in the amount of TRPC3 channels at the cell surface and/or interactions with PLC- γ since PLC- γ regulates agonist controlled Ca 2+ influx through TRPC3 (Caravero et al., 2006). Caraveo et al. found that reduced expression of TFII-I did increase the amount of TRPC3 channels at the cell surface whereas overexpression of TFII-I reduced the number of TRPC3 channels. This control of TFII-I on the number of TRPC3 channels was dependent however upon the binding of TFII-I to PLC- γ at both PH domain and/or SH2 domain. When TFII-I was bound to PLC- γ, a decrease in TRPC3 channels at the cell surface was observed. Furthermore, phosphorylation of TFII-I by Btk or other kinases could be responsible for activating TFII-I to interact with PLC- γ. These findings supported previous suggestions of TFII-I as a negative regulator of agonist-controlled calcium entry by competing with TRPC3 for binding to PLC- γ (Caraveo et al., 2006). Aside from TFII-I transcriptional function in the nucleus, TFII-I is also present in the dendrites of Purkinje cells in the cerebellum suggesting abundance in the cytosol (Danoff et al., 2004). Proteins, such as TFII-I, may coordinate the overall ability of a cell to respond to stimuli as well as activate gene expression through their functions in the nucleus and cytoplasm (Park and Dolmetsch, 2006) TFII-I Expression in the Brain In the adult mouse brain, Gtf2i mrna is expressed in specific brain regions (Allen Brain Atlas; High expression was observed in cerebellar purkinje cells, pyramidal cells of the ventral hippocampus, as well as in the ventral hypothalamus. The high expression of TFII-I in the cerebellum is suggested to be related to cerebellum anatomical abnormalities in individuals with WBS (Danoff et al., 2004). The high expression of TFII-I in the

47 30 hippocampus and hypothalamus is also noteworthy. The hippocampus is highly implicated in emotional processing and neuropsychological accounts of the role of the hippocampus and lesion studies in rats have emphasized its importance for anxiety (reviewed in Barkus et al., 2009). The ventral hypothalamus is part of the hypothalamo-pituitary-adrenal axis (HPA). HPA is involved in the neurobiology of anxiety disorders through the release of stress hormones by the adrenal cortex after stimulation such as by stress. Furthermore, HPA stress responses are intertwined with anxiety responses through findings of activations of some of the same brain regions (reviewed in Shin et al., 2010). 1.7 Anxiety Introduction to Anxiety Disorders According to the World Health Organization, anxiety disorders are among the ten most important public health concerns (Thase et al., 2006). Anxiety is a common and typically adaptive behavior experienced by the general population; however, excessive anxiety is very debilitating. Anxiety disorders are marked by excessive fear, often in response to specific objects or situations in the absence of a real threat (Shin et al., 2010). Such disorders are associated with significant personal distress, reduced quality of life, inefficient workplace performance and are recognized as risk factors for many diseases, including neuropsychiatric and cardiovascular diseases (Greenberg et al., 1999, and Cryan et al., 2005 and Garner et al., 2009). Furthermore, anxiety disorders show comorbidity with other psychiatric illnesses, particularly, depression (Ravindran et al., 2010). Anxiety disorders are the most common form of psychopathology in childhood, with separation anxiety disorder (SAD) among the most frequent anxiety diagnoses, and with a prevalence of ~2.5% in children (Beesdo et al., 2009 and Shaffer et al., 1996). Despite evidence of strong heritability in anxiety disorders and several efforts to identify responsible genes, no consistently replicated molecular genetic associations have yet been demonstrated in humans (Smoller et al., 2008). Rare genetic disorders that involve overlapping symptoms with common disorders help provide more easily identifiable genetic causes that can be used as a starting point for identifying biological pathways amenable to treatment. In particular, the study of WBS and Dup7q11.23

48 31 here, may help establish links with genes and pathways that play a role in anxiety disorders due to anxiety phenotypes observed in these two disorders Anxiety Neurocircuitry Anxiety disorders are marked by excessive fear elicited in response to specific objects or situations, in anticipation of a threatening experience or in the absence of true danger. Therefore, fear circuitry and brain responses to emotional stimuli have been the focus of studies investigating the neurocircuitry underlying anxiety disorders (Shin et al., 2010). The amygdala connections with the prefrontal cortex are believed to be involved in enabling the representation of emotional salience of a stimulus as well as the top-down control mechanisms to influence interpretive processes (Bishop, 2007). The amygdala is thought of as an emotion processing brain region, important for threat detection. It receives higher cortical input from the prefrontal cortex which regulates its activity (Garner et al., 2009, Mohler, 2012)). Hyperactivity of the amygdala together with hypoactivity of the medial prefrontal cortex (mpfc) has been implicated in the majority of, if not all, anxiety disorders (Garner et al., 2009). Specifically, disorders that involve fear and panic, such post traumatic stress disorder (PTSD), panic disorder, and phobias, are characterized by under-activity of the prefrontal cortex therefore disinhibition of the amygdala (Garner et al., 2009). On the other hand, disorders that involve worry and over thinking, such as seen in GAD and obsessive compulsive disorder (OCD), are characterized by over-activity in the prefrontal area (Berkowitz et al., 2007; and reviewed in Garner et al., 2009). The amygdala is the site for formation and storage of fear memories (Davis et al., 2000). Normally, there is low neuronal firing activity in the amygdala due to the strong inhibition from other areas such as the mpfc. However, when a signal indicates threat, the activity of the amygdala increases and downstream targets are activated to cause fear and anxiety (reviewed in Mohler, 2012). The activity of the amygdala is regulated by a balance between glutamate induced excitation and GABA-mediated inhibition (reviewed in Shekhar et al., 2005). This balance between excitatory and inhibitory neurotransmission is particularly important for the regulation of anxiety responses. Within the amygdala, the basolateral (BLA) and the central medial (CeM) nuclei have long been known to regulate affective responses. It has been proposed that BLA is a major receiver and integrator of sensory input. Disruption of BLA has been shown

49 32 to inhibit both the acquisition of conditioned fear/negative affective responses as well as retrieval of information necessary for the expression of emotion (Campeau and Davis, 1995). The CeM on the other hand, is proposed to be the primary output site of the amygdala (reviewed in Shekhar et al., 2005). Furthermore, serotonergic neurons, originating at the dorsal raphe nuclei, project to and activate GABA interneurons in the basolateral amygdala to provide modulatory input for amygdala activation (Stutzmann and LeDoux, 1999) (Figure 1.11). Figure A schematic representation of an extended emotional network hypothesized to be involved in anxiety responses. Projection neurons are presented as circles within the specific brain regions. Green arrows indicate excitatory projections (mediated by glutamatergic neurons) whereas red arrows indicate inhibitory projections (mediated by GABA neurons. It has been hypothesized that BLA is a major receiver and integrator of sensory input whereas CeM is a major output source. mpfc provides top-down regulation to the amygdala either directly at the BLA or indirectly through GABA neurons found in IC. Serotonin neurons provide modulatory input to neurons in the BLA. Abbreviations: mpfc, medial prefrontal cortex; BLA, basolateral amygdala; CeM, central medial nucleus of amygdala; IC, intercalated cells of amygdala; RN, raphe nuclei.

50 Pharmacology and Neurotransmitter Hypothesis of Anxiety Current Pharmacological Treatment of Anxiety Pharmacological treatments of anxiety disorders have included, amongst others, drugs that target the serotonergic, GABAergic, and glutamatergic systems, due to a hypothesized low level of serotonin and/or imbalance between excitatory and inhibitory neurotransmission in the pathological state (Connolly et al., 2011). Current treatments for anxiety disorders have shown modest efficacy, with response rates about 50-60% for most anxiety disorders and many patients requiring trials of multiple medications before an effective treatment is identified Neurotransmitter Hypothesis of Anxiety Although much is still to be learned about the neurocircuitry of anxiety disorders, it has been proposed that serotonin, GABA, and glutamate neurotransmitters contribute. One hypothesis has suggested low serotonin levels in the brain as the cause of anxiety (reviewed in Ravindran et al., 2010). These conclusions have mainly arrived from observations that drugs that increase serotonin levels in the brain, such as selective serotonin reuptake inhibitors (SSRIs), provide therapeutic relief to those affected by anxiety disorders (Thase et al., 2006). Another hypothesis has suggested an imbalance between GABAergic mediated inhibition in the brain and glutamatergic mediated excitation in the brain (Wieronska et al., 2011). More specifically, hyperexcitability of the presumed circuits, whether due to reduced GABAergic inhibition and/or increased glutamatergic excitation, may be responsible for the anxious state (Fish et al., 2000, and Takahashi et al., 2009) Serotonin Hypothesis of Anxiety One proposed theory of anxiety implicates low serotonin levels in the brain due, to the positive therapeutic results in reducing anxiety of pharmacological compounds that increase serotonin levels (Schafer, 1999, Ressler and Nemeroff, 2000, Fernandez and Gaspar, 2012). Selective serotonin reuptake inhibitors (SSRIs) increase extracellular serotonin (5-HT; 5- hydroxytryptamine) in the brain by blocking the serotonin transporter (SERT), thereby inhibiting reuptake of serotonin from the extrcellular space at the synapse (Figure 1.12) (Garner et al., 2010). The broad efficacy of SSRIs is observed in the acute and long-term treatment of patients with GAD (Baldwin and Polkinghorn, 2005), PTSD (reviewed in Garner et al., 2010), OCD

51 34 (Fineberg et al., 2005) and social anxiety disorder (Stein et al., 2003). There are currently six SSRIs available for clinical use: escitalopram, fluoxetine, sertraline, paroxetine, fluvoxamine, and citalopram (Ravindran et al., 2010). Each SSRI has different indications for specific anxiety disorders, but as a class of drugs, SSRIs are considered the first line of treatment for all anxiety disorders due to their overall levels of efficacy, safety, tolerability, and more favorable sideeffect profile compared to other treatment options (Hidalgo et al., 2000, and Ravindran et al., 2010). Furthermore, SSRIs have the advantage of treating comorbid depression, and lack the potential for abuse or dependence (Hoffman et al., 2008). Despite the 50% success rate of SSRIs in treating anxiety disorders (Katzman et al.,2009), such treatment has also been associated with a wide range of side effects including, but not limited to, insomnia, drowsiness, weight changes, fatigue, headache, dry mouth and sexual dysfunction (Dording et al., 2002 and Mohler, 2012). Furthermore, SSRIs have efficacy limitations that include a lack of response in some patients, a delay of at least four weeks before symptoms relief, and risk of relapse (Katzman et al., 2009). Therefore, the need for new drugs with improved response rates, shorter latency of effect, and fewer side effects is pressing. Figure Schematic representation of a serotonergic synaptic terminal. Sert, the serotonin (5-HT) transporter, reuptakes serotonin from the synapse and ultimately reduces

52 35 serotonin levels in the brain. Blockers of this transporter, such as the selective serotonin reuptake inhibitor (SSRI) will increase serotonin levels in the synapse and prove therapeutic in states of anxiety (Adapted from Cedarlane website) GABA and Glutamate Hypothesis of Anxiety Another proposed mechanism of anxiety involves an imbalance between GABAergic mediated inhibition and glutamatergic mediated excitation (Fish et al., 2000, and Takahashi et al., 2009). Modulation of GABAergic neurotransmission is a potential therapeutic approach for anxiety (Mohler, 2012). Benzodiazepines (BZs) allosterically modulate gamma-aminobutyric acid A (GABA-A) receptors, resulting in an increase of GABAergic neurotransmission (Figure 1.13). BZs were first introduced in the 1960s and are still widely used for the treatment of various anxiety disorders (Shader et al., 1993) due to good tolerability and the rapid onset of effects (Swanson et al., 2005; and Ravindran et al., 2010). Reduced BZ receptor-binding in certain forebrain areas has also been observed in individuals with GAD, panic disorder and PTSD further implicating a reduction of GABAergic neurotransmission in these disorders (Bremner et al., 2000). Although BZs are potent anxiolytics, they have been associated with many side effects, such as sedation, memory problems, tolerance, psychomotor incoordination, and discontinuation symptoms (reviewed in Garner et al., 2009; and Ravindran et al., 2010). Moreover, anxiety disorders are often co-morbid with other psychiatric illnesses, especially depressive disorders. Since BZs do not have antidepressant effects similar to SSRIs, the use of SSRIs is favored (reviewed in Ravindran et al., 2010). Overall, BZ use is often limited to short term treatment of anxiety disorders, given their rapid onset of action and ability to be used on an as-needed basis (Hoffman et al., 2008) and SSRIs are preferred when longer term treatment is required.

53 36 Figure Schematic representation of a GABA synaptic terminal. GABA is the major inhibitory neurotransmitter in the brain. Stimulation of the post-synaptic receptors will inhibit the neuron. Post-synaptic GABA-A receptors are the major targets of anxiolytic drugs such as benzodiazepines (adapted from Clap et al., 2008). Although drugs that target the serotonergic and GABAergic systems are some of the most widely prescribed anxiety medication, glutamatergic targets are becoming more recognized and better understood. For instance, a genetic association has been reported between a variant of the glutamate NMDA receptor subtype 2B gene (GRIN2B) and OCD diagnosis (Arnold et al. (2004). Glutamate levels in cerebral spinal fluid are also significantly higher in OCD, which adds to the growing evidence in support of a potential role for glutamate in anxiety disorders (Chakrabarty, 2005). Glutamatergic drugs currently under investigation as anxiolytics target either the ionotropic or metabotropic glutamate receptors to reduce glutamate neurotransmission. Ionotropic receptors, such as the excitatory NMDA receptor, and metabotropic group II receptors, such as the inhibitory mglu2/3 receptors, have been major targets of these experimental anxiolytic drugs (Figure 1.14) (Swanson et al., 2005). For instance, ketamine is an NMDA receptor antagonist that results in anxiolytic activity in both human patients (Krystal et al., 1994) and animal models (Li et al., 2011). Additionally, an mglu2/3 receptor antagonist, LY354740, also reduced anxiety levels in patients (Grillon et al., 2003, and Levine et al., 2001).

54 37 Figure Schematic representation of a glutamate synaptic terminal. Glutamate is the major excitatory neurotransmitter in the brain. Two types of receptors are present in this synapse: ionotropic (NMDA and AMPA) and metabotropic (mglur) receptors. mglur can be found in both the presynaptic and post-synaptic neuron whereas ionotropic receptors are located on the postsynaptic neuron exclusively. Both ionotropic and metabotropic glutamate receptors have been the target of anxiolytics (adapted from Clap et al., 2008) Prevalence of Anxiety in WBS and Dup7q11.23 Anxiety phenotypes occur in WBS in both children and adults (Udwin et al., 1998, Davis et al., 1998, Dykens, 2003, Einfel et al., 2001, Leyfer et al., 2009, Martens et al., 2012). More children with WBS experience excessive anxiety compared to controls matched for age, sex, verbal intelligence, and social class (Udwin et al., 1991). A cohort of 23 WBS children of 8-10 years old were described as more tense when their personality features were compared to those of IQmatched children with developmental disabilities (Klein-Tasman and Mervis, 2003). A larger cohort of 119 individuals with WBS, ranging from 4 to 16 years old, was studied by Leyfer et al. (2006) using the DSM-IV criteria for diagnosis of anxiety disorders. The study reported a diagnosis rate of 54% for specific phobia, 12% for generalized anxiety disorder (GAD), and 7% for separation anxiety. In 2009, Leyfer conducted a subsequent study with another cohort of 132

55 38 WBS individuals, aged 4-16 years old, and compared the prevalence of anxiety disorders in WBS individuals to those previously reported by Shaffer et al. (2000) in the general population and to developmentally disabled children previously reported by Dekker and Koot (2003). The authors reported much higher rates for WBS individuals compare to developmentally delayed children for specific phobias (56 %), GAD (8%), and separation anxiety (6%) (Leyfer et al., 2009). Studies of anxiety prevalence in the WBS population have not only focused on children, but also on adults. In a study of 51 adults with WBS ranging from 5 to 49 years old, 16% of them met DSM-based diagnostic criteria for an anxiety disorder (reviewed in Woodruff-Borden et al., 2010). Additionally, 19 of 20 WBS participants over the age of 30 had clinically significant problems with anxiety (Cherniske et al., 2004). A high prevalence of anxiety-related symptoms and anxiety disorders themselves exist among all aged individuals with WBS and persists over time relative to the general population (Woodruff-Borden et al., 2010). Although non-social anxiety is common in individuals with WBS, separation anxiety is present only in 4% to 7% of these individuals (Leyfer et al., 2006, Leyfer et al., 2009, Woodruff-Borden et al., 2010, Mervis et al., 2012). Individuals with Dup7q11.23 are also commonly diagnosed with one or more anxiety disorders, such as social anxiety and separation anxiety (Berg et al., 2007, Depienne et al., 2007, Torniero et al., 2008 and Van der Aa et al., 2009, Velleman and Mervis, 2012, and Mervis et al, 2012). 75% of children with Dup7q11.23 syndrome meet the DSM-IV criteria for at least one anxiety disorder; with over 50% having phobias and/or social anxiety and more than 25% suffering with separation anxiety (Velleman and Mervis, 2012). Attention deficit hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD) are also common in individuals with Dup7q11.23 (Velleman and Mervis, 2012). A 30% incidence rate of separation anxiety disorder was reported in another cohort of 27 individuals with Dup7q11.23 syndrome (Mervis et al., 2012). Taken together, non-social anxiety is highly prevalent, chronic and not limited to one type of anxiety disorder over time in individuals with WBS (Woodruff-Borden et al., 2010). Anxiety is also highly prevalent in individuals with Dup7q11.23 and disruptive to their social skills

56 39 (Velleman and Mervis, 2012). Despite a high rate of anxiety disorder diagnosis in these two rare neurodevelopmental disorders, a difference is clear in the specific types of anxiety these two populations are affected by, particularly in terms of separation anxiety. Whereas separation anxiety is only present in 4% to 7% of individuals with WBS, a much higher rate of 25% to 30% is observed in individuals with Dup7q11.23 (Leyfer et al., 2006, Leyfer et al., 2009, Woodruff- Borden et al., 2010, Velleman and Mervis., 2012, and Mervis et al., 2012) (Table 1.4). Despite these findings, treatment of anxiety in these individuals remains largely ignored. Therefore, studies investigating methods of anxiety prevention and intervention in WBS and Dup7q11.23 syndrome are necessary. Table 1.4. Prevalence of DSM-IV disorders in individuals with WBS and Dup7q11.23 compare to the general populations and individuals with developmental delays. (*p<.05, **p.01, ***p.001. Asterisks to the left of the percentages indicate significant differences between the WBS and Dup7 groups; asterisks to the right indicate significant differences between the WS or Dup7 groups and the Population (left of the /) or DD samples (right of the /). Note: Age range for WS and Dup7 samples: years. a From Shaffer et al., b From Dekker and Koot, 2003, c From Kessler et al., 2005, adjusted for the age distribution of the Dup7 sample. d Prevalence not reported. NA: Not assessed by ADIS-IV.

57 Current Treatment of Anxiety in WBS and Dup7q11.23 Although anxiety medications are prescribed for both children and adults with WBS (Stinton et al., 2010, Thornton-Wells et al., 2011, Woodruff-Borden et al., 2010), studies of their efficacy have been limited. The most common anti-anxiety medications prescribed in WBS individuals are SSRIs, used by 24% of individuals (Martens et al., 2012). Twelve percent (12%) of WBS individuals were prescribed non-ssri based anxiolytics and/or antidepressants (Martens et al., 2012). Some of the most commonly prescribed anxiolytics were sertraline, citalopram, fluoxetine, and paroxetine from the SSRI class of drugs. Buspirone, a 5-HT1A receptor partial agonist, was also used. Lastly, benzodiazepines, such as lorazepam and clonazepam, were also commonly prescribed in WBS individuals for treatment of anxiety disorders (Martens et al., 2012). The efficacy and side effects of these anti-anxiety medications were also analyzed. SSRIs were reported by 81% of the participants to be either helpful or somewhat helpful with 34% of these individuals reporting some type of side effect when taking an SSRI. Of those taking another form of antidepressant or non-ssri based anxiolytic medication, 64% reported it to be either helpful or somewhat helpful. Thirty percent (30% ) of these individuals reported a side effect when taking the medication. However, reported incidence of anxiety disorders in individuals with WBS has often been higher than 50% (Woodruff-Borden et al., 2010). Therefore, when considering the number of individuals with WBS taking anxiety medications, as well as the wide range of side effects associated with current medications, the need for better understanding of the anxiety symptoms and targeted therapeutics becomes clear Animal Models of Anxiety Animal models allow the investigation of brain-behavior correlations in both normal and pathological states. Genetically modified animal models have helped identify the role of specific neurotransmitters and receptors in anxiety responses, and changes in brain neurobiology that underlie and confer risk for anxiety. Pharmacological animal models of anxiety have helped to validate pharmacological interventions. These models have revealed the anxiolytic properties of neurotransmitter and neuropeptide receptor manipulations, some of which have been validated in clinical trials. For example, anxiolytic properties of receptor agonists including 5-HT, GABA-A, oxytocin, and adrenergic receptors, as well as receptor antagonists including glutamate and vasopressin, were shown through the use of animal models (reviewed in Garner et al., 2009).

58 41 Animal models of anxiety typically employ ethologically based behavioural tasks developed from the natural behavioural patterns of the animal (Rodgers et al., 1997). Some of these behavioural tasks include approach-avoidance, as tested in open-field and elevated-plus-maze paradigms (Cryan and Holmes, 2005), social interactions (File and Seth., 2003), predator stress (Blanchard et al., 1971), and ultrasonic vocalizations induced by maternal-separation (Sanchez, 2003). The pathogenesis of anxiety needs to be better understood in order to develop new or more effective treatment options. Developing animal paradigms, however, that more accurately model specific human anxiety disorders remains a challenge. This is especially difficult for disorders in which cognitive components of human anxiety, such as anticipatory anxiety, cannot be assessed in animal models (Garner et al., 2009). Also, some studies of anxiety in animal models have focused on the fear conditioning paradigm due to the component of excessive fear in anxiety. However, parallels between these animal studies and anxiety disorders in humans have not always been clear. In human anxiety disorders, a clear stimulus as seen in animal models is often absent (Shin et al., 2010). Nonetheless, animal models of anxiety have been critical in allowing a better understanding of the neurocircuitry of anxiety and validating pharmacological interventions Maternal Separation-Induced USVs The neonatal house mouse (Mus musculus) emits high-frequency acoustic emissions, known as ultrasonic vocalizations (USVs) that are beyond the upper limit of human hearing (Sales, 1979). Mouse pups emit USVs in the range of khz in response to maternal separation, as well as to other various stressful physical and social stimuli such as male odors, cooling, and rough handling (Lions, 1982). Pup USVs emitted following maternal separation follow a clear ontogenic profile, peaking around the eighth day after birth and decreasing thereafter (Branchi et al., 2001). These USVs increase mother-infant social contact and prompt the retrieval of the pup by the mother. The dam displays approach behaviours, retrieval, and contact towards the pups (Nelson, 1998). Maternal separation induces emission of USVs by pups whereas maternal cues suppress pup USVs (Oswalt, 1975). Panksepp et al. (1982) proposed that maternal stimuli induce endogenous opioid release in pups, which comforts the pups (Nelson, 1998). Maternal separation -induced USVs are strongly associated with separation anxiety and have been used as one of the

59 42 ethologically validated measures for preclinical characterization of anxiolytic drugs (Brunelli et al. 1994; Gardner 1985; Miczek et al. 1995, 2008; Noirot 1972; Hodgson, 2008). The unconditioned nature of vocalization emissions following maternal separation and the generality of these vocalizations to several rodent species distinguish this test from many other preclinical measures of anxiety (Sanchez, 2003) USVs, Serotonin, and Anxiety After maternal separation, SSRIs have been shown to attenuate production of USVs in mouse pups (Fish et al., 2004). Escitalopram is a widely used SSRI in animal models of anxiety, where it reduces maternal separation-induced USVs in seven-day old mouse pups. In clinical trials, escitalopram was reported to have fewer adverse effects than other SSRIs (Fish et al., Ravindran et al., 2010). The maternal separation-induced USVs test has been included in behavioural phenotyping of mouse models with targeted mutations of the serotonin receptor genes. The 5- HT1A serotonin receptor has received particular attention as a target for the treatment of anxiety. 5ht1a KO mice exhibit increased anxiety whereas agonists of the 5-HT1A receptor have anxiolytic effects in humans and animal models (reviewed in Kusserow et al., 2004). Agonists of the 5-HT1A receptor have been also tested for their anxiolytic properties. 8-OH-DPAT, a 5- HT1A agonist, reduced maternal separation-induced USVs in a dose-dependent manner in sevenday old mouse pups (Fish et al., 2000). Similarly, mice that overexpress 5-HT1A receptors show a reduction in anxiety-like behaviors (Kusserow et al., 2004) USVs, GABA, and Anxiety Modulators of the GABA-A receptors that increase GABA mediated neurotransmission, such as BZs and GABA-A positive allosteric modulators, have been shown to have strong anxiolytic effects in pre-clinical studies of anxiety disorders. Distress-like calls are inhibited in several animal species following administration of these drugs. The effectiveness of these drugs as anxiolytics is especially evident in decreasing isolation-induced neonatal mouse USVs ( Bento and Nastiti, 1988, Nastiti et al., 1991 and Cirulli et al., 1994). The classic BZ chlordiazepoxide has been found to reduce USVs in mouse pups in a dose-dependent manner (Takahashi et al., 2009). In addition to the classic BZs, positive allosteric modulators of GABA-A receptors, such

60 43 as allopregnanolone, were shown to dose-dependently reduce maternal separation-induced USVs in seven-day old mouse pups (Fish et al., 2000) USVs, Glutamate, and Anxiety Antagonists of glutamate receptors have emerged as potential anxiolytic compounds due to the enhanced glutamatergic excitation noted in anxiety. These glutamate receptors have included metabotropic II glutamate receptors (mglu) and post-synaptic NMDA receptors. NMDA receptor antagonists have been shown to reduce separation-induced USVs in rat pups (Winslow and Insel, 1991, and Kehne et al., 1991). MK-801, a non-competitive antagonist of NMDA receptor, was reported to dose-dependently reduce USVs in mouse pups following maternal separation (Takahashi et al., 2009). mglu2/3 receptor agonists limit the release of glutamate and ultimately demonstrates anxiolytic properties (reviewed in Swanson et al., 2005). LY379268, a mglu2/3 receptor agonist, dose-dependently reduced maternal separation-induced USVs in postnatal day (PND) 7 mice further implicating glutamatergic drugs as effective anxiety treatments Role of Maternal Care on Mouse Pups Behavior Animal studies have shown a significant impact of maternal care and maternal environment, both pre- and post- natal, on the development of behaviors indicative of risk for psychiatric disorders (Meaney, 2001). Environmental factors have been studied extensively whereas studies on the genetic basis of maternal care have been limited (reviewed in Gleason et al., 2011). Specifically, maternal care has been shown to modulate ultrasonic calling in rodents. Studies of acute and short-term effects of maternal care on pup vocalizations have shown that the mere presence of the dam acutely inhibits ultrasonic calling (reviewed in Hofer, 1996). Furthermore, after brief interactions between the pup and the dam, ultrasonic calling by the pup is greatly intensified in subsequent isolation periods (reviewed in Shair, 2007). Studies have also looked at the long-term effects of maternal behavior on pup vocalizations. A sustained level of maternal care is soothing and yields anxiolytic- like effects in mouse pups (D Amato and Populin, 1987). When comparing vocalizations of pups from mothers of different strains showing different extent of maternal care, pups raised by mothers from the more responsive C57BL/6 strain emitted fewer calls than those raised by the less responsive BALB/c strain when separated from the mother (D Amato et al., 2005).

61 44 Maternal care is indicated by maternal behaviors such as pup retrieval and nurturing, and by the ability of pups to thrive, and by pup mortality rates (Kuroda et al., 2008). The effects of maternal genotype on the offspring in terms of maternal care have been reported for various genes. Mouse dams with a null mutation of cyclic AMP response element binding protein (CREB)-alpha-delta exhibit impaired pup retrieval and pups with heterozygous mutation of CREB-alpha-delta fail to thrive (Jin et al., 2005). Similarly, mouse dams with a null mutation of FosB exhibit impaired pup retrieval and nurturing behavior (Kuroda et al., 2008). Dams carrying a null allele of Peg-3 exhibit reduced nurturing behavior followed by an associated reduction in offspring survival (Li et al., 1999). Wild-type pups of Peg-3 mutant dams gained weight less rapidly and reached puberty later than controls. The Peg-3 mutant dams failed to increase caloric intake during pregnancy and had reduced milk let-down, both important for nurturing of offspring. These effects were additive and led to increased pup mortality when both dam and offspring carried the Peg-3 mutation (Curley et al., 2004 and 2008). The corticotropin releasing factor I gene (CRF1) has also been implicated in maternal behavior. Dams carrying a null deletion of CRF1 exhibit reduced nursing behaviour (licking and grooming) and spend more time off the nest (Gammie et al., 2007). Similarly, mouse dams deficient in Pet-1, a gene encoding for a serotonergic transcription factor, neglected their offspring (Lerch-Haner et al., 2008). The neuropeptide arginine vasopressin (AVP) is also important for maternal care. Mouse dams overexpressing AVP, after vector-mediated up-regulation of AVP -V1a receptors, exhibit higher levels of maternal care. Dams with a reduction in AVP on the other hand, following either local blockade of AVP-V1a expression or central AVP-V1a antagonism, showed reduced maternal care towards their offsprings (Bosch and Neumann, 2008). The oxytocin receptor gene is also important for maternal care due to findings of dams with a null mutation of the oxytocin receptor gene demonstrating defects in lactation and maternal nurturing (Takayanagi et al., 2005). The D2 dopamine receptor (D2R) has also been shown to be highly important in mother-infant interactions. Specifically, D2R knockout in the dam reduced maternal care shown by delayed pup-retrieval and nest-building, as well as lack of an increase in plasma prolactin levels induced by USV-emitting pups. D2R knockout pups emitted fewer USVs

62 45 than their wild-type littermates. Furthermore, heterozygous D2R pups emitted even fewer USVs if their dam was D2R knockout than if their dam was a wild-type. This suggests an additive effect of maternal D2R genotype on offspring phenotype with a D2R knockout (Curry et al., 2013). Therefore, both offspring and maternal genotype need to be considering when assesing mouse pup behavior. The serotonin 5-HT1A receptor has been highly implicated in anxiety and has been the target of many pharmacological compounds (Fish et al., 2000, Ravindran et al., 2010). The 5-HT1A receptor knockout (5-HT1A -/- ) mouse demonstrates heightened anxiety in several anxietyrelated behavioral assays (Heisler et al., 1998, Ramboz et al., 1998). Heterozygote (5-HT1A +/- * 5-HT1A +/- ) breeding pair crosses are typically used to generate F1 offspring to be tested. However, this set up exposes offspring, both wild-type and 5-HT1A receptor-deficient mice, to a 5-HT1A receptor-deficient maternal environment (Gleason et al., 2011). In a study looking at the interaction between maternal and offspring 5-HT1A receptor genotype, reduced adult anxiety was reported for heterozygous offspring (5-HT1A +/- ) of 5-HT1A knockout dams (5-HT1A -/- ) compared to offspring of the same genotype but from wild-type dams (5-HT1A +/+ ) (Weller et al., 2003). In Swiss Webster mouse dams, partial or complete 5-HT1A receptor deficiency increased anxiety-related behavior and stress reactivity in wild-type and 5-HT1A receptor deficient offspring. Therefore, the authors concluded that a maternal genotype effect on the anxiety level in offspring was independent of offspring genotype (Gleason et al., 2010). Maternal genotype effects on offspring s phenotype have also been shown in specific neurodevelopmental disorders on phenotypes other than anxiety. Fragile X syndrome (FXS) is a neurodevelopmental disorder that causes intellectual disability and autism (Wijetunge et al., 2013). Inactivation of the fragile X mental retardation gene (FMR1) is responsible for FXS. This gene encodes a protein, fragile X mental retardation protein (FMRP), which is an RNA-binding protein that plays a multifunctional role in protein synthesis and neuronal development (Bagni and Greenough 2005, Kao et al., 2010). The Fmrp knockout mouse model of Fragile X syndrome exhibits a number of the phenotypes observed in humans,, such as locomotor hyperactivity, cognitive defects, macroorchidism, and sensory hyper-reactivity (Spencer et al., 2005, Yun et al., 2006, reviewed in Gleason et al., 2011). However, such studies have mostly ignored a potential maternal genotype effect on these behaviors. Gleason et al (2011) reported

63 46 increased constitutive locomotor activity in wild-type adult male mice offspring of Fmrp heterozygote dams (Fmrp +/- ). Furthermore, an additive effect of the Fmr1deficiency on locomotor activity was observed when both offspring and dam were heterozygotes (Fmrp +/- ) (Zupan and Tooth, 2008). Therefore, a deficit in Fmrp in the dam was sufficient to cause longterm effects on offspring behavior, at least in the hyperactivity phenotype as assessed by the locomotor activity test. This work therefore points at a potential maternal genotype effect not only in affected offspring, but also in non-affected offspring through increased susceptibility of offspring to psychiatric disease Anxiety, HPA axis, and Neuronal Activation Activation of some of the same brain regions in both anxiety and HPA stress responses, such as medial prefrontal cortex, insula, amygdala, hippocampus, and bed nucleus of the stria terminalis (BNST), suggest that the two are intertwined and can influence one another (Liberzon and Martis, 2006 and Shin et al., 2010). For example, Grillon et al. (2007) studied the role of acute stress on a subsequent anxiety phenotype in healthy controls. Prior exposure to a social stressor (such as speech or counting task) potentiated the acoustic startle response in the dark. This preexposure to a stressor also induced increases in stress hormones, as indicated by higher salivary cortisol levels, and subjective distress. Therefore, stress potentiated an anxiety related response and was paralleled by physiological changes in stress hormone levels (Grillon et al., 2007). In animal studies, a potentiation of the anxiety response has been noted immediately after both acute and chronic stressors (Zangrossi et al., 1992 and Korte et al., 2003). Furthermore, delayed effects of stress during vulnerable developmental periods have also been extensively studied in animal models. Early maternal separation in rodents caused long-term alterations in HPA stress responses and key neurotransmitter systems (Plotsky et al., 1993). Pohl et al. (2007) exposed rats repeatedly to stress during their childhood-adolescent period and found altered anxiety-like behaviors in adulthood. Stress is thus an important player in the development and maintenance of anxiety disorders, with a critical role in the potentiation of the anxiety-like responses. Therefore, when examining the long-term effects that stress has on anxiety phenotypes, the character of the stress exposure needs to be clearly identified. The stress exposure may be mild or severe, short or prolonged, predicted or non-predicted, and the sex of the individual might play an important role.

64 47 Behavioural manifestations of distress, such as the emission of USVs following maternal separation, are accompanied by physiological responses. Such physiological responses may include an increase in HPA axis activity and subsequent release of adrenocorticotropin (ACTH) from the pituitary, thereby leading to elevations of plasma corticosterone levels (Cirulli et al., 1994). Thus, the effectiveness of pharmacological agents on anxiety has been measured not only through changes in anxiety-related behavior such as USVs, but also through changes in plasma corticosterone levels. In PND 9 mice, an increase in plasma corticosterone levels was observed following maternal separation (Howard et al., 2012). Anxiolytic compounds, such as benzodiazepines, attenuate the stress-induced elevations of corticosterone (reviewed in Mikkelsen et al., 2005). The expression of immediate early genes (IEGs) has been particularly useful in determining neuronal activation. Among the many IEGs, c-fos has become the most widely used IEG for mapping neuronal activation induced by a stimulus (Martinez et al., 2002). Acute stressful stimuli, whether internal or external, will induce a pattern of activation in the brain that can be detected by c-fos expression. Basal expression of c-fos is low, if present at all, in most brain areas (Martinez et al., 2002). Stressful stimuli such as maternal separation and/or injection of a pharmacological compound cause a rapid and transient increase in c-fos expression in the brain. There is growing evidence suggesting that pathophysiology of anxiety disorders is associated with disruption of multiple brain regions and/or neurotransmitter systems accompanied by overactivation of the stress response circuits (Troakes et al., 2009). Animal studies of different anxiolytics have measured c-fos mrna expression to investigate the pattern of neuronal activation. Results have been mixed with some anxiolytics increasing and others decreasing overall neuronal activation. Immunohistochemistry studies staining for c-fos have been more informative in reporting the pattern of neuronal activation by looking at specific brain regions (Troakes et al., 2009). For example, LY354740, a selective agonist at presynaptic mglu2/3 receptors, showed anxiolytic properties in the elevated plus maze test of anxiety in mice as well as a suppression of stress-induced c-fos expression in the hippocampus and an increase in c-fos expression in stress-sensitive brain regions such as the amygdala (Linden et al., 2005). The paradoxical stress-induced anatomical changes found in the hippocampus and amygdala, whereby a suppression of activity in the hippocampus but an increase of activity in the amydala

65 48 is observed, derive from the different involvement of these two neuroanatomical areas in the neurocircuitry of stress. Behavioural studies utilizing stress-induced paradigms and changes in early gene expression to measure neuronal activity have emphasized the role of stress in impairing hippocampal-dependent learning and facilitating amygdala-dependent aversive learning (reviewed in Cortese et al., 2005). Furthermore, the hippocampus inhibits the HPA, which is important for release of stress-related hormones such as corticosterone, whereas the amygdala activates the HPA (Herman et al., 1997 and Cortese et al., 2005). Therefore, in a stress-inducing situation, an increase in amygdala activity accompanied by a decrease in hippocampal activity is expected Anxiety and Rare Disorders Neurodevelopmental disorders, such as anxiety disorders, have major impacts on affected individuals, their families, and society at large. Genetic factors play an important role in these disorders however, most disorders often involve multiple loci, and to this date only a few chromosomal regions have been linked to specific disorders using targeted and genome-wide association studies (GWAS) (Greenberg et al., 1999, Smoller et al., 2008, Franke et al., 2009, Newbury et al., 2010). An understanding of the molecular mechanisms of such common disorders would assist in the development of targeted therapeutic interventions. Recently, genetic research has focused particularly on finding gene candidates that may be responsible for the many childhood neuropsychiatric and behavioral disorders that exist. Emerging data from these studies suggest that common genetic variants are unlikely to explain the majority of risk for developing these disorders as well as the phenotypic variance of these disorders. The search for genetic variants that contribute to these common disorders is often hampered by the need for large sample sizes to account for the complex pattern of inheritance of these common disorders, and their genetic heterogeneity. Rare disorders that share overlapping phenotypes with common disorders offer a window into better understanding common diseases, as they provide more easily identifiable genetic causes, do not require a large sample size, and show a simpler pattern of inheritance. Identification of the underlying genetic causes of these rare disorders can then be used as a starting point for understanding the neuromolecular underpinnings of phenotypes that are shared between common disorders.

66 49 The complex neurodevelopmental disorders that arise from the deletions or duplications in the 7q11.23 chromosome region may provide better understanding of human cognition, speech, language, and behavior. WBS and Dup7q11.23 are both rare neurodevelopmental disorders with unique phenotypic spectra that include disorders common in the general population, such as anxiety. Furthermore, evidence for the dosage sensitivity of the 7q11.23 genes potentially involved in anxiety comes from contrasting the phenotypes of individuals with WBS to those with Dup7q Individuals with WBS are described as hypersocial and are diagnosed nonsocial anxiety whereas those with Dup7q11.23 are described as shy and are diagnosed with separation anxiety disorder, social phobias and ADHD (Mervis and Velleman, 2011 and Velleman and Mervis, 2012). Due to the small set number of genes responsible for these two rare disorders, WBS and Dup7q11.23 provide a starting point for the identification of genetic variants and molecular pathways underpinning anxiety Conclusion The creation of three new Gtf2i single gene mouse mutants allow tests of Gtf2i gene-dosage effects. In particular, we testedgtf2i gene-dosage effects on anxiety phenotypes and stress hormone corticosterone levels. Finally, we tested different types of anxiolytics to evaluate linkage between Gtf2i gene-dosage and pharmacology of anxiety.

67 50 Chapter 2 Behavioral Analysis of Mouse Models with Altered Gtf2i Copy Number 2.1.Effects of Injection Stress and Gtf2i Pup Genotype on Maternal Separation-Induced USVs Introduction Research Aims To help dissect genotype-phenotype correlations in WBS and Dup7q11.23, single-gene Gtf2i mouse mutants with one to four Gtf2i gene copies were used to investigate the role that GTF2I plays in the anxiety phenotype of individuals with WBS and Dup7q Using the maternal separation-induced USV paradigm, separation anxiety was characterized in post-natal day 8 (PND8) mice with a single copy of Gtf2i (Gtf2i +/- ), and PND8 mice with one and two extra copies of Gtf2i (Gtf2i +/dup and Gtf2i dup/dup, respectively) Maternal Separation-Induced USVs Rodents emit vocalizations, known as ultrasonic vocalizations (USVs), that are beyond the upper limit of human audition (Sales, 1979). Mouse pups will vocalize when separated from their dam. Separation anxiety can be assessed through such USVs emitted after maternal separation (Scattoni et al., 2009). Mouse pups USVs are not only produced in response to maternal separation but to other stressful stimuli as well (Scattoni et al., 2009). These maternal separation-induced USVs are an ethologically validated measure for pre-clinical characterization of anxiolytic drugs (reviewed in Takahashi et al., 2009). USVs emitted following maternal separation are known to peak on day 8 after birth, and as such, PND8 mouse pups are used to assess the anxiolytic properties of drugs (Branchi et al., 2001, Fish et al., 2000, Fish et al., 2003, Takahashi et al., 2009).

68 51 A screening period of 30 sec is usually included prior to the maternal separation test in mouse pups. Pups that emit 6 or less USVs during this screening period are excluded from further tests. This corresponds to approximately 25% of the data being excluded (Fish et al., 2000, Fish et al., 2003, and Takahashi et al., 2009). In section , results on whether screening is applicable to our study will be included Duplication of Gtf2i Results in Separation Anxiety in Mice and Humans We have previously shown that an increase in Gtf2i genomic copy number is linked to separation anxiety in both mice and humans (Mervis et al., 2012). Children with altered GTF2I dosage (either with WBS or Dup7q11.23) as well as mice with different Gtf2i gene-dosages were tested for separation anxiety. Postnatal day 8 (PND8) mouse pups with either heterozygous or homozygous duplication of Gtf2i showed increased maternal separation-induced ultrasonic vocalizations, in contrast to PND8 pups with a heterozygous deletion of Gtf2i, which exhibited a tendency for reduced maternal separation-induced USVs (Figure 2.1). This pattern suggests that Gtf2i has a dose-dependent effect on maternal separation-induced anxiety in mice. To determine if a similar effect is present in humans, we measured separation anxiety in children with Dup7q11.23 (3 copies of GTF2I) and children with WBS (1 copy of GTF2I). Twenty-seven children [ages 4-13 years] with Dup7q11.23 and 214 age-matched children with WBS were assessed using the Anxiety Disorders Interview Schedule for DSM-IV-Parent Interview (ADIS- P). In addition, parental responses for 14 children with Dup7q11.23 aged 2 5 years and 189 age-matched children with WBS were compared on the separation anxiety question of the Child Behavior Checklist (CBCL) for Ages 1 1 / 2-5. Based on the ADIS-P, 29.6% of children with Dup7q11.23 were diagnosed with separation anxiety disorder, compared with only 4.2% of those with WBS (p <.0001) (Table 2.1). CBCL findings indicated that 33.3% of children with Dup7q11.23, but only 1.1% of children with WBS, had unusual difficulty separating from their parents (p <.0001) (Table 2.2). These results suggest that GTF2I plays a significant role in the contrasting separation anxiety phenotypes seen in children with Dup7q11.23 and WBS.

69 52 Figure 2.1. Test of maternal separation anxiety in mice with altered Gtf2i genomic copy number. When measuring maternal separation-induced USVs, mice with increased Gtf2i copy number (Gtf2i +/dup and Gtf2i dup/dup mice with 3 and 4 copies respectively) produced significantly more USVs than those with normal (Gtf2i +/+ mice with 2 copies) or fewer (Gtf2i +/- mice with 1 copy) Gtf2i copy number. (Gtf2i +/- : n=11, Gtf2i +/+ : n=49, Gtf2i +/dup : n=30, Gtf2i dup/dup : n=18, p<.001) (Mervis et al., 2012). Table 2.1. Incidence of Separation Anxiety Disorder (SAD) in children with WBS and Dup7q11.23 compare to the general population. Children with Dup7q11.23 had a high incidence of SAD compared to both the WBS population and controls (***p<0.001 and ****p< respectively as shown by asterisks). DSM-IV criteria for SAD based on the Anxiety Disorders Interview Schedule for DSM-IV: -Parent version was used for diagnosis of SAD. Age range: years (Mervis et al., 2012).

70 53 Table 2.2. Separation difficulties in children with WBS and Dup7q11.23 compared to the general population. Child Behavior Checklist for Ages (CBCL 1.5 5) assessed separation difficulties based on parental responses to item 37 ( Gets too upset when separated from parents ). Parents rated the item on a 3 point scale 0 (not true), 1 (somewhat or sometimes true), or 2 (very true or often true) on the basis of their child s behavior during the preceding 2 months. When difficulty with separation was defined as a score of 2, a significantly higher proportion of children with Dup7q11.23 (0.333) than the proportion of children with WBS (0.011) was observed (****p<0.0001). Similarly, when difficulty with separation was defined as a score of 1 or 2, proportion of those with Dup7q11.23 (0.611) was higher than proportion of those with WBS (0.182) (p<0.0001). Age range: years (Mervis et al., 2012) Hypothesis Studies of individuals with atypical deletions of the WBS region and mice with copy number changes in Gtf2i, have implicated the GTF2I gene in the behavioral phenotypes of WBS and Dup7q GTF2I encodes the transcription factor, TFII-I, which has multiple functions in the brain, and as such, it may regulate the expression of other genes during development (reviewed in Cheriyath and Roy, 2001). We hypothesized that Gtf2i copy number influences anxiety phenotypes in mice and humans through neurochemical and physiological pathways already known to play a role in anxiety Materials and Methods Contributions: I performed all behavioral tests, genotyping and sexing of test animals, and statistical analysis Generation of Mice with Altered Gtf2i Copy Number A mouse model with increased copy number of Gtf2i was generated, with either 1 extra copy (Gtf2i +/dup ) or two extra copies of the gene (Gtf2i dup/dup ) (Mervis 2012). Gtf2ird1 Gt(XS0608)Wtsi mice

71 54 with a genetrap insertion within intron 4 of Gtf2ird1 were generated as described previously (Proulx, 2010). Clones from a 5 /3 phage library were used to generate the Gtf2i 3 UTRloxP ES cell line by gap repair (Zheng, 1999). The targeting vector included a 9 kb fragment of Gtf2i spanning exon 25 to the 3 UTR, resulting in duplication of these exons downstream of Gtf2i after recombination between the vector and the endogenous locus. Correctly targeted ES cell clones were used to generate germline-transmitting chimeric mice after aggregation with morula-stage embryos (Nagy, 2002). The resulting chimeras were bred to CD1 females to produce Gtf2i +/3 UTRloxP Gt (XS0608) mice. To generate the intra-chromosomal duplication of Gtf2i, Gtf2ird1 Wtsi mice were crossed with Gtf2i 3 UTRloxP mice that also carried a Cre transgene under the control of the Sycp1 promoter (Sycp1-Cre) (Vidal, 1998). Trans-loxer males carrying both the Gtf2ird1 Gt(XS0608)Wts and Gtf2i 3 UTRloxP alleles as well as the Sycp1-Cre transgene were crossed with CD1 females (Figure 2.2A) and offspring were screened by PCR. Mice carrying the duplication (Gtf2i +/Dup ) were further characterized using qpcr to identify changes in copy number of Gtf2i exons 5 exon 30. ES cell clone YTA365 carrying an insertion of the gene trap vector pgt0lxf in intron 3 of the Gtf2i gene (available from the Mutant Mouse Regional Resource Centers, UC Davis) was used to generate mutant mice after injection into C57BL/6 blastocysts. The resulting chimeras were bred to CD1 females to produce Gtf2i Gt(YTA365)Byg/+ mice, referred to in this thesis as Gtf2i +/del (Figure 2.2B).

72 55 Figure 2.2. Generation of mice with decreased or increased Gtf2i dosage. A) Schematic representation of the two embryonic stem cell lines used to generate mice with different copy number of Gtf2i. XS0608 contains a loxp site inserted into intron 4 of the Gtf2ird1 gene, while the G7 3 UTR-loxP line contains a loxp site downstream of the last coding exon of Gtf2i. Recombination between the loxp sites in vivo resulted in duplication of the entire Gtf2i gene. The centromere of the mouse chromosome is represented by the circle at the left end of each diagram. B) ES cell line YTA365 carrying an insertion of a gene trap cassette in intron 3 of the Gtf2i gene used to generate Gtf2i +/del mice (Mervis et al., 2012) Animal Housing Animals were maintained on a mixed CD1/129 background. All experimental animals were housed at the Medical Sciences Building of the University of Toronto in polycarbonate cages (30 x 22 x 15 cm) under standard animal housing conditions. Animals were maintained in a lightcontrolled room on a 12:12 light-dark cycle (with lights on at 6 am) at a controlled temperature (23 ± 2 C) and humidity (approximately 50-60%.) Pups were born in litters of 4 to 19 pups and lived with both parents in their home cage. Standard rodent chow and water were available to the adult mice ad libitum except during behavioural testing. Date of birth was considered PND0 and pups were marked on their toes using a non-alcoholic marker for identification purposes

73 56 immediately prior to testing. All experimental protocols using animals were performed in accordance with the Guide to the Care and Use of Experimental Animals (Canada), and approved by the Animal Care Committee of the University of Toronto. Mice with heterozygous duplication of Gtf2i (Gtf2i +/dup) were paired to generate F1 pups of three different genotypes for USVs testing (Figure 2.3A). Mice with heterozygous deletion of Gtf2i (Gtf2i +/del) were paired with wild-type mice of opposite sex (Gtf2i +/+) to generate F1 pups with a deletion of Gtf2i (Figure 2.3B).

74 57 Figure 2.3. Schematic diagram depicting the parental crosses set up to generate F1 offspring used for testing the effects of anxiolytics on maternal separation-induced USVs. A) Breeding pairs set up to generate mice with duplication of Gtf2i. Mice with heterozygous duplication of Gtf2i (Gtf2i +/dup ) were paired. B) Breeding pairs set up to generate mice with deletion of Gtf2i. Mice with heterozygous deletion of Gtf2i (Gtf2i +/del ) were paired with wildtype mice of opposite sex (Gtf2i +/+ ). Therefore, either the dam or the sire carried the deletion in a specific breeding pair. Pups were tested on post-natal day Apparatus and Measurements The testing apparatus was located in the procedure room, separate from the animal housing colony. USVs were obtained with a D1000X ultrasound recorder (Pettersson Elektronik AB, Uppsala, Sweden) for 4 minutes at a sampling frequency of 250 khz. The microphone was suspended 13 cm above the floor from a sound attenuated chamber (40cm x 25cm x 30 cm). The apparatus was illuminated by one 40 watt red bulb in the procedure room. Spectrographs ( khz) were generated by discrete Fourier transform (256 bins) and analyzed with Avisoft

75 58 SASLab Pro Software v4.39 (Avisoft Bioacoustics, Berlin, Germany). Data were analyzed blind to genotype and drug treatment. Mean number of USVs were calculated in minute bins. The mean number of total USVs emitted by pups receiving a drug is shown as a percentage of the number of total USVs emitted by pups receiving a control saline injection. For the screening procedure, correlations between the number of USVs emitted during the 30- second screening period and the total number of USVs emitted during the 4-minute trial were calculated for PND8 pups receiving saline. The number of pups excluded once screening is taken into account was also calculated and shown as a percentage of the original number of pups tested prior to screening. Lastly, the overall drug effect on USVs was calculated before and after screening was taken into account Maternal Separation-Induced USVs Procedure Test sessions were conducted between 10 am and 6 pm. On PND8, the home cage containing the litter of pups and their parents was transported to the procedure room which was kept at a room temperature of 22 C - 25 C. Each pup was separated from the litter one at a time in random order, and placed in a shallow plastic beaker (height = 6 cm, diameter = 4 cm) in a sound attenuating chamber for recording of USV emissions. After a 5-second habituation period, a 30- second screening trial of USV emissions was recorded at sampling frequency of 250 khz. Following a 30-second screening period, each pup was weighed, toe-marked, and received a subcutaneous injection of either drug (anxiolytic) or saline (control), before being returned to their home cage with the rest of the litter. 30 to 45 minutes after the time of the injection, each pup was separated from the litter again, one at a time, in the same order as before, and placed in a new shallow plastic beaker in a sound attenuating chamber for recording of USV emissions for 4 minutes at a sampling frequency of 250 khz. At the end of the trial, each pup was sacrificed and tissue from tails was collected for genotyping and determination of sex. Note: A specific anxiolytic drug and saline were tested in the same litter counterbalancing between saline and drug condition. Anxiolytic effects on USVs however are presented in Section 3.1.

76 Statistical Analysis Results are expressed as means ± SEM and were analyzed by SPSS. The Shapiro-Wilk test of normality was performed on all of the USV data to assess the hypothesis of normal distribution. Due to violations of the assumption of normality and unequal number of mouse pups across genotype groups, nonparametric statistics were used for the analysis of maternal separationinduced USVs. A Kruskal-Wallis test assessed differences among groups in the median number of vocalizations produced over the 4-minute trial. The Mann-Whitney test was used to assess differences between two genotype groups Genotyping and Sexing of PND8 Mice DNA Extraction from Tails Genomic DNA was isolated from tail clips. Tail clip tissues were incubated in 400μl of lysis buffer (0.5% SDS, 0.1M NaCl, 50mM Tris, 2.5 μm EDTA) and 100μg/ml proteinase K at 65 C until tissue was no longer visible. Potassium acetate was then added to purify the DNA, which was then followed by chloroform. After vigorous shaking, the solution was stored at -20 C for a minimum of 20 minutes. Samples were centrifuged at 12,000 g for 5 minutes at room temperature to separate the DNA, aqueous phase, from the rest. DNA was transferred to a new tube and precipitated with 2 volumes of 100% ethanol. Samples were centrifuged again at 12,000 g for 5 minutes at room temperature. The DNA pellets were washed with 1 volume of 70% ethanol before being resuspended in 100µl of nuclease free water Genotyping of Gtf2i +/- Litters Gtf2i+/-litters were genotyped using conventional PCR. 1 µl of DNA sample was added to a PCR Master Mix (19.95 µl H2O, 1.8 µl 25Mm MgCl, 3 µl 10X buffer, 3 µl 2mM dntp, 0.5 µl Forward Primer, 0.5 µl Reverse Primer, and 0.25 µl Taq Polymerase; Thermo Scientific) for a total volume of 30 µl. Each sample was run in two primer sets; m2igti3-g forward and reverse and mgt-del-g forward and reverse (Table 2.3). The m2igti3 primer set is used as a control to detect the presence of Gtf2i intron3, present in both wild-type and Gtf2i +/del mice, and generate a band. The mgt-del primer set detects the

77 60 presence of the two targeting vectors used to generate mice with a deletion of Gtf2i and gives rise to a band during PCR due to the Cre-Lox reaction excising the gene. Therefore, wild-type mice were identified when only a single band was produced, whereas Gtf2i+/del mice were identified when two bands were generated. Reactions were incubated at 94 C for 5 min, followed by 35 cycles of 94 C for 30s, 60 C for 30sec, and 72 C for 30 s, followed by a decrease to 10 C (PCR machine). Following thermal cycling, 5 μl from each PCR product were mixed with 5 μl 2 loading dye solution before being loaded onto a 3% agarose gel (BioBasic., Inc. Canada) next to 0.5 μg 100-bp DNA ladder (GeneRuler ; Thermo Fisher Fermentas) and electrophoresed in 1 TAE buffer (40 mm Trisacetate, 2 mm EDTA) at 8 V/cm for 35 min. After staining the gel with ethidium bromide (BDH, Toronto, ON, Canada), UV light transillumination revealed either a single band or double bands for each of the samples. If only primer set 1 (m2igti3) had a product (~550bp), then the mouse was wild type. If both primer sets revealed a product (one ~550bp and the other ~200bp), then it was concluded that the mouse was heterozygous for a deletion of Gtf2i (Gtf2i+/-). Table 2.3. List of Primers. A) Primers for PCR amplification from DNA for genotyping of mice with deletion of Gtf2i. B) Primers for quantitative real-time PCR (RT-PCR) amplification of DNA for genotyping of mice with duplication of Gtf2i. C) Primers for PCR amplification from DNA for sexing of mice.

78 Genotyping of Gtf2i +/dup Litters Gtf2i +/dup litters were genotyped using real-time PCR (RT-PCR), which allowed for determination of Gtf2i genomic copy number. ABI Prism7900HT sequence detection system was used with 11μl reactions, containing 5ng template and Power SYBR Master Mix (LifeTech). Samples were diluted 1/100 with sterile water and run in triplicate. Samples were run in four different primer sets for the following genes: Hmbs, Sdha, Gtf2i, and Gtf2ird1. The sequence of the primers is included in Table 2.3. Each plate contained a No Template Control (i.e. water) and serially diluted concentrations of control genomic DNA to generate a standard curve for genomic quantification. Results were normalized to the housekeeping genes Hmbs and Sdha Sexing of PND8 Mice A new technique was recently described to distinguish mouse pups bearing two X-chromosomes from those bearing one X- and one Y- chromosome (Clapcote and Roder, 2005). The authors utilized the high degree of sequence similarity on the interval between exons 9 and 10 of an X- chromosome-specific gene (Jarid1c) to that of a Y-chromosome-specific gene (Jarid1d), as well as the similar length of the corresponding exons of both genes (120 bp and 159 bp respectively). Furthermore, the introns between exons 9 and 10 in Jarid1c and Jarid1d show a difference of 29 bp with the Jarid1c intron 9 having a length of 114 bp and the Jarid1d intron 9 having a length of 85 bp. This allowed for the design of a pair of primers that would simultaneously amplify different sized fragments from both Jarid1c and Jarid1d (Clapcote and Roder, 2005). The sequence of the primers, Jarid1c/d forward and reverse, are included in Table 2.3. To identify the sex of PND8 mouse pups, conventional PCR was used as described above for genotyping of Gtf2i +/- litters but instead with the Jarid1c/d primer set and different thermal steps. Reactions were incubated at 94 C for 5 min, followed by 35 cycles of 94 C for 20 s, 54 C for 1 min, and 72 C for 40 s, followed by 72 C for 10 min. UV light transillumination revealed either single bands, which indicated the presence of two X-chromosomes and therefore a female pup, or two bands, which indicated the presence of an X- and Y- chromosome and therefore a male pup.

79 Results Subcutaneous Injection Alters USV Production in Mice with Altered Gtf2i Gene Copy Number The effects of saline injection on USVs were included as a control for the injection of anxiolytics. USVs emitted by pups receiving the control saline injection were pooled within genotypes since there was no litter effect (p>0.05). These were compared to USVs emitted by pups that did not receive an injection (no saline group) from our previous work (Mervis et al., 2012). A significant effect of genotype and injection group was observed (p<0.001). When comparing mean number of total USVs emitted in the 4 min trial following maternal separation, wild-type pups (Gtf2i +/+ ) produced similar number of USVs independent of whether they received a subcutaneous injection (Figure 2.4). However, mutant mice with altered Gtf2i genomic copy number did not. In pups that did not receive a subcutaneous injection, those with 1 copy of Gtf2i (Gtf2i +/- ) emitted the least number of USVs and those with 4 copies of Gtf2i (Gtf2i dup/dup ) emitted the most (Mervis et al., 2012, Figure 2.4). In pups that received a subcutaneous injection of saline min prior to the USVs test, the opposite trend was observed. That is, pups with 1 copy of Gtf2i (Gtf2i +del ) emitted the highest number of USVs and those with 4 copies of Gtf2i (Gtf2i dup/dup ) emitted the lowest (Figure 2.4). This suggests that the subcutaneous injection affected mice with altered Gtf2i copy number differently than control wild-type pups.

80 63 ** * Figure 2.4. Effects of subcutanous injection in maternal separation-induced USVs. Bar graphs show mean number of total USVs emitted by PND8 pups that did not receive a subcutaneous injection prior to undergoing the 4 min maternal separation-induced USVs trial (No saline) and those that did receive a subcutaneous injection of saline minutes prior to being tested for USVs emission (Saline S.C.). A significant effect of genotype and condition is observed (p<0.01 by Kruskal-Wallis test ). (n1,n2,n3, n4 in the x-axis is showing # of pups tested for each genotype group: Gtf2i +/del, Gtf2i +/+, Gtf2i +/dup, and Gtf2i dup/dup respectively in each treatment group) (*p<0.05, **p<0.01 by Kruskal-Wallis test). 2.2 Injection Stress, Plasma Corticosterone, and Altered Gtf2i Gene Copy Number Introduction The stress and anxiety circuits have been described as tightly intertwined due to findings that anxiety phenotypes are accompanied by a stress response often mediated by the activation of the HPA (reviewed in Grillon et al., 2007). Stress is important not only for the maintenance of anxiety-like responses through paralleled changes in stress hormone levels, but it can also potentiate anxiety phenotypes (reviewed in Grillon et al., 2007). USVs emitted after maternal separation are accompanied by physiological responses that include an increase in HPA axis

81 64 activity and immediate subsequent elevations of plasma corticosterone levels (Cirulli et al., 1994). Therefore, separation anxiety-like phenotypes can be assessed through both behavioral measures, such as via maternal separation induced USVs, and subsequent changes in stress hormone levels Research Aims To dissect the role of corticosterone in Gtf2i and anxiety in individuals with WBS and Dup7q11.23 by studying the Gtf2i mouse models Hypothesis We hypothesized that the physiological basis of the anxiety phenotype in the mouse models of WBS and Dup7q11.23 involved changes in plasma corticosterone concentrations. Furthermore, injection stress may also alter immediate plasma corticosterone levels. Thus, we hypothesized that both differential gene-dosage of Gtf2i and stress may play a role in modulating corticosterone concentrations in plasma Materials and Methods Contributions: I performed all assays and analysis of plasma corticosterone concentrations Animals Animals tested were the same as those described in Section for maternal separationinduced USVs. Wild-type PND8 pups (Gtf2i +/+ ) and those with a duplication of Gtf2i (Gtf2i +/dup and Gtf2i dup/dup ) were tested. Three sets of controls were included for plasma corticosterone testing. Naïve PND8 pups did not receive a subcutaneous injection or undergo the maternal separation-induced USVs test. Pups were removed from the litter and blood was collected immediately after sacrificing. A second set of control PND8 pups underwent the USV trial but did not receive a subcutanous injection (USVs pups). Lastly, saline controls were pups that received control saline subcutanous injections and then underwent the maternal separation USV trial. The rest of the pups were those that received a subcutanous injection of an anxiolytic (one of the 5 drugs), underwent the USV trial, and were then sacrificed so that blood could be

82 65 collected for plasma corticosterone measurements. Gtf2i +/+, Gtf2i +/dup, and Gtf2i dup/dup PND8 pups were tested Plasma Collection Plasma corticosterone levels were measured to determine whether increased expression of Gtf2i affects corticosterone levels in the plasma. Blood was collected in PND8 mouse pups immediately after decapitation using Microvette CB300 capillary tubes containing 30ug of EDTA. The microvettes containing blood were left to clot for min at room temperature. Plasma was obtained by centrifugation at 13000g for 15 min. Plasma (upper phase) was transferred to eppindorf tubes and stored at -80 C until the day of the assay. Plasma corticosterone concentration was measured as previously described (Monique, 2012 & Pang, 2009) using an ELISA kit and according to manufacturer s instructions (Cayman Chemicals #500655, MI, USA) Corticosterone Assay Following plasma extraction, corticosterone levels were measured according to the manufacturer s instructions (Cayman Chemicals #500655, MI, USA). The assay is based on the competition between corticosterone and a corticosterone acetylcholinesterase (AChE) conjugate (AChE tracer) for a limited number of corticosterone-specific sheep antiserum binding sites. A specific amount of Corticosterone Tracer was added to each well, which maintained the tracer at a constant concentration. Meanwhile, the concentration of corticosterone varied in each sample. As such, the amount of the tracer that was able to bind to the sheep antiserum was inversely proportional to the concentration of corticosterone in each well. The sheep antiserum corticosterone complex (whether free corticosterone or tracer corticosterone) bound to the rabbit polyclonal antisheep IgG that was previously attached to the well. The plate was then washed to remove unbound reagents. Following washing with a buffer, Ellman s Reagent (containing the substrate to corticosterone acetylcholinesterase) was added to each well. The product of the above enzymatic reaction produced a distinct yellow color that absorbed strongly between 405 and 420 nm. The intensity of the color was determined through spectrophotometry. This intensity, proportional to the amount of corticosterone tracer bound to the well, is inversely proportional to the amount of free corticosterone present from the plasma sample.

83 Statistical Analysis Results are expressed as means ± SEM and were analyzed by SPSS. The Shapiro Wilk test of normality was performed on the corticosterone data to assess the hypothesis of normal distribution. Violations of the assumption of normality and unequal number of mouse pups across groups determined the use of nonparametric statistics. A Kruskal-Wallis test assessed differences among groups in the concentration of plasma corticosterone immediately after the behavioral test. The Mann-Whitney test was used to assess differences between the saline and drug conditions within a genotype group. The Mann-Whitney test also assessed differences between genotype groups within the same condition. The test of Kruskal-Wallis also checked for litter effect. Since there was no litter effect, saline data were pooled together across litters. After pooling, data showed a normal distribution and, as such, parametric tests could be used. However, to keep the tests of significance consistent, nonparamentric tests were used for the data even after pooling Results Maternal Separation and Subcutaneous Injection Induced Changes in Plasma Corticosterone Levels in a Gtf2i Gene-Dosage Dependent Manner Plasma corticosterone levels differed between genotype and condition (p<0.05, Figure 2.5).Due to no litter effect (p>0.05), data from pups of the same genotype receiving saline were pooled together across litters. Three sets of controls were included: naïve, USVs, saline pups. No difference between genotypes was observed in naïve pups (p>0.05), as expected due to the lack of stressful stimuli in these pups. An up-regulation of plasma corticosterone levels is noted across genotypes in mouse pups that underwent the maternal separation-induced USVs trial (USVs controls) compare to naïve pups (Figure 2.5A and B). This up-regulation is as expected due to the presence of a stressful stimuli in the second group; the maternal separation. When

84 67 comparing between genotypes within the USVs group, there was a tendency for Gtf2i +/dup pups to have higher corticosterone concentrations than Gtf2i +/+ ones (Figure 2.5A). This indicates a Gtf2i gene-dosage dependent effect whereby pups with increased Gtf2i gene copy number show higher levels of plasma corticosterone. No data for Gtf2i dup/dup pups was collected. However, this trend was completely reversed in pups receiving a saline injection where pups with higher Gtf2i gene copy number (Gtf2i dup/dup ) had lower plasma corticosterone levels (Figure 2.5A). A Gtf2i gene-dosage dependent effect is still present in these pups receiving saline except reversed when compared to those that did not receive an injection (Figure 2.5A).This suggests that changes in Gtf2i gene copy number may influence the plasma corticosterone response of mouse pups to a saline injection.

85 68 Figure 2.5. Plasma corticosterone concentrations measured using Corticosterone EIA Assay. Naive PND8 mouse pups were used as controls to measure baseline plasma corticosterone levels. USVs controls were PND8 pups that underwent the maternal separationinduced USVs trial only. Saline controls were PND8 pups that underwent the maternal separation-induced USVs trial minute after a subcutaneous injection of saline. A difference in plasma corticosterone levels was observed across genotypes and between control groups (p<0.01 by Kruskal Wallis test). A) Data is grouped by experimental condition for better comparison between genotypes within a condition B) Same data as in Fig A but grouped by genotype for better visualization for comparison between different sets of controls within a genotype. {n1,n2,n3 in the x-axis is showing # of pups tested for each genotype group: Gtf2i +/+, Gtf2i +/dup, and Gtf2i dup/dup respectively in each control group) Maternal Separation-Induced USVs Predict Plasma Corticosterone Concentrations Behavioural manifestations of distress are accompanied by physiological responses that include an increase in HPA axis activity and subsequent release of stress hormones (Cirulli et al., 1994). Maternal separation-induced USVs, a measure of anxiety in rodents, have been shown to be accompanied by an increase in plasma levels of the stress hormone corticosterone (Howard et al., 2012). To see whether the same was true in our study, mean number of total USVs previously reported in PND8 mice following maternal separation (Mervis et al., 2012) were compared to plasma corticosterone levels in PND8 pups that underwent the same 4 min trial of maternal separation-induced USVs. The same comparison was conducted for PND8 pups in this study that received a saline injection prior to the USVs trial. The Gtf2i gene-dosage dependent effect seen

86 69 with USVs is also present when looking at changes in plasma corticosterone levels. For PND8 pups that underwent the USV trial but did not receive an injection, as Gtf2i gene copy number increases, mean number of total USVs and plasma corticosterone concentration increase (Figure 2.6A and B). Plasma corticosterone data for Gtf2i +/- and Gtf2i dup/dup have not been included. This parallel between USVs and plasma corticosterone levels as Gtf2i gene copy number changes is also observed in pups that received a subcutaneous injection of saline prior to the USVs test. In this set of data, as Gtf2i genomic copy number increases, mean number of total USVs and plasma corticosterone concentrations decrease (Figure 2.6A and B). A ** *

87 B 70 Figure 2.6. Maternal separation-induced USVs correlate with plasma corticosterone concentrations. Bar graphs show A) mean number of total USVs emitted in the 4 min trial of maternal separation and B) plasma corticosterone concentrations in PND8 mouse pups that underwent the USVs trial but did not receive an injection (Gene group (no saline)) and in mouse pups that received a subcutaneous injection of saline prior to the USVs trial (gene group (saline S.C.) The Gtf2i gene-dosage-dependent effect observed with USVs is also present when looking at changes in plasma corticosterone levels for both gene groups. Plasma corticosterone data for Gtf2i +/del have not been included {n1,n2,n3, n4 in the x-axis is showing # of pups tested for each genotype group: Gtf2i +/del, Gtf2i +/+, Gtf2i +/dup, and Gtf2i dup/dup respectively in each treatment group}. (*p<0.05, **p<0.01 by Kruskal Wallis test). Note that figure 2.6A is a replicate of figure 2.4 presented in section Effect of Gtf2i Maternal Genotype on Maternal Separation- Induced USVs Introduction Maternal genotype effects have been elucidated for a number of genes such as 5-HT1A, Fmrp, Peg-3, AVP, and oxytocin receptor gene to name a few (Gleason et al., 2010, Zupan and Tooth, 2008, Li et al., 1999, Bosch and Neumann, 2008, Takayanagi et al., 2005). Dams carrying these mutations have shown an effect on offsprings phenotypes independent on whether the offsprings carried the mutation themselves (Gleason et al., 2010, Zupan and Tooth, 2008, Li et al., 1999, Bosch and Neumann, 2008, Takayanagi et al., 2005). These maternal genes are suspected to affect maternal care and therefore subsequent behavior of offsprings. Therefore, it is important to

88 71 study potential Gtf2i maternal genotype effect in the anxiety phenotype observed in PND8 mouse pups with altered Gtf2i gene copy number Research Aims To investigate the effect of maternal Gtf2i genotype on anxiety phenotypes in the Gtf2iduplication mouse model Hypothesis Due to the known role of maternal care in mouse anxiety (reviewed in Hofer, 1996), we hypothesized that a difference in the number of USVs emitted following maternal separation in PND8 mice might depend on whether the duplication was inherited from the dam or the sire Materials and Methods Contributions: I performed all behavioral tests, genotyping and sexing of test animals, and statistical analysis Animals Animals tested were the same as those described in Section for maternal separationinduced USVs. Two types of crosses were set up for this experiment. In the first one, the dam carried the duplication of Gtf2i (Gtf2i +/dup or Gtf2i dup/dup ) and was paired with a wild-type sire (Gtf2i +/+ ). The second crossing was reciprocal to the previous one with the sire carrying the duplication for Gtf2i (Gtf2i +/dup ) and paired with a wild-type dam (Figure 2.7).

89 72 Figure 2.7. Schematic diagram depicting the parental crosses set up to generate F1 offspring used for testing the effect of maternal Gtf2i genotype on maternal separationinduced vocalizations in post-natal day 8 pups. Mice with heterozygous duplication of Gtf2i (Gtf2i +/dup ) were paired with wild-type mice of opposite sex (Gtf2i +/+ ). Therefore, either the dam or the sire carried the duplication in a specific breeding pair Apparatus and Measurements The testing apparatus was the same as described above for the maternal separation-induced USVs trial in Section Data were analyzed blind to genotype and parental crossing. Mean number of USVs were calculated in minute bins Maternal Separation-Induced USVs Procedure The procedure was identical to the one described above in Section with the exception that these pups did not receive a subcutaneous injection prior to the USVs trial.

90 Statistical Analysis Results are expressed as means ± SEM and were analyzed by SPSS. The Shapiro Wilk test of normality was performed on all of the USVs data to assess the hypothesis of normal distribution. Nonparamatric tests of significance were used due to violations of the assumption of normality and unequal number of mouse pups across groups. A Kruskal-Wallis test assessed differences among the four groups in the median number of vocalizations produced over the 4 min trial. The Mann-Whitney test was used to assess differences between two different groups. The test of Kruskal-Wallis also assessed differences in body weight, sex, and litter effect Results Maternal Genotype Effect on Offspring s Maternal Separation- Induced USVs A significant effect of maternal genotype was observed on total number of USVs (p<0.05, Figure 2.8) whereby PND8 pups inheriting the duplication from the dam emitted on average more USVs than pups inheriting the duplication from the sire. When comparing pups of different genotypes, there was a tendency for Gtf2i +/dup mouse pups, independent of which parent they inherited the duplication from, to produce less USVs than their wild-type littermates. Although a significant difference was observed across all genotype groups (p<0.005), when using Mann-Whitney test to compare between two groups, a significant difference was observed only between Gtf2i +/+ and Gtf2i +/dup mouse pups inheriting the duplication from the dam (p<0.05, Figure 2.8A). The sex of each pup was determined to assess whether a particular sex was more likely to show this maternal genotype effect. A Kruskal-Wallis test indicated an effect of sex in the mean number of USVs emitted by pups of different genotype (p<0.05). More specifically, in the Gtf2i +/+ pups from crossings where the dam was wild-type and the sire carried the Gtf2i duplication, females emitted more USVs than males (p<0.05). Similarly, in Gtf2i +/+ pups from crossings where the dam carried the Gtf2i duplication and the sire was wild-type, there was a tendency for females to produce more USVs. This is contrary to Gtf2i +/dup pups, independent of which parental crossings they came from, where there was a tendency for males to emit more vocalizations (Figure 2.8B).

91 A 74 B Figure 2.8 Maternal separation-induced USVs in PND8 mouse pups generated from different breeding pups where the Gtf2i duplication is inherited from either the dam or the sire. A) An effect of maternal Gtf2i genotype is observed (p<0.05 by Kruskal Wallis test). PND8 pups inheriting the duplication from the dam had a tendency to emit on average more USVs than pups inheriting the duplication from the sire. B) Maternal separation-induced USVs are differentially affected by maternal genotype in male and female PND8 mouse pups (p<0.05 by Kruskal Wallis test). In Gtf2i +/+ pups from crossings where the dam was wild-type and the sire carried the Gtf2i duplication, females emitted more USVs than males (p<0.05 by Mann Whitney test). Similarly, in Gtf2i +/+ pups from crossings where the dam carried the Gtf2i duplication and the sire was wild-type, there was a tendency for females to produce more USVs.

92 75 This is contrary to Gtf2i +/dup pups, independent of which parental crossings they came from, where there was a tendency for males to emit more vocalizations. {n in the legend is showing sample size for each genotype group}. (*p<0.05, **p<0.01). 2.4 Discussion and Conclusions Injection Stress Stimulated Changes in Maternal Separation -Induced USVs in a Gtf2i Gene-Dosage Dependent Manner A Gtf2i gene-dosage-dependent effect on separation anxiety, as assessed by maternal separationinduced USVs in mouse pups, was previously reported (Mervis et al., 2012). PND8 mouse pups with either a heterozygous or homozygous duplication of Gtf2i (Gtf2i +/dup and Gtf2i dup/dup respectively) emitted a higher number of maternal separation-induced USVs, in contrast to PND8 pups with a heterozygous deletion of Gtf2i (Gtf2i +/del ) that exhibited a tendency for reduced maternal separation-induced USVs (Mervis et al., 2012). In this study, we reported the opposite trend when PND8 mouse pups received a saline injection min prior to the USVs test, that is, mice with a heterozygous deletion of Gtf2i (Gtf2i +/del ) emitted the highest number of calls. As Gtf2i gene copy number increased, the number of USVs emitted decreased. Therefore, maternal separation-induced USVs are affected by the stress induced by both separation from the dam and the injection. This interaction between stress and USVs appears to be at least in part dependent on Gtf2i gene dosage. The mechanism of how Gtf2i attenuates injection-stress- induced USVs after maternal separation is yet to be elucidated Maternal Separation and Subcutaneous Injection Elevate Plasma Corticosterone Levels in a Gtf2i Gene-Dosage Dependent Manner Anxiety-related behaviors, as measured in stress-inducing environments, are accompanied by physiological changes. Such physiological responses may include an increase in HPA axis activity, which may then lead to elevations in plasma corticosterone levels (Cirulli et al., 1994). In our study, maternal separation-induced USVs were accompanied by an increase in plasma corticosterone concentrations. However, this increase differed between groups depending on whether any additional stimuli affected corticosterone concentrations. Naïve PND8 pups showed low levels of plasma corticosterone. Since corticosterone is a stress-induced hormone, it was

93 76 expected that naïve pups would show minimal levels of plasma corticosterone due to the lack of stressful stimuli in their everyday environment. An increase in plasma corticosterone concentrations was evident in pups undergoing the USV trials compared to naïve pups. This increase in corticosterone levels is due to the stress associated with maternal separation. Pups receiving a saline injection prior to the USVs trial showed even higher levels of plasma corticosterone across all genotypes. This increase is believed to be due to the added stress that the injection itself imposes on these pups, which is compounded by the stress induced by the maternal separation. It is important to note that the subcutaneous injection occurred min prior to the USVs test and plasma collection. This suggests that the injection has a strong effect on pups with altered Gtf2i gene copy number that is maintained even min after the injection. Since changes in plasma corticosterone levels are known to begin extremely quick following a stimuli such as stress induced by maternal separation, we can deduce that the effect of the subcutaneous injection is strong enough to elicit changes in both the number of maternal separation induced USVs and associated plasma corticosterone changes. When comparing plasma corticosterone concentrations among pups with different Gtf2i genomic copy number, the opposite trend was observed in those receiving a saline injection versus those that did not. In pups that did not receive an injection, those with more copies of Gtf2i (Gtf2i +/dup ) had a tendency to show higher levels of plasma corticosterone than wild-type pups with two copies of Gtf2i (Gtf2i +/+ ). In pups that received a saline injection prior to the USV trials, lower levels of corticosterone were measured as Gtf2i gene copy number increased. This suggests that, when an injection is included, the response between pups with altered Gtf2i genomic copy number differs Maternal Separation-Induced USVs Predict Plasma Corticosterone Concentrations A clear correlation is observed between separation anxiety, as measured by the number of maternal separation-induced USVs, and physiological changes, as measured by plasma corticosterone levels. This correlation is evident in both PND8 pups that underwent the USV trials but did not receive an injection, and in pups that did receive a saline injection. Furthermore, a Gtf2i gene-dosage dependent effect on plasma corticosterone levels was observed in both

94 77 groups. In pups that did not receive an injection, the total number of USVs and plasma corticosterone concentrations increased as Gtf2i gene copy number increased. In pups that received a saline injection, the total number of USVs and plasma corticosterone levels decreased as Gtf2i gene copy number increased. This suggests that behavioral manifestations of anxiety correlate with physiological changes. Despite this clear correlation, one drawback of the plasma corticosterone data is the small sample size of control pups that did not receive a subcutaneous injection. Furthermore, only two groups were included, Gtf2i +/+ and Gtf2i +/dup. Therefore, future studies should aim to increase the sample sizes and include studies of pups with a single copy of Gtf2i (Gtf2i +/del ) and four copies of Gtf2i (Gtf2i dup/dup ). In addition, changes in plasma corticosterone levels in Gtf2i +/del pups that receive a saline injection prior to the USVs test also need to be assessed. Despite these limitations, the current data show a clear correlation between the total number of USVs and plasma corticosterone levels, which indicates a tight link between behavioral measures of anxiety and physiological responses. The mean number of total USVs across all genotypes is comparable between the injection group and the group without the injection, although the change in number of USVs as Gtf2i gene copy number increases differs between the two groups. However, plasma corticosterone levels seem to be higher overall in all of the pups that received a saline injection, regardless of genotype. Our data are in agreement with previous studies indicating that subcutaneous injections of saline alone induce a marked increase in plasma corticosterone levels when compared to naïve animals (Benedetti et al., 2012). This may be due to the added stress associated with the subcutaneous injection, in addition to the stress associated with the maternal separation. Furthermore, pain due to the injection likely enhances the corticosterone response since it has been previously shown that pain is associated with intense recruitment of the HPA axis and downstream corticosterone release (Aubrun et al., 2004, and reviewed in Benedetti et al, 2012) Hypothesized Changes in Pain Sensitivity in the Gtf2i Mouse Models Injection stress affects both total number of maternal separation-induced USVs and plasma corticosterone levels in a Gtf2i gene-dosage-dependent way. In mice that do not receive a subcutaneous injection of saline, we reported an increase in the number of USVs (Mervis et al., 2012) and a trend of increase in plasma corticosterone concentrations as Gtf2i gene copy number

95 78 increases. However, the opposite was reported when PND8 pups received a subcutaneous injection of saline. That is, as Gtf2i gene copy number increases, the total number of USVs and plasma corticosterone levels decrease. It is important to note that in the second group of pups, saline group, USVs are measured minutes after the injection. Therefore, the response to the subcutaneous injection is maintained at times of stress such as during maternal separation, even though it is min after the initial injection. This indicates of a strong effect of injection specifically in pups with altered Gtf2i gene copy number since the number of USVs in wild-type mouse pups remains the same independent of whether they receive an injection. Changes in pain sensitivity in our mutant mice may be a cause for this reversal of trend in USVs. We hypothesize that hypersensitivity to pain characterizes mice with a deletion of Gtf2i (Gtf2i +/del ) and hyposensitivity to pain characterizes those with a heterozygous and homozygous duplication of Gtf2i (Gtf2i +/dup and Gtf2i dup/dup respectively). If an underlying difference in pain sensitivity exists in mice with altered Gtf2i gene copy number, then the perceived pain associated with the subcutaneous injection would differ between Gtf2i genotype groups. Furthermore, pain in itself is associated with intense recruitment of the HPA axis and downstream corticosterone release (reviewed in Benedetti et al., 2012). Therefore, increased sensitivity to pain in mice with a deletion of Gtf2i (Gtf2i +/del ) should be accompanied by elevated plasma corticosterone concentrations whereas reduced sensitivity to pain in mice with a duplication of Gtf2i (i.e. Gtf2i +/dup and Gtf2i dup/dup ) would be expected to correlate with lower concentrations of plasma corticosterone. Our findings support this hypothesis and show that plasma corticosterone levels are affected by both the stress associated with maternal separation and the stress/pain associated with the subcutaneous injection in a Gtf2i dose-dependent manner. Behavioral characterizations of the current mouse models of WBS have not generally focused on the assessment of pain sensitivity. In the current literature, only one mouse model for which a pain-related phenotype has been reported, which is the model consisting of a deletion that spans half of the syntenic WBS region and encompasses the genes between and including Limk1 to Gtf2i (defined as PD mice). Increased sensitivity to painful stimuli was reported for PD mice using the hot plate test (Li et al., 2009). This change in pain sensitivity was not observed in mice carrying a deletion of the other half of the WBS region that does not include Gtf2i (defined as

96 79 DD mice). The hot plate test provides insight into pain sensitivity by using a thermal stimulus to assess thermal threshold (Hargreaves et al., 1988). A shorter latency to respond to the painful stimuli of high temperature was reported for PD mice (Li et al., 2009). This accelerated response to thermal pain suggests that mice with a deletion of the WBS region are hypersensitive to pain. Although this phenotype cannot be mapped to a specific gene within the PD deleted region, Gtf2i is an excellent candidate gene. Our Gtf2i single gene mouse models provide a tool with which to assess whether gain or loss of Gtf2i alters pain sensitivity, and as such, future studies of the Gtf2i mouse models should also assess pain sensitivity. To date, no clinical studies have assessed sensitivity to pain in individuals with WBS or with Dup7q11.23 syndrome. However, anecdotal reports of high pain thresholds in people with 7q11.23 duplication are common (personal communication by C. Mervis and C. Morris). For example, one child had a broken arm and another child a broken foot, both of which were only discovered days after the actual injury ocurred. Neither child complained of any pain in the broken limb. The finger of another child was caught in a car door at the age of almost two years old. The child was not bothered by the pain, on the contrary, he was excited to show it off to his father. The incident thus formed the reason for the referral to a geneticist, which led to a subsequent microarray that revealed a duplication of the 7q11.23 region. Another report of high pain threshold comes from a child with 7q11.23 duplication syndrome who had to be stuck about 8 times with a needle to retrieve blood. The child appeared unbothered by this, as he did not cry or try to get away, but instead put his thumb in his mouth and went to sleep (C. Mervis, University of Louisville, Personal communication, May 3, 2011). Lastly, another individual was characterized with high pain threshold after breaking his collarbone and not realizing that he had done so for several days (C. Morris, University of Nevada, Personal communication, May 3, 2011). These anecdotal reports suggest reduced pain sensitivity in individuals with Dup7q Therefore, it would be of interest to formally assess pain sensitivity in Dup7q11.23 syndrome individuals as well as those with WBS. Altered pain sensitivity has also been reported in individuals with other disorders with overlapping phenotypes. Specifically, reduced pain sensitivity has been reported in individuals with autism spectrum disorder (Nader et al., 2004). These individuals share common phenotypes

97 80 with individuals with Dup7q11.23 syndrome, and studies have argued that duplication of 7q11.23 is strongly associated with autism (Levy et al., 2011, and Sanders et al., 2011). Fragile X syndrome (FXS) is another disorder where changes in pain sensitivity have been reported. FXS has been linked with alterations in pain processing due to the self-injurious behaviors observed in individuals with this disorder (Price et al., 2007). The mouse model for this disorder, an Fmr1 knock-out, has also been studied and showed reduced sensitivity to pain, linking a specific gene to changes in pain sensitivity (Price et al., 2007). Therefore, pain threshold in individuals with WBS and Dup7q11.23 needs to be formally addressed. Mouse models could then be used to elucidate the genetic and molecular mechanisms responsible for this phenotype and the potential role of Gtf2i in pain sensitivity. Assessment of pain sensitivity in humans however is not quite as clear-cut as in animal models. Currently, there are no standardised pain measures available to clinicians for children or individuals with cognitive impairment (Breau et al., 2002). This makes assessment of pain sensitivity difficult to measure directly in individuals with WBS or Dup7q One of the common methods of assessing sensitivity to pain in children is using an indirect approach-parent report (reviewed in Nader et al., 2004). However, one confounding factor that arises when assessing pain sensitivity in individuals with WBS specifically is the finding that these individuals are described as hypersensitive in general, making it difficult to dissociate emotional and physical hypersensitivity (Zarchi et al., 2010). Pain sensitivity in individuals with WBS and Dup7q11.23 may be assessed by using a painful medical procedure such as venepuncture. Studies of children with autism have shown the utility of this procedure in measuring pain-related behaviors (Nader et al., 2004). During the venepuncture procedure, the child is videotaped. The video is then analysed using the Observational Scale of Behavioral Distress (OSBD) which assesses behavioral distress in children undergoing medical procedures that inflict pain. The OSBD is a behavioral coding system that consists of 8 operationally defined behaviors indicative of anxiety and/or pain responses. It has been widely used to measure children s distress in medical situations such as venepuncture and injections. The Non-Communicating Children s Pain Checklist (NCCPC) is another pain measurement tool specifically designed to assess pain sensitivity in children with

98 81 cognitive impairments through parent reports (reviewed in Breau et al., 2002). The checklist consists of 30-items that assess behaviors of the child by the parent or caregiver. When parents complete the items of this checklist, they are asked to think of previous incidents when their child had been in pain. Lastly, the Faces Pain Scale (FPS) is used to provide observer measures of pain. It consists of 7 faces showing gradual increases in expression of pain (reviewed in Nader et al., 2004). Parental reports of pain through FPS scores are then correlated with facial pain responses of children to characterize the relationship between parental report and child behavioral measures of pain Potential Confounding Variables on the Sensitivity of Plasma Corticosterone Changes Changes in plasma corticosterone levels have been shown to be time-dependent and affected by the type of mouse pain model, the nature of manipulations to the animal, and the strain of the mouse (Benedetti et al., 2012). Noxious stimulation increases plasma corticosterone concentrations in mice. Changes in plasma corticosterone concentrations were investigated in a study of acute and chronic pain in two different mouse strains: C57Bl/6 and Balb/C. Acute pain, induced by stimulation of peripheral nociceptive afferents via subcutaneous capsaicin, was accompanied by a remarkable increase in corticosterone levels in both mouse strains, with higher levels reported in the Balb/C mice. Furthermore, although time-dependent changes in plasma corticosterone levels were observed, such changes differed between mouse strains (Benedetti et al., 2012). This difference between mouse strains might reflect primarily the influence of genetic factors. This difference also emphasizes the importance of considering the genetic background when examining variables that affect behavioral test results. To address such concerns, mice of the same, mixed strain background were tested for all the measurements included in this work: CD1/129. Furthermore, plasma corticosterone levels were measured in all subjects from blood collected at the same time point in the experiment, immediately after the sacrificing of the animal. The manipulations and the strain imposed on the animals were minimized and maintained constant across subjects to avoid between-subject differences. Therefore, we believe that the changes we observed in plasma corticosterone levels are due to the Gtf2i genetic manipulations and/or experimental group of the animals.

99 82 In-vivo research has often been praised for the opportunities that it provides scientists to assess a hypothesis at the level of the whole organism. However, a major issue facing scientists who conduct in-vivo research is whether their experimental protocol imposes significant stress on their subjects. Such stress could introduce confounding factors in interpreting the study results. However, in this study, we employed the maternal separation-induced USVs paradigm which is a validated measure of separation anxiety in the neonatal mouse (Scattoni et al., 2009). Furthermore, we specifically tested stress induced by the maternal separation and, therefore, stress introduced by the experimental protocol is captured through measures of USVs. Lastly, variables such as lighting, time of the day, humidity, noise, diet, and animal handling can also greatly affect sensitivity in behavioral tests and subsequent measures of physiological changes. However, in our study, by maintaining these external variables constant across all subjects, we controlled variability between subjects. Factors such as animal handling and injections may also influence the change in plasma corticosterone levels. Animal handling specifically has been shown to induce a marked increase in circulating corticosterone levels (Benedetti et al., 2012). In a previous study, animals subjected only to the manoeuvre used to subcutaneously inject the paw with capsaicin exhibited increased circulating corticosterone levels, indicating that even short periods of handling can activate the HPA axis (Gariepy et al., 2002, Benedetti et al., 2012). In our study, all animals were subjected to the same degree of handling and therefore, handling-induced increases in plasma corticosterone levels are expected to be comparable between all subjects. Similarly, subcutaneous injections of a control vehicle such as saline can also induce increases in plasma corticosterone concentrations in addition to the elevation due to handling. In a study of acute pain, subcutaneous injections of saline induced a marked increase in plasma corticosterone levels when compared to naïve animals, albeit not to the same magnitude as changes induced by injection of the drug (Benedetti et al., 2012). Our findings parallel those previously reported. Similarly, we observed much higher levels of plasma corticosterone in pups that received a subcutaneous injection of saline than in naïve animals. The subcutaneous injection itself appear to be sufficient to induce a marked stress response, as measured by plasma corticosterone concentrations. The novel finding in our study is the induction of plasma corticosterone levels

100 83 following the injection in a Gtf2i gene-dosage-dependent way. Therefore, an interaction between stress-inducing paradigms, corticosterone stress hormone levels and Gtf2i gene dosage can be presumed. The within-cage order of testing is another factor that may influence plasma corticosterone levels. Due to the procedural set up, potential influences from the first mouse undergoing the stress-inducing test on the yet-to-be-tested animals that remain in the same cage may potentially exist. It is widely accepted that mice can vocalize when restrained and may use such vocalizations or pheromone release for social communication (Scattoni et al., 2009). In our study, injected and handled mice had brief contact with non-injected mice. Furthermore, this period of contact was the same for all pups due to the specific set up of the procedure. However, this potential within-cage order effect was previously investigated and found to not occur, at least when looking at the influence on plasma corticosterone levels (Benedetti et al, 2012). Within-cage order did not interfere with plasma corticosterone levels where tested animals had contact with non-tested animals. Furthermore, within-cage order was also reported to not be relevant for naïve mice since no changes were observed in plasma corticosterone concentrations between the first and the last naïve mouse (Benedetti et al., 2012) Role of Maternal Gtf2i Genotype and Parental Genotype Interaction on Offspring Anxiety and Maternal Care Rodent studies have shown a significant impact of maternal care on ultrasonic vocalizations in pups (reviewed in Hofer, 1996). The extent of the influence of maternal genetic background on maternal care is unclear. Some studies have reported differences in maternal care between mothers of different strains, thereby indicating the importance of maternal genetic background in this behavior (D Amato et al., 2005). Maternal genotype has been excluded from most association studies with the majority of these studies focusing on the genotype of the offspring. Therefore, it is likely that the prevalence of maternal genotype effects is largely underestimated and could account for some of the high heritability in psychiatric diseases that is currently unaccounted for (reviewed in Gleason et al., 2011). Despite this potential underestimation, maternal genotype effects have been elucidated for a number of genes. In our study, we assessed the effect of maternal Gtf2i genotype on maternal separation-induced USVs.

101 84 We report a maternal Gtf2i genotype effect on separation anxiety as measured by maternal separation-induced USVs. Wild-type mouse pups raised by dams carrying the Gtf2i duplication showed a tendency towards emitting a higher number of USVs compared to wild-type pups raised by a wild-type dam. This suggests a maternal Gtf2i genotype effect on maternal separation-induced USVs in wild-type mouse pups, which parallels the findings from maternal genotype effect studies of the Fmrp and 5-HT1A mouse models (Zupan and Tooth, 2008, Weller et al., 2003, Gleason et al., 2010). However, the difference is not evident in mouse pups carrying the Gtf2i duplication themselves. Mouse pups with a duplication of Gtf2i (i.e. Gtf2i +/dup ) emitted a similar number of USVs independent of whether they were raised by a dam carrying the Gtf2i duplication or a wild-type dam. Contrary to previous work with other genes of interest, Fmrp and 5-HT1A, this suggests that, if the offspring carries the mutation themselves, the mutation carried by the dam does not provide any additive effect on the phenotype. Furthermore, when comparing pups of wild-type dams to pups of Gtf2i +/dup dams, results show that, independent of the offspring genotype, pups raised by dams carrying the Gtf2i duplication had a tendency to emit a higher number of USVs. This may be a consequence of reduced maternal care by Gtf2i +/dup dams. Therefore, the interaction between maternal-offspring genotype cannot be ignored due to the potential role of maternal genotype in the pup s rearing. Parental genotype interaction is another important factor that needs to be considered when assessing differences in maternal separation-induced USVs. When comparing wild-type pups to Gtf2i +/dup pups, independent of maternal genotpe, wild-type pups emitted more USVs on average than Gtf2i +/dup pups. This contrasts with our previous findings where Gtf2i +/dup pups vocalized more (Mervis et al., 2012). Since strain and other environmental factors were maintained constant, we can hypothesize that an interaction between parental genotypes may play a role. Breeding pairs for this experiment were set up with either the dam or the sire carrying the duplication. This is different from our previous work where heterozygous crosses (Gtf2i +/dup * Gtf2i +/dup ) were set up to produce F1 offspring (Mervis et al., 2012). A potential interaction between Gtf2i genotype of the dam and the sire, and associated stress hormone levels may influence offspring behavior in pups carrying a duplication of Gtf2i. The sex of pups was also important for the number of maternal separation-induced USVs. Firstly, when comparing USVs emitted by sex- and genotype- separated groups, a significant

102 85 difference between male and female pups is only observed for wild-type pups raised by wildtype dams but not for any of the other groups carrying the Gtf2i duplication: the offspring, the dam, or both. This suggests that the sex of the offspring, even when testing wild-type animals, is an important factor to be considered when looking at maternal separation-induced USVs. Adult mice of both sexes produce complex vocalizations in social settings that differ in their purpose between the sexes (Scattoni et al., 2009). As such, studies of adult mice USVs have focused independently on male and female USVs. The same is not seen in mouse pups however due to a lack of differentiation in the function of USVs in mouse pups of a different sex. Whether there is an anatomical or evolutionary basis for this sex-dependent difference in maternal separationinduced USVs is still to be elucidated. A tendency for sex-dependent segregation in the number of maternal separation-induced USVs is also observed when comparing pups of different genotypes raised by dams of different genotypes. In wild-type (Gtf2i +/+ ) mouse pups, there was a tendency for females to emit more USVs than males independent of maternal genotype. In contrast, the male Gtf2i +/dup pups had a tendency to emit more USVs independent of maternal genotype. This suggests that Gtf2i may play a role in the sex-dependent differences in USVs.

103 86 Chapter 3 Characterizing the Separation Anxiety Phenotype in Mouse Models of Varying Gtf2i Copy Number 3.1 Effects of Anxiolytics and Gtf2i Pup Genotype on Maternal Separation-Induced USVs Introduction Research Aims To help dissect neurocircuitry involved in the anxiety phenotypes in WBS and Dup7q11.23, single-gene Gtf2i mouse mutants with one to four Gtf2i gene copies were used. Using the maternal separation induced USVs paradigm, the anxiolytic effects of several drugs were assessed to help characterize the involvement of specific neurotransmitter systems that may give rise to the Gtf2i gene copy number mediated separation anxiety phenotype Hypothesis Studies of individuals with WBS and Dup7q11.23 have reported a high prevalence of anxiety in these two disorders (Mervis et al., 2012, Pober, 2010). Although anxiety medications are prescribed for both children and adults with WBS, their effectiveness has been limited (Stinton et al., 2010, Thornton-Wells et al., 2011, Woodruff-Borden et al., 2010). We hypothesize that the anxiety phenotype observed in our mouse models with altered Gtf2i gene copy number can be further studied to identify effectiveness and potential mechanism of action of anti-anxiety medications for WBS and Dup7q Maternal Separation-Induced USVs Maternal separation-induced USVs are an ethologically validated measure for pre-clinical characterization of anxiolytic drugs (reviewed in Takahashi et al., 2009). USVs emitted following maternal separation are known to peak on day 8 after birth, and as such, PND8 mouse

104 87 pups are used to assess the anxiolytic properties of drugs (Branchi et al., 2001, Fish et al., 2000, Fish et al., 2003, Takahashi et al., 2009) Pharmacological Compounds with Anxiolytic Properties Treatments for anxiety disorders have targeted serotonin, GABA and glutamate receptors and transporters (Connolly et al., 2011). The effects of six different anxiolytic drugs on maternal separation-induced USVs in PND8 pups were examined in this study Serotonergic Targeting Drugs Escitalopram and 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) are drugs that work to increase levels of serotonin (5HT) or mimic effects of 5HT. Escitalopram is a selective serotonin reuptake inhibitor (SSRI) that blocks the reuptake of serotonin from the synapse by blocking the serotonin transporter, SERT (Fish et al., 2000, Ravindran et al., 2010, Figure 3.1). 8-OH-DPAT is a 5HT1A receptor agonist. As an agonist, 8-OH-DPAT binds to this post-synaptic receptor and mimics the role of 5HT, thereby increasing serotonergic-mediated neurotransmission (Fish et al., 2000 Figure 3.1).

105 88 Figure 3.1. Schematic representation of a serotonergic synaptic terminal. SSRIs block the serotonin transporter (SERT) which is responsible for reuptake of serotonin neurotransmitter from the synapse. 5-HT1A is a postsynaptic receptor activated by the agonists 8-OH-DPAT to increase serotonergic mediated neurotransmission (adapted from Fernandez and Gaspar, 2012) GABAergic Targeting Drugs Chlordiazepoxide and allopregnanolone are GABA agonists. Chlordiazepoxide, a classic benzodiazepine (BZ), works on the GABA-A receptor to increase GABAergic-mediated neurotransmission. Specifically, the α1 and γ2 subunits of the GABA-A receptor are important for the effects of chlordiazepoxide (Reddy and Woodward, 2004, Belelli and Lambert, 2005, and Lopez-Munoz et al., 2011 Figure 3.2). Allopregnanolone works on the GABA-A β2 subunit as a positive allosteric modulator of the receptor (Belelli and Lambert, 2005, and Reddy, 2004 Figure 3.2). Both drugs increase GABAergic-mediated inhibition via interactions with different subunits of the GABA-A receptor.

106 89 Figure 3.2. Schematic representation of pentameric GABA-A receptors with five protein subunits that comprise the chloride ion channel.different pharamcological compounds interact with specific binding sites. The benzodiazepine binding site is hypothesized to be located in between the α1 and γ2 subunits. Neurosteroids such as allopregnanolone bind on the ß2 subunit instead (Reddy, 2013) Glutamatergic Targeting Drug MK-801 is a glutamate inhibiting drug. MK-801 targets the postsynaptic NMDA receptor. As a non-competitive antagonist, MK-801 binds to the phencyclidine (PCP) site of the NMDA receptor and blocks channel activity (Kornhuber and Muller, 1997, and Takahashi et al., 2009 Figure 3.3). Ultimately, MK-801 reduces glutamatergic-mediated neurotransmission by blocking postsynaptic receptor activity.

107 90 Figure 3.3. Schematic representation of a glutamatergic synaptic terminal. Both ionotropic, AMPA and NMDAR, and metabotropic (mglur) receptors have been the target of anxiolytics. MK-801 is a non-competitive antagonist of the postsynaptic NMDA receptor thereby inhibiting glutamatergic mediated neurotransmission (adapted from Snyder and Murphy, 2008) Materials and Methods Contributions: I performed all behavioral tests, genotyping and sexing of test animals, and statistical analysis Animals/Housing Animals were the same as those described in Section Apparatus and Measurements The testing apparatus was the same as described in Section Maternal Separation-Induced USVs Procedure The procedure was identical to the one described in Section After the number of USVs emitted were calculated, we compared USVs between the first and the rest of the animals tested from the same cage to test for effects of within-cage order of testing. Within-cage order testing effects would suggest that tested animals may influence the behavior of the yet-to-be tested animals.

108 Drugs Escitalopram (Sigma-Aldrich), (+)-8-hydroxy-2-(dipropylamino) tetralin hydrobromide (8-OH- DPAT; Sigma-Aldrich), chlordiazepoxide (Toronto Research Chemicals Inc., Canada), and MK- 801(Sigma-Aldrich) were dissolved in 0.9% saline. 5α-3α-pregnan-ol-20-one (allopregnanolone; Steraloids, Inc., Newport, R.I., USA) was suspended with the aid of sonification in 20% hydroxypropyl-betacyclodextrin (Biobasic, Inc., Canada) in distilled H 2 O solution. All drugs were injected subcutaneously in a volume of 0.1 ml/10 g body weight. Injection doses were selected based on previous tests of these drugs on maternal separation-induced USVs in PND7 mouse pups (Fish et al., 2000, 2003, Takahashi et al., 2009). The selected doses were most effective in reducing USVs and had the least side effects on locomotion and/or sedation. A list of all the doses tested is included in Table 3.1. All drugs were injected minutes prior to the separation test. Escitalopram, 8-OH-DPAT, and chlordiazepoxide were tested in Gtf2i +/+, Gtf2i +/dup, and Gtf2i dup/dup PND8 pups. Allopregnanolone and MK-801 were tested in the previously mentioned three genotype groups, plus Gtf2i +/del pups. Table 3.1. Summary of the doses used for subcutaneous injections of the drugs targetting either the serotonergic, GABAergic, or glutamatergic system. Subcutaneous injections of either saline or drug were given in 0.1ml/10g body weight Statistical Analysis Results are expressed as means ± SEM and were analyzed by SPSS. The Shapiro-Wilk test of normality was performed on all of the USV data to assess the hypothesis of normal distribution. Due to violations of the assumption of normality and unequal number of mouse pups across

109 92 genotype groups, nonparametric statistics were used for the analysis of maternal separationinduced USVs. A Kruskal-Wallis test assessed differences among groups in the median number of vocalizations produced over the 4-minute trial. The Mann-Whitney test was used to assess differences between the saline and drug condition within a genotype group. In cases where sample sizes were too small to perform a nonparametric Mann-Whitney test, the analogous t-test with Welch correction for small sample sizes was performed. The test of Kruskal-Wallis assessed differences in body weight as a function of genotype as well as litter effect. The Kruskal-Wallis test also assessed the effect of sex in the median number of USVs among sexseparated genotype groups Genotyping and Sexing of PND8 Mice The procedure for genotyping was identical to the one described in Section Results Screening is not Applicable A screening procedure is common in studies of anxiolytics in mouse pups when utilizing the maternal separation-induced USV paradigm, as discussed above. In our study, however, number of USVs emitted during the 30 sec trial did not predict number of USVs emitted during the 4 min test for PND8 mouse pups receiving a saline injection across genotypes (r=0.328, Figure 3.4). Correlations for pups receiving an anxiolytic drug were not included due to the differences in anxiolytic effects on USVs between drugs.

110 93 Figure 3.4. A scatter plot representing correlation between number of USVs emitted during the 30 sec screening period and total number of USVs emitted during the 4 min trial for all pups receiving a saline injection. Although slightly positive, # of USVs during screening did not predict total # of USVs during the 4 min trial (r=0.328). Furthermore, a similar percentage of pups were excluded from all genotype groups when screening was considered (p>0.05, Figure 3.5). Lastly, no significant differences were observed in overall drug effects on USVs when pups that emitted 6 or less USVs during screening were excluded (data not shown). Thus, screening was not taken into account in our data analysis.

111 94 Figure 3.5. Number of pups excluded from analysis after removing pups that produced 6 or less USVs during the 30 sec screening period. Number of pups excluded is expressed as a percentage (%) of the original number of pups prior to screening. No difference in the number of pups excluded after screening is observed between genotype groups (p>0.05 by Kruskal Wallis test). [n1,n2 in the legend shows # of pups prior to exclusion (n1) and after exclusion (n2)] No Within-Cage Order of Testing Effects on USVs The potential influence of within-cage order testing on USVs was also investigated. It is possible that mice may vocalize and use such vocalizations to convey information to each other (Scattoni et al., 2009), such as to warn other mice of what is to come. We compared USVs between the first and the rest of the animals tested from the same cage. The order of testing did not influence the number of USVs emitted by pups during maternal separation (data not shown) Anxiolytic Effects on Maternal Separation-Induced USVs Based on the Neurotransmitter System Targeted and Gtf2i Gene Copy Number. Figure 3.6, 3.7, and 3.8 show mean number of total USVs emitted by PND8 mouse pups receiving a drug injection as a percentage (%) of number of USVs emitted by pups of same genotype receiving a control saline injection. Due to an effect of subcutaneous injection described in Section , the number of USVs emitted by pups receiving a drug will be

112 95 expressed relative to the number of USVs emitted by pups receiving control saline. A value of 100 means there was no difference between drug and saline condition in the total number of USVs emitted in the 4 min trial Gtf2i Gene-Dosage Effect of Serotonergic Targeting Drugs on USVs Escitalopram attenuated the total number of maternal separation-induced USVs (p<0.05, Figure 3.6A). A Gtf2i gene-dosage-dependent effect was revealed whereby escitalopram was most effective in reducing USVs in Gtf2i +/+ pups (2 copies of Gtf2i), less effective in Gtf2i +/dup pups (3 copies of Gtf2i), and ineffective in Gtf2i dup/dup pups (4 copies of Gtf2i), where escitalopram in fact increased USVs. No sex or weight effect was observed (p>0.05). 8-OH-DPAT did not reduce maternal separation-induced USVs in PND8 mouse pups (p>0.05, Figure 3.6B). However, a trend of a Gtf2i gene-dosage dependent effect was observed whereby the drug was most effective at reducing USVs in Gtf2i +/+ pups (2 copies of Gtf2i) and increased USVs instead as Gtf2i copy number increased. No sex or weight effect was observed (p>0.05).

113 A B 96 Figure 3.6. Gtf2i gene-dosage effect of serotonergic targeting drugs on USVs. Bar graphs show mean number of total USVs emitted by pups receving a drug injection expressed as a percentage of mean number of total USVs emitted by pups of same genotype receiving a control saline injection. A) Escitalopram attenuated the number of USVs in PND8 pups (p<0.05 by Kruskal-Wallis test). Escitalopram was more potent at attenuating USVs in wild-type mouse pups(p<0.05 by Mann Whitney test) than those with duplication of Gtf2i (Gtf2i+/+ and Gtf2i+/dup respectively). In mice with homozygous duplication of Gtf2i (Gtf2idup/dup), escitalopram enhanced USVs. B) 8-OH-DPAT did not reduce the number of USVs significantly across genotype groups (p>0.05 by Kruskal Wallis test). 8-OH-DPAT did have a tendency however to attenuateusvs in wild-type mouse pups. With an increase in the copy number of Gtf2i, 8-OH-DPAT had a tendency towards an anxiogenic effect in these pups where it increased number of USVs. {n1,n2 in the x-axis is showing # of pups receiving a drug (n1) and number of pups receiving saline (n2)}. (*p<0.05 by Mann Whitney test when comparing mean number of USVs emitted by pups receiving a drug with those receiving saline within a genotype group Anxiolytic Effects of GABAergic Targeting Drugs on USVs Chlordiazepoxide did not attenuate overall maternal separation-induced USVs across genotypes (p>0.05, Figure 3.7A). When comparing between genotypes, chlordiazepoxide reduced USVs in Gtf2i +/dup pups to ~64% but increased USVs in Gtf2i dup/dup pups to ~151% respectively. Note that although data point for Gtf2i +/+ pups is shown, it is excluded from further discussion due to the small number of 2 pups receiving saline. No sex or weight effect was observed (p>0.05). Allopregnanolone reduced total number of maternal separation-induced USVs across all genotypes (p<0.01). The drug reduced USVs the least in Gtf2i +/dup pups (~47%) but was more

114 97 potent in Gtf2i +/-, Gtf2i +/+, and Gtf2i dup/dup pups (~13%, 8%, and 8% respectively) (Figure 3.7B). No sex or weight effect was observed (p>0.05). A B Figure 3.7. Anxiolytic Effects of GABAergic Targeting Drugs on USVs Bar graphs show mean number of total USVs emitted by pups receving a drug injection expressed as a percentage of mean number of total USVs emitted by pups of same genotype receiving a control saline injection. A) Chlordiazepoxide did not have an effect on the number of USVs across genotypes (p>0.05 by Kruskal Wallis test). Within genotype group, chlordiazepoxide had a tendency to reduce number of USVs in pups with a heterozygous duplication of Gtf2i (Gtf2i+/dup) whereas it increased number of USVs in wild-type pups and those with a homozygous duplication of Gtf2i (Gtf2i+/+ and Gtf2i dup/dup respectively). B) Allopregnanolone attenuated total number of USVs in PND 8 mouse pups of all Gtf2i genotypes (p < 0.01by Kruskal Wallis test). Allopregnanolone was more potent at attenuating USVs in wild-type mouse pups and those with a deletion and homozygous duplication of Gtf2i (Gtf2i +/+, Gtf2i +/del, and Gtf2i dup/dup respectively). {n1,n2 in the x-axis is showing # of pups receiving a drug (n1) and number of pups receiving saline (n2)}. (*p<0.05, **p<0.01 by Mann Whitney test when comparing mean number of USVs emitted by pups receiving a drug with those receiving saline within a genotype group. Note: means the data point was excluded from the discussion due to the small number of pups of that genotype receiving saline) Anxiolytic Effects of Glutamatergic Targeting Drug on USVs MK-801 reduced total number of maternal separation-induced USVs across all genotypes (p<0.01). The drug revealed a Gtf2i gene-dosage dependent effect whereby MK-801 was most effective in attenuating USVs in pups with the fewest Gtf2i gene copy number (Gtf2i +/- ), and least effective in pups with the highest Gtf2i gene copy number (Gtf2i dup/dup ) (Figure 3.8). No sex or weight effect was observed (p>0.05).

115 98 G Figure 3.8. Anxiolytic Effects of GlutamatergicTargeting Drugs on USVs. Bar graphs show mean number of total USVs emitted by pups receving a drug injection expressed as a percentage of mean number of total USVs emitted by pups of same genotype receiving a control saline injection. MK-801 attenuated the total number of USVs in PND8 mouse pups across genotype groups (p<0.01 by Kruskal Wallis test). A Gtf2i gene-dosage dependent effect is observed where MK-801 was most potent at reducing number of USVs in mouse pups with 1 copy of Gtf2i (Gtf2i+/del) and less potent as Gtf2i gene copy number increased (in Gtf2i+/+, Gtf2i+/dup, and Gtf2i dup/dup mice with 2, 3 and 4 copies of Gtf2i respectively). {n1,n2 in the x-axis is showing # of pups receiving a drug (n1) and number of pups receiving saline (n2)}. (*p<0.05, **p<0.01 by Mann Whitney test when comparing mean number of USVs emitted by pups receiving a drug with those receiving saline within a genotype group.

116 Stress, Immediate Early Gene Expression, and Altered Gtf2i Gene Copy Number Introduction Research Aims To dissect changes in early gene expression levels that link Gtf2i to anxiety in individuals with WBS and Dup7q11.23, by studying the Gtf2i mouse models Hypothesis We hypothesized that the molecular basis of the anxiety phenotype in people with WBS and Dup7q11.23 involved changes in c-fos expression, which may explain the anxiety phenotype observed in our mouse models. Changes in c-fos expression can help elucidate gross changes in brain activity. Furthermore, studies have implicated a role for Gtf2i in the activation of the c-fos promoter (Kim et al., 1998). Treatment with anxiolytics also induce changes in brain activity as measured by c-fos expression (Troakes et al., 2009, and Linden et al., 2005). Thus, we hypothesized that differential gene-dosage of Gtf2i and anxiolytics may play a role in modulating c-fos expression levels in the brain and provide a way to elucidate whole brain changes Materials and Methods Contributions: I performed all assays and analysis of brain c-fos expression Animals Animals tested were the same as those described above for maternal separation-induced USVs. Two sets of controls were included for the c-fos data. One set included PND8 pups that underwent the USV trial but did not receive a subcutaneous injection. Following the behavioral test, brain was removed and collected. Saline controls were pups that received a control saline subcutaneous injection and then underwent the maternal separation USV trial. The rest of the pups received a subcutaneous injection of an anxiolytic (either allopregnanolone or MK-801), underwent the USVs trial, and were then sacrificed with brain tissue collected. Gtf2i +/-, Gtf2i +/+,

117 100 Gtf2i +/dup, and Gtf2i dup/dup PND8 pups were tested. Note that allopregnanolone and MK-801 were the two drugs chosen for looking at c-fos brain expression due to their strong anxiolytic effects in reducing maternal-separation induced USVs across genotypes described in sections and Furthermore, allopregnanolone and MK-801 target two different neurotransmitters; GABA and glutamate respectively. As such, studies of c-fos expression following injection of one of the two drugs might indicate a stronger involvement of one neurotransmitter over the other in the expression of c-fos Dissection of Mouse Brain Tissues and RNA Isolation PND8 mice (Gtf2i +/-, Gtf2i +/+, Gtf2i +/dup, and Gtf2i dup/dup ) were sacrificed immediately after the behavioral test, and brain was dissected and cut in half in a sagittal orientation along the midline. One-half of the brain was immediately submerged into TrizolReagent (Sigma-Aldrich) and stored at -80 C for c-fos expression analysis. The other half was flash frozen in liquid nitrogen and stored at -80 C for future protein analysis. Total RNA was extracted following the manufacturer s protocol (TrizolReagant) c-fos Expression Analysis Using Quantitative Real-Time PCR Following extraction, total RNA samples were treated with DNase (TurboDNase). 15μg of RNA was converted to cdna using the Superscript III First Strand Synthesis and random hexamer primers (Invitrogen). cdna samples were diluted 1/100 with sterile water and run in triplicate. Real-time PCR analysis used the Power SYBR Green PCR Master mix (Applied Biosystemts) to detect changes in expression. Different c-fos primers spanning exon-exon junctions were tested before an optimal one spanning exon3 and exon4 was selected: mcfosrt3/4a (Table 3.2).C-fos data was normalized to the housekeeping gene succinate dehydrogenase (SDHA). Absolute quantification was used. Therefore, a value of higher than 1 would indicate an increase in c-fos levels whereas a value less than 1 would indicate a decrease in c-fos levels. Each plate included a No Template Control (water) and serially diluted concentrations of control genomic cdna to generate a standard curve for transcript quantification. Negative controls were also run for each sample to

118 101 ensure that there was no genomic contamination of the samples. Negative controls contained RNA and all of the reagents needed to make cdna except for reverse transcriptase Statistical Analysis Results are expressed as means ± SEM and were analyzed by SPSS. The Shapiro Wilk test of normality was performed to assess the hypothesis of normal distribution on the c-fos data. Due to violations of the assumption of normality and unequal number of mouse pups across groups, nonparametric statistics were used to assess differences. A Kruskal-Wallis test compared c-fos data between mice with altered Gtf2i gene copy and different conditions. The Mann-Whitney test was used to assess differences between two genotype groups within the same condition and between two different conditions with same genotype Results Injection Stress Induces c-fos Expression in a Gtf2i Gene- Dosage Dependent Manner Differences in immediate early gene c-fos expression levels compared to the housekeeping gene Sdha were observed between genotypes and conditions (p<0.05 Figure 3.9). In control mouse pups that underwent the USVs trial but did not receive an injection, a difference was observed between mice with altered Gtf2i genomic copy number (p<0.05, Figure 3.9A). Mouse pups receiving saline showed a Gtf2i gene-dose dependent trend whereby pups with less copies of Gtf2i (Gtf2i +/del ) had lower levels of c-fos expression in the brain and those with more copies (Gtf2i +/dup and Gtf2i dup/dup ) had higher levels of c-fos expression compare to wild-type pups (Gtf2i +/+ ) (p<0.01, Figure 3.9A). This suggests a Gtf2i gene-dosage dependent induction of brain c-fos expression by the subcutaneous injection Effective Inhibition of c-fos Expression by Allopregnanolone but not MK-801 When comparing the two anxiolytics, allopregnanolone and MK-801, pups receiving allopregnanolone showed similar levels and pattern of c-fos expression among genotypes as

119 102 control pups that did not receive an injection. There was no difference in c-fos expression between pups of different Gtf2i genotype receiving allopregnanolone (p>0.05, Figure 3.9A) Pups receiving MK-801 showed similar pattern among genotypes of levels of c-fos expression as pups receiving control saline injection although higher absolute quantities (p<0.01, Figure 3.9A). This could be indicative of a stronger anxiolytic effect of allopregnanolone than MK-801. When comparing within each genotype group, a significant difference in c-fos levels between different treatment groups was observed only for Gtf2i +/dup and Gtf2i dup/dup mice (p<0.05 for both, Figure 3.9B) A