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)
ii
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
GABAA 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.
iii
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 clinician-
scientist 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.
iv
Table of Contents
Table of Contents iv
List of Abbreviations ix
List of Tables xiv
List of Figures xv
Chapter 1: Introduction 1
1.1. Williams-Beuren Syndrome 1
1.1.1. History 1
1.1.2. Clinical Profile 3
1.1.3. Cognitive Profile 6
1.1.4. Behavioural Profile 8
1.2. 7q11.23 Duplication Syndrome 9
1.2.1. History 9
1.2.2. Clinical and Cognitive Profile 11
1.2.3. Behavioral Profile 12
1.2.4. Triplication of Dup7q11.23 13
1.3. The Genetics of 7q11.23 Rearrangements 14
1.3.1. Genetic Basis of Williams-Beuren Syndrome 14
1.3.2. Mutational Mechanism in WBS and Dup7q11.23 15
1.3.3. Inheritance Incidence 18
1.4. Genotype-Phenotype Correlation 19
1.5. Mouse Models 21
1.5.1. Previously Studied Mouse Models 23
1.5.2. Contiguous Gene Deletion Fkbp6 to Gtf2i 24
1.5.3. Gtf2ird1- Knockout Mouse 24
1.6. GTF2I Family of Transcription Factors 25
1.6.1. GTF2I (TFII-I) 26
1.6.1.1. TFII-I in the Nucleus 26
1.6.1.1.1. TFII-I in c-fos Promoter Activity 27
1.6.1.2. TFII-I in the Cytoplasm 28
1.6.1.3. TFII-I Expression in the Brain 29
1.7. Anxiety 30
1.7.1. Introduction to Anxiety Disorders 30
1.7.2. Anxiety Neurocircuitry 31
1.7.3. Pharmacology and Neurotransmitter Hypothesis of Anxiety 33
1.7.3.1. Current Pharmacological Treatment of Anxiety 33
1.7.3.2. Neurotransmitter Hypothesis of Anxiety 33
1.7.3.2.1. Serotonin Hypothesis of Anxiety 33
1.7.3.2.2. GABA and Glutamate Hypothesis of Anxiety 35
1.7.4. Prevelance of Anxiety in WBS and Dup7q11.23 37
1.7.4.1. Current Treatment of Anxiety in WBS and Dup7q11.23 40
1.7.5. Animal Models of Anxiety 40
v
1.7.5.1. Maternal Separation-Induced USVs 41
1.7.5.1.1. USVs, Serotonin, and anxiety 42
1.7.5.1.2. USVs, GABA, and anxiety 42
1.7.5.1.3. USVs, Glutamate, and anxiety 43
1.7.6. Role of Maternal Care on Mouse Pups Behavior 43
1.7.7. Anxiety, HPA axis, and Neuronal Activation 46
1.7.8. Anxiety and Rare Disorders 48
1.8. Conclusions 49
Chapter II: Behavioral Analysis of Mouse Models with Altered Gtf2i Copy Number 50
2.1. Effects of Injection Stress and Gtf2i Pup Genotype on Maternal
Separation-Induced USVs 50
2.1.1. Introduction 50
2.1.1.1. Research Aims 50
2.1.1.2. Maternal Separation-Induced USVs 50
2.1.1.3. Duplication of Gtf2i Results in Separation Anxiety in Mice and Humans 51
2.1.1.4. Hypothesis 53
2.1.2. Materials and Methods 53
2.1.2.1. Generation of Mice with Altered Gtf2i Copy Number 53
2.1.2.2. Animal Housing 55
2.1.2.3. Apparatus and Measurements 57
2.1.2.4. Maternal Separation-Induced USVs Procedure 58
2.1.2.5. Statistical Analysis 59
2.1.2.6. Genotyping and Sexing of PND8 Mice 59
2.1.2.6.1. DNA Extraction from Tails 59
2.1.2.6.2. Genotyping of Gtf2i+/-
Litters 59
2.1.2.6.3. Genotyping of Gtf2i+/dup
Litters 61
2.1.2.6.4. Sexing of PND8 Mice 61
2.1.3. Results 62
2.1.3.1. Subcutaneous Injection Alters USV Production in Mice with Altered
Gtf2i Gene Copy Number 62
2.2. Injection Stress, Plasma Corticosterone, and Altered Gtf2i Gene Copy Number 63
2.2.1. Introduction 63
2.2.1.1. Research Aims 64
2.2.1.2. Hypothesis 64
2.2.2. Materials and Methods 64
2.2.2.1. Animals 64
2.2.2.2. Plasma Collection 65
2.2.2.3. Corticosterone Assay 65
2.2.2.4. Statistical Analysis 66
2.2.3. Results 66
2.2.3.1. Maternal Separation and Subcutaneous Injection Induced Changes in Plasma
Corticosterone Levels in a Gtf2i Gene-Dosage Dependent Manner 66
2.2.3.2. Maternal Separation-Induced USVs Predict Plasma Corticosterone
Concentrations 68
2.3. Effect of Gtf2i Maternal Genotype on Maternal Separation-Induced USVs 70
2.3.1. Introduction 70
2.3.1.1. Research Aims 71
vi
2.3.1.2. Hypothesis 71
2.3.2. Materials and Methods 71
2.3.2.1. Animals 71
2.3.2.2. Apparatus and Measurements 72
2.3.2.3. Maternal Separation-Induced USVs Procedure 72
2.3.2.4. Statistical Analysis 73
2.3.3. Results 73
2.3.3.1. Maternal Genotype Effect on Offspring’s Maternal Separation-Induced
USVs 73
2.4. Discussion and Conclusions 75
2.4.1. Injection Stress Stimulated Changes in Maternal Separation-Induced USVs
in a Gtf2i Gene-Dosage Dependent Manner 75
2.4.2. Maternal separation and Subcutaneous Injection Elevate Plasma
Corticosterone Levels in a Gtf2i Gene-Dosage Dependent Manner 75
2.4.3. Maternal Separation-Induced USVs Predict Plasma Corticosterone
Concentrations 76
2.4.4. Hypothesized Changes in Pain Sensitivity in the Gtf2i Mouse Models 77
2.4.5. Potential Confounding Variables on the Sensitivity of Plasma Corticosterone
Changes 81
2.4.6. 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 86
3.1 Effects of Injection Stress and Gtf2i Pup Genotype on Maternal Separation-Induced
USVs 86
3.1.1 Introduction 86
3.1.1.1 Research Aims 86
3.1.1.2 Hypothesis 86
3.1.1.3 Maternal Separation-Induced USVs 86
3.1.1.4 Pharmacological Compounds with Anxiolytic Properties 87
3.1.1.4.1 Serotonergic Targeting Drugs 87
3.1.1.4.2 GABAergic Targeting Drugs 88
3.1.1.4.3 Glutamatergic Targeting Drug 89
3.1.2 Materials and Methods 90
3.1.2.1 Animals/Housing 90
3.1.2.2 Apparatus and Measurements 90
3.1.2.3 Maternal Separation-Induced USVs Procedure 90
3.1.2.4 Drugs 91
3.1.2.5 Statistical Analysis 91
3.1.2.6 Genotyping and Sexing of PND8 Mice 92
3.1.3 Results 92
3.1.3.1 Screening is not Applicable 92
3.1.3.2 No Within-Cage Order of Testing Effects on USVs 94
3.1.3.3 Anxiolytic Effects on Maternal Separation-Induced USVs Based on the
Neurotransmitter System Targeted and Gtf2i Gene Copy Number 94
3.1.3.3.1 Gtf2i Gene-Dosage Effect of Serotonergic Targeting Drugs on
USVs 95
vii
3.1.3.3.2 Anxiolytic Effects of GABAergic Targeting Drugs on USVs 96
3.1.3.3.3 Anxiolytic Effects of Glutamatergic Targeting Drugs on USVs 97
3.2 Stress, Immediate Early Gene Expression, and Altered Gtf2i Gene Copy Number 99
3.2.1 Introduction 99
3.2.1.1 Research Aims 99
3.2.1.2 Hypothesis 99
3.2.2 Materials and Methods 99
3.2.2.1 Animals 99
3.2.2.2 Dissection of Mouse Brain Tissues and RNA Isolation 100
3.2.2.3 c-fos Expression Analysis Using Quantitative Real-Time PCR 100
3.2.2.4 Statistical Analysis 101
3.2.3 Results 101
3.2.3.1 Injection Stress Induces c-fos Expression in a Gtf2i Gene-Dosage
Dependent Manner 101
3.2.3.2 Effective Inhibition of c-fos Expression by Allopregnanolone but not
MK-801 101
3.3 Discussion and Conclusions 103
3.3.1 Screening of Mice regarding USVs 103
3.3.2 Potency of Serotonergic, GABAergic and Glutamatergic Drugs on USVs 105
3.3.2.1 Strong Anxiolytic Properties of Glutamatergic- and GABAergic Targeting
Drugs 105
3.3.2.2 Inhibitory Role of Gtf2i on Serotonergic Transmission 105
3.3.2.3 Effects of Anxiolytics in Wild-Type PND8 Mice 107
3.3.2.4 A Shared Route for Allopregnanolone and MK-801 107
3.3.2.5 Environmental and Other Factors Influencing USVs 108
3.3.2.6 Stress- and Pain- Induced Elevations in Brain c-fos Expression with a
Change in Gtf2i Gene Copy Number 109
3.3.2.7 Anxiolytics Induced Changes in Brain c-fos Expression with a Change
in Gtf2i Gene Copy Number 111
3.3.2.8 TFII-I Activation of the c-fos Promoter versus Anxiolytic Mediated
Reduction of c-fos Expression 112
Chapter IV: Conclusions and Future Directions 113
4.1 Summary 113
4.1.1 Overview 113
4.1.2 Stronger Anxiolytic Effects of Drugs Targeting the Glutamate and GABA
Systems than Serotonin System 114
4.1.3 Hypothesized Changes in Pain Sensitivity with Change in Gtf2i Gene Copy
Number 114
4.1.4 Stress- and Pain- Induced Elevations of Brain c-fos Expression with a
Change in Gtf2i Gene Copy Number 115
4.1.5 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 116
4.2 Future Directions 117
4.2.1 Assessment of Pain Sensitivity in WBS and Dup7q11.23 117
4.2.2 Additional Measures During and After Maternal Separation-Induced USVs
Test 118
viii
4.2.3 Assessment of c-fos Changes in Specific Brain Regions 119
4.3 Conclusion 120
ix
Abbreviations
5-HT Serotonin
5-HT1A Serotonin Receptor 1A
8-OH-DPAT 8-Hydroxy-N,N-dipropyl-2-Aminotetralin
AchE Acetylcholinesterase
ACTH Adrenocorticotropin Hormone
ADHD Attention Deficit Hyperactivity Disorder
ADIS-P Anxiety Disorders Interview Schedule for DSM-IV-Parent Interview
ANOVA Analysis of Variance
ASD Autism Spectrum Disorder
AVP Arginine Vasopressin Peptide
BLA Basolateral Amygdala
BNST Bed Nucleus of the Stria Terminalis
Bp Base Pairs
Btk Bruton’s Tyrosine Kinase
BZ Benzodiazepine
CA1 Cortical Area 1 of the Hippocampus
CA2 Cortical Area 2 of the Hippocampus
CBCL Child Behavior Checklist
CeM Central Medial Nuclei of Amygdala
x
CNV Copy Number Variation
CORT Corticosterone
CRE Calcium Cycle AMP Response Element
CREB Cyclic AMP Response Element Binding Protein
CRF1 Corticotropin Releasing Factor I Gene
CSF Cerebral Spinal Fluid
D2R D2 Dopamine Receptor
DD Distal Deletion
DNS Down syndrome
D/P Deletion of WBS Syntenic Region on Mouse Chromosome 5
DSM-IV Diagnostic and Statistical Manual of Mental Disorders 4th
edition
Dup7q11.23 Duplication 7q11.23 Syndrome
EGF Epidermal Growth Factor
ELN Elastin Gene
ERK Extracellular Signal-Related Kinase
ES Embryonic Stem
FPS Faces Pain Scale
FISH Fluorescence In-Situ Hybridization
FOXP2 Forkhead Box Protein P2
GABA Gamma Aminobutyric Acid
xi
GAD Generalized Anxiety Disorder
GPCR G Protein Coupled Receptor
GRIN2B Glutamate NMDA Receptor Subtype 2B Gene
GTF2I General Transcription Factor 2I
GTF2IRD1 General Transcription Factor 2I Repeat Domain 1
GTF2IRD2 General Transcription Factor 2I Repeat Domain 2
GWAS Genome Wide Association Study
HLH Helix-Loop-Helix Structure
HMBS Hydroxymethylbilane Synthase
HPA Hypothalamic-Pituitary-Adrenal Axis
ID Intellectual Disability
IIH Idiopathic Infantile Hypercalcemia
Inr Transcription Start Site Initiator Element
LCR Low Copy Repeats
LHPA Limbic-Hypothalamo-Pituitary-Adrenal Axis
LI Language Impairment
LoxP Loxus of X-Over P1
LZ Leucine Zipper
Mb Megabase (Million Base Pair)
MCI Minimal Critical Interval
xii
mGlur2/3 Metabotropic Glutamate Receptors Type 2 and 3
mPFC Medial Prefrontal Cortex
mRNA Messenger RNA
NAHR Non Allelic Homologous Recombination
NCCPC Non-Communicating Children’s Pain Checklist
NMDA N-Methyl-D-Aspartate
OSBD Observational Scale of Behavioral Distress
OCD Obsessive Compulsive Disorder
ODD Oppositional Defiant Disorder
PCR Polymerase Chain Reaction
PD Proximal Deletion
PDGF Platelet Derived Growth Factor
PLC Phospholipase C
PND Post Natal Day
PTSD Post Traumatic Stress Disorder
RNA Ribonucleic Acid
RTK Receptor Tyrosine Kinase
RT-PCR Reverse-Transcription Polymerase Chain Reaction
SAD Separation Anxiety Disorder
SDHA Succinate Dehydrogenase
xiii
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
SERT Serotonin Transporter
SH2 Src Homology 2
SINE Short Interspersed Nuclear Elements
SRE Serum Response Element
SRF Serum Response Factor
SSRI Selective Serotonin Reuptake Inhibitor
SVAS Supravalvular Aortic Stenosis
TFII-I Transcription Factor 2I
TRPC3 Transient Receptor Potential C3
US Unconditioned Stimulus
WBS Williams-Beuren Syndrome
WT Wild Type
xiv
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
xv
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 1.10. Schematic representation of the structure of the four TFII-I isoforms and their
respective amino acids length
Figure 1.11. A schematic representation of an extended “emotional” network hypothesized to be
involved in anxiety responses
Figure 1.12. Schematic representation of a serotonergic synaptic terminal
Figure 1.13. Schematic representation of a GABA synaptic terminal
Figure 1.14. Schematic representation of a glutamate synaptic terminal
Figure 2.1. Test of maternal separation anxiety in mice with altered Gtf2i genomic copy number
xvi
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
xvii
Figure 3.9. Expression of immediate early gene c-fos using RT-PCR
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 gene-
dosage 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
1.1.1 History
Williams-Beuren syndrome (WBS)(OMIM: 194050) is a neurodevelopmental disorder caused by
the hemizygous deletion of 26 genes on chromosome 7q11.23. 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
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).
3
1.1.2 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 multi-
systemic 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).
4
Table 1.1. Clinical features of individuals with WBS (Adapted from Eronen et al., 2002,
Leyfer et al., 2006, Morris, 2010, and Pober, 2010).
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).
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).
1.1.3 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
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).
8
Figure 1.2. Visuospatial construction difficulties in individuals with WBS compare to IQ-
and 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.
1.1.4 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).
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).
1.2 7q11.23 Duplication Syndrome
1.2.1 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
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
11
duplication of the 7q11.23 chromosomal region, is now known as Dup7q11.23 syndrome
(OMIM: 609757).
1.2.2 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
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).
1.2.3 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).
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).
1.2.4 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 3q25.3-29 chromosomal regions have shown that the resulting phenotype is
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
1.3.1 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.
15
1.3.2 Mutational Mechanism in WBS and Dup7q11.23
WBS is caused by the hemizygous deletion of 26 - 28 genes on chromosome 7q11.23. 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
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).
17
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).
1.3.3 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.
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
20
individuals with atypical deletions have been identified and assessed to determine genotype-
phenotype 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, non-
deleted 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
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).
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
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.
1.5.1 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.
24
1.5.2 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).
1.5.3 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,
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.
26
1.6.1 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 1.10. 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
1.6.1.1 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
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).
1.6.1.1.1 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 cGMP-
dependent 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
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 signal-
related 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).
1.6.1.2 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
Ca2+
influx (reviewed in Caraveo et al., 2006). PLC- γ activation increases Ca2+
influx through
both the triggering of calcium release from intracellular stores, as well increase in cell surface
expression of transient receptor potential C3 (TRPC3) Ca2+
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 Ca2+
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 Ca2+
influx (Rossum et al., 2005). PLC-γ contains multiple domains, including the PH
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 Ca2+
influx observed after reduction of
TFII-I levels. Similarly, TFII-I overexpression reduced the Ca2+
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 Ca2+
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 Ca2+
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).
1.6.1.3 TFII-I Expression in the Brain
In the adult mouse brain, Gtf2i mRNA is expressed in specific brain regions (Allen Brain Atlas;
http://mouse.brain-map.org/static/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
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
1.7.1 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
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.
1.7.2 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
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 1.11. 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.
33
1.7.3 Pharmacology and Neurotransmitter Hypothesis of Anxiety
1.7.3.1 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.
1.7.3.2 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).
1.7.3.2.1 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
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 side-
effect 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 1.12. Schematic representation of a serotonergic synaptic terminal. Sert, the
serotonin (5-HT) transporter, reuptakes serotonin from the synapse and ultimately reduces
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).
1.7.3.2.2 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.
36
Figure 1.13. 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).
37
Figure 1.14. 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).
1.7.4 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 IQ-
matched 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
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
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: 4.00 – 12.99 years. aFrom
Shaffer et
al., 1996. bFrom Dekker and Koot, 2003,
cFrom Kessler et al., 2005, adjusted for the age
distribution of the Dup7 sample. dPrevalence not reported. NA: Not assessed by ADIS-IV.
40
1.7.4.1 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.
1.7.5 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).
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.
1.7.5.1 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 50-120 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
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).
1.7.5.1.1 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 seven-
day 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).
1.7.5.1.2 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
43
as allopregnanolone, were shown to dose-dependently reduce maternal separation-induced USVs
in seven-day old mouse pups (Fish et al., 2000).
1.7.5.1.3 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 post-
natal day (PND) 7 mice further implicating glutamatergic drugs as effective anxiety treatments.
1.7.6 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).
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
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 anxiety-
related 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
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 long-
term 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.
1.7.7 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 pre-
exposure 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.
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 over-
activation 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
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.
1.7.8 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.
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 Dup7q11.23. Individuals with WBS are described as hypersocial and are diagnosed non-
social 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.
1.8. 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.
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
2.1.1 Introduction
2.1.1.1 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 Dup7q11.23. 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 Gtf2idup/dup
, respectively).
2.1.1.2 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).
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 3.1.3.1, results on whether screening is applicable to
our study will be included.
2.1.1.3 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 11/2 -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.
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 Gtf2idup/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, Gtf2idup/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<0.0001 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: 4.07-12.96 years (Mervis et al., 2012).
53
Table 2.2. Separation difficulties in children with WBS and Dup7q11.23 compared to the
general population. Child Behavior Checklist for Ages 1.5–5 (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: 2.03-5.81 years (Mervis et al., 2012).
2.1.1.4 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
Dup7q11.23. 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.
2.1.2 Materials and Methods
Contributions: I performed all behavioral tests, genotyping and sexing of test animals, and
statistical analysis.
2.1.2.1 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 (Gtf2idup/dup
) (Mervis 2012). Gtf2ird1Gt(XS0608)Wtsi
mice
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 Gtf2i3’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
mice. To generate the intra-chromosomal duplication of Gtf2i, Gtf2ird1Gt (XS0608)
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
Gtf2ird1Gt(XS0608)Wts
and Gtf2i3’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 Gtf2iGt(YTA365)Byg/+
mice, referred to in this thesis as Gtf2i
+/del
(Figure 2.2B).
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).
2.1.2.2 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 light-
controlled 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
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).
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 wild-
type 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 8.
2.1.2.3 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 (20-125
kHz) were generated by discrete Fourier transform (256 bins) and analyzed with Avisoft
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.
2.1.2.4 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.
59
2.1.2.5 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 separation-
induced 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.
2.1.2.6 Genotyping and Sexing of PND8 Mice
2.1.2.6.1 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.
2.1.2.6.2 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
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 Tris-
acetate, 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.
61
2.1.2.6.3 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.
2.1.2.6.4 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.
62
2.1.3 Results
2.1.3.1 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
(Gtf2idup/dup
) emitted the most (Mervis et al., 2012, Figure 2.4). In pups that received a
subcutaneous injection of saline 30-45 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 (Gtf2idup/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.
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 30-45 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
2.2.1 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
**
*
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.
2.2.1.1 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.
2.2.1.2 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.
2.2.2 Materials and Methods
Contributions: I performed all assays and analysis of plasma corticosterone concentrations.
2.2.2.1 Animals
Animals tested were the same as those described in Section 2.1.2.2 for maternal separation-
induced USVs. Wild-type PND8 pups (Gtf2i+/+
) and those with a duplication of Gtf2i (Gtf2i+/dup
and Gtf2idup/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
65
collected for plasma corticosterone measurements. Gtf2i+/+
, Gtf2i+/dup
, and Gtf2idup/dup
PND8
pups were tested.
2.2.2.2 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 30-60 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).
2.2.2.3 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.
66
2.2.2.4 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.
2.2.3 Results
2.2.3.1 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
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.
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 separation-
induced USVs trial only. Saline controls were PND8 pups that underwent the maternal
separation-induced USVs trial 30-45 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).
2.2.3.2 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
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
**
*
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 2.1.3.
2.3 Effect of Gtf2i Maternal Genotype on Maternal Separation-
Induced USVs
2.3.1 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
B
71
study potential Gtf2i maternal genotype effect in the anxiety phenotype observed in PND8
mouse pups with altered Gtf2i gene copy number.
2.3.1.1 Research Aims
To investigate the effect of maternal Gtf2i genotype on anxiety phenotypes in the Gtf2i-
duplication mouse model.
2.3.1.2 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.
2.3.2 Materials and Methods
Contributions: I performed all behavioral tests, genotyping and sexing of test animals, and
statistical analysis.
2.3.2.1 Animals
Animals tested were the same as those described in Section 2.1.2.2 for maternal separation-
induced 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 Gtf2idup/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).
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 separation-
induced 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.
2.3.2.2 Apparatus and Measurements
The testing apparatus was the same as described above for the maternal separation-induced
USVs trial in Section 2.1.2.3. Data were analyzed blind to genotype and parental crossing. Mean
number of USVs were calculated in minute bins.
2.3.2.3 Maternal Separation-Induced USVs Procedure
The procedure was identical to the one described above in Section 2.1.2.4 with the exception that
these pups did not receive a subcutaneous injection prior to the USVs trial.
73
2.3.2.4 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.
2.3.3 Results
2.3.3.1 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).
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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.
A
B
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
2.4.1 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 separation-
induced 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 Gtf2idup/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 30-45 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.
2.4.2 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
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 30-45 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 30-45 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.
2.4.3 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
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).
2.4.4 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
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 30-45 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 30-45 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 Gtf2idup/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 Gtf2idup/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
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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 Dup7q11.23.
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
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 Dup7q11.23. 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
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.
2.4.5 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.
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
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).
2.4.6 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.
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
85
difference between male and female pups is only observed for wild-type pups raised by wild-
type 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 separation-
induced 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.
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
3.1.1 Introduction
3.1.1.1 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.
3.1.1.2 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 Dup7q11.23.
3.1.1.3 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
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).
3.1.1.4 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.
3.1.1.4.1 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).
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).
3.1.1.4.2 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.
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).
3.1.1.4.3 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.
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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).
3.1.2 Materials and Methods
Contributions: I performed all behavioral tests, genotyping and sexing of test animals, and
statistical analysis.
3.1.2.1 Animals/Housing
Animals were the same as those described in Section 2.1.2.2.
3.1.2.2 Apparatus and Measurements
The testing apparatus was the same as described in Section 2.1.2.3.
3.1.2.3 Maternal Separation-Induced USVs Procedure
The procedure was identical to the one described in Section 2.1.2.4.
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.
91
3.1.2.4 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 H2O 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 30-45 minutes prior to the
separation test.
Escitalopram, 8-OH-DPAT, and chlordiazepoxide were tested in Gtf2i+/+
, Gtf2i+/dup
, and
Gtf2idup/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.
3.1.2.5 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
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genotype groups, nonparametric statistics were used for the analysis of maternal separation-
induced 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 sex-
separated genotype groups.
3.1.2.6 Genotyping and Sexing of PND8 Mice
The procedure for genotyping was identical to the one described in Section 2.1.2.6.
3.1.3 Results
3.1.3.1 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.
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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.
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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)].
3.1.3.2 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).
3.1.3.3 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 2.1.3.1, the number of USVs emitted by pups receiving a drug will be
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.
3.1.3.3.1 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 Gtf2idup/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).
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.
3.1.3.3.2 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 Gtf2idup/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
A B
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potent in Gtf2i+/-
, Gtf2i+/+
, and Gtf2idup/dup
pups (~13%, 8%, and 8% respectively) (Figure 3.7B).
No sex or weight effect was observed (p>0.05).
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).
3.1.3.3.3 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 (Gtf2idup/dup
) (Figure 3.8). No
sex or weight effect was observed (p>0.05).
B A
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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.
G
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3.2 Stress, Immediate Early Gene Expression, and Altered Gtf2i
Gene Copy Number
3.2.1 Introduction
3.2.1.1 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.
3.2.1.2 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.
3.2.2 Materials and Methods
Contributions: I performed all assays and analysis of brain c-fos expression.
3.2.2.1 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+/+
,
100
Gtf2i+/dup
, and Gtf2idup/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 3.1.3.3.2 and
3.1.3.3.3. 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.
3.2.2.2 Dissection of Mouse Brain Tissues and RNA Isolation
PND8 mice (Gtf2i+/-
, Gtf2i+/+
, Gtf2i+/dup
, and Gtf2idup/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).
3.2.2.3 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
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.
3.2.2.4 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.
3.2.3 Results
3.2.3.1 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.
3.2.3.2 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
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 Gtf2idup/dup
mice (p<0.05 for both,
Figure 3.9B)
A
103
Figure 3.9. Expression of immediate early gene c-fos using RT-PCR. mRNA was extracted
from whole brains of PND8 mic. Expression values are shown relative to the housekeeping gene
Sdha. Statistically significant differences in expression were deteced between genotypes and
experiemental conditions (p<0.001 by Kruskal Wallis test). Control PND8 mouse pups
underwent the 4 min maternal-separation induced USVs trial but did not receive a subcutaneous
injection. The other 3 groups, saline, allopregnanolone and MK-801, received a subcutanous
injection (control saline or drug) 30-45 min prior to the USVs 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, 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 group). (*p<0.05, **p<0.01 by Kruskal Wallis test).
3.3 Discussion and Conclusions
3.3.1 Screening of Mice regarding USVs
Screening has often been included in previous work with mouse pup USVs in order to exclude
pups that emit few USVs in the first place from analysis (Fish et al., 2000, 2003, Takahashi et al.,
2009, and Scattoni et al., 2009). Such studies examine the efficacy of potential anxiolytic
treatments and are therefore only interested in the overall change in USVs due to anxiolytic
treatment but not in the baseline levels of USVs. Therefore, pups that emit 6 or less USVs during
an initial screening procedure are excluded from future tests. Furthermore, when testing the
effects of anxiolytics on maternal separation-induced USVs, wild-type mouse pups are often
B
104
used and potential genotype effects on USVs are not relevant (Fish et al., 2000, 2003, Takahashi
et al., 2009).
However, such screening was not usable in our study. Firstly, correlations between the number
of USVs emitted during screening and the number of USVs emitted during the 4-minute trial in
control saline pups showed that USVs emitted during screening did not predict USVs emitted
during the maternal separation test. Secondly, we tested pups of different Gtf2i gene copy
number, and as such, we might have excluded pups showing a particular phenotype if we had
carried out the screening process. To assess this, the percentage of pups excluded after screening
was taken into account and was found to be similar across different genotype groups as described
in the Results section (~ 30%). Furthermore, this percentage of pups that was excluded is
comparable to previous work (Fish et al., 2000, 2003, and Takahashi et al., 2009). Lastly, overall
trends and significance of the effects of anxiolytics on USVs did not change when comparing
results before and after screening was carried out. Therefore, we concluded that screening was
not applicable to this study.
An implicit assumption is made that anxiety can be investigated in an unselected general
population. However, although a random sample of the human population will have varying
levels of anxiety, only a certain number will be diagnosed with anxiety. Those who are
diagnosed usually present with symptoms of anxiety that cause distress and disruption of
everyday life. Whether the anxiety that these individuals are diagnosed with differs
quantitatively and/or qualitatively from anxiety in the general population is still unclear.
Furthermore, the therapeutic effects of an anxiolytic are often not noticed in the control subjects,
but only evident in clinically anxious populations (reviewed Lister et al., 1990). Therefore, a
dilemma arises on whether all subjects are suitable for a study on anxiety.
105
3.3.2 Potency of Serotonergic, GABAergic and Glutamatergic Drugs on
USVs
3.3.2.1 Strong Anxiolytic Properties of Glutamatergic- and GABAergic
Targeting Drugs
In this study, we showed that the glutamatergic- targeting drug, MK-801, and the GABAergic-
targeting drug, allopregnanolone, are more effective than the more widely used serotonergic-
targeting drugs in attenuating separation anxiety-like behavior in mice as measured by the
maternal separation-induced USV test. This attenuation was observed across pups of all Gtf2i
genotypes where the drugs reduced USVs to anywhere between 2%-50% of the USVs emitted by
pups of same genotype receiving control saline. Our findings emphasize the importance of drugs
targeting the glutamate and GABA neurotransmitter systems in anxiety disorders and their
potential for becoming the target of future therapeutic interventions for anxiety disorders in
general or separation anxiety specifically as we address in this study.
Commonalities between anxiety disorders include excessive or inappropriate brain excitability
within specific brain circuits (reviewed in Swanson et al., 2005). Since glutamate is the major
excitatory neurotransmitter in the mammalian brain, it is reasonable that new approaches for
anxiety treatment might aim to modulate glutamatergic functions. However, despite this
expectation, the contribution of glutamatergic neurotransmission in anxiety disorders is a
relatively unexplored field due to the lack of clearly safe approaches to modulate glutamate
neurotransmission.
3.3.2.2 Inhibitory Role of Gtf2i on Serotonergic Transmission
The two serotonergic targeting drugs, escitalopram and 8-OH-DPAT, had similar effects on
maternal separation-induced USVs, although escitalopram was more potent overall at reducing
USVs, and 8-OH-DPAT had a tendency to attenuate USVs. Both drugs exhibited a Gtf2i gene-
dosage dependent effect whereby the drugs were most effective at reducing USVs in wild-type
PND8 mouse pups (Gtf2i+/+
) and less effective as Gtf2i gene copy number increased. In mice
with 4 copies of Gtf2i (Gtf2idup/dup
), both serotonergic targeting drugs exerted an anxiogenic
106
effect whereby they increased maternal separation-induced USVs. Therefore, PND8 mice with a
duplication of Gtf2i, appear to be resistant to the anxiolytic effects of drugs targeting the
serotonin neurotransmitter system.
One potential hypothesis for the effect seen in mice with a duplication of Gtf2i is an attenuation
of serotonergic neurotransmission in the Gtf2i duplication mouse model. Gtf2i, together with
Gtf2ird1, code for members of the TFII-I family (reviewed in Roy et al., 2005). All three
proteins contain the same structural elements: DNA-binding I-repeat domains, a helix-loop-
helix-like structure, a putative leucine zipper, and a nuclear localization signal (Hinsley et al.,
2004). Although Gtf2i is the most studied of the three members, the role of these proteins as
transcription factors and their similar, almost identical, structure makes it likely for some overlap
in the functions of these proteins. Studies of mouse models with a targeted knockout of Gtf2ird1
have suggested a potential function of Gtf2ird1 in serotonergic neurotransmission. Gtf2ird1
knockout mice exhibit decreased fear in the cued fear conditioning test (Young et al., 2008).
These phenotypes were correlated with increased levels of the 5-HT metabolite in specific brain
regions, such as the amygdala and the prefrontal cortex, as well as enhanced inhibitory 5-HT
currents mediated by the 5-HT1A receptor in layer V pyramidal neurons of the prefrontal cortex
(Young et al., 2008, and Proulx et al., 2010). These results suggest enhanced serotonergic
transmission in Gtf2ird1+/-
mice and therefore a potential inhibitory function of Gtf2ird1 on
serotonergic neurotransmission.
Extending these findings, it can be hypothesized that if Gtf2ird1 is duplicated, reduced
serotonergic neurotransmission would occur, assuming that serotonergic transmission is
dependent on Gtf2ird1 control in a dose-dependent manner. To our knowledge, studies of mice
with Gtf2ird1 duplication have not been carried out. One can hypothesize that reduced
serotonergic neurotransmission might also be demonstrated in mice with a duplication of Gtf2i as
a consequence of the overlap in structure and potential functions of Gtf2i and Gtf2ird1. If that
were the case, it would explain why the two serotonergic targeting drugs, escitalopram and 8-
OH-DPAT, had a tendency to increase the number of USVs in mice with a homozygous
duplication of Gtf2i (Gtf2idup/dup
). Since low levels of serotonergic transmission would be present
in mice with a homozygous duplication of Gtf2i, it is possible that the serotonergic targeting
107
drugs are ineffective at overcoming this low state of serotonergic transmission. Future studies
should therefore assess 5-HT metabolite levels and 5-HT currents in the Gtf2i duplication mouse
model to test the hypothesis of an inhibitory role of Gtf2i on serotonergic transmission.
3.3.2.3 Effects of Anxiolytics in Wild-Type PND8 Mice
Overall, all anxiolytics reduced maternal separation induced USVs in wild-type PND8 mouse
pups (Gtf2i+/+
). Stronger anxiolytic effects of both drugs targeting serotonin neurotransmitter,
escitalopram and 8-OH-DPAT specifically, were previously reported in maternal-separation
induced USVs in wild-typePND7 mouse pups (Fish et al., 2004 and Fish et al., 2000).
On the other hand, the strong anxiolytic effects of allopregnanolone, a GABA-ergic targeting
drug, reported in this thesis parallel previous studies with PND7 mouse pups (Fish et al., 2000).
Similar results to previous work were also obtained with the less effective GABA-ergic targeting
drug, chlordiazepoxide, tested using the maternal-separation induced USVs paradigm in PND7
mouse pups (Takahashi et al., 2009). Lastly, even stronger than previously reported anxiolytic
effects on USVs were observed with MK-801 (Takahashi et al., 2009). Comparing between
drugs in this study, the glutamatergic targeting drugs were most effective, followed by
GABAergic and then serotonergic targeting drugs. Although SSRIs, which are serotonergic
targeting drugs, are considered the first line of treatment for anxiety disorders, they have also
been associated with a wide range of side effects (Hidalgo et al., 2000, Ravindran et al., 2010,
Dording et al., 2002 and Mohler, 2012). Considering that maternal separation-induced USVs
have been used as one of the ethologically validated measures for pre-clinical characterization of
anxiolytic drugs (Hodgson, 2008, and Sanchez, 2003), our findings of lower effectiveness of
SSRIs in wild-type mouse pups, indicate that studies of anxiolytics need to extend beyond SSRIs
and the serotonergic system.
3.3.2.4 A Shared Route for Allopregnanolone and MK-801
Allopregnanolone, a GABAergic targeting drug, MK-801, a glutamatergic targeting drug,
produced similar effects in USVs with a change in Gtf2i gene copy number. The similar effect of
these two drugs targeting different neurotransmitter systems suggests that glutamate, a major
excitatory neurotransmitter, and GABA, a major inhibitory neurotransmitter, work in
combination to maintain a “healthy balance” of excitation and inhibition in the brain. One of the
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main hypotheses of anxiety has suggested an imbalance between GABAergic-mediated
inhibition and glutamatergic-mediated excitation in the brain. Specifically, hyperexcitability of
the circuit, whether due to reduced GABAergic inhibition and/or increased glutamatergic
excitation, may give rise to anxiety-like behaviors (Wieronska et al., 2011, Fish et al., 2000, and
Takahashi et al., 2009). Furthermore, an overlap in GABA and glutamate neurons is present in
the amygdala and other neuroanatomical areas that are highly implicated in anxiety (reviewed in
Shekhar et al., 2005). Therefore, modulation of either the GABAergic mediated inhibition via
allopregnanolone, or the glutamatergic mediated excitation via MK-801, can prove therapeutic in
anxiety by reducing the hyperexcitability of the proposed anxiety neurocircuitry. Such drugs may
be targeting different neuron populations within the same or different brain areas implicated in
anxiety to produce anxiolytic effects.
3.3.2.5 Environmental and Other Factors Influencing USVs
Although maternal separation-induced vocalizations are ethologically validated measures for
preclinical characterization of anxiolytic drugs, potentially confounding variables should be
considered. Maternal separation-induced USVs are a measure of anxiety that is highly sensitive
to the age of the pups. Mouse pup USVs emitted following maternal separation display a clear
ontogenic profile, peaking approximately eight days after birth, and decreasing thereafter
(Branchi et al., 2001). This demands the use of a between-subjects experimental design, and
thus, littermates were used as controls for the number of USVs emitted by PND8 mouse pups.
Hypothermia may represent a confounding variable in the interpretation of the USVs since low
body temperature can elicit USV emission (Hofer et al., 1993). However, rodents are known to
primarily use USVs during thermoregulation in extreme hypothermia (Sokoloff and Blumberg,
1997 and Hofer et al., 1993). Furthermore, one would expect an increase in the number of USVs
as the pup gets colder over the duration of the test. This was however not noted with USVs
produced by our pups. Therefore, it is highly unlikely that hypothermia is reached in a 4-minute
trial of maternal separation and as such, we believe that temperature does not play a role in USVs
emitted by our mice.
Maternal deprivation, as defined by an extended period of separation from the dam, affects
development and later-life behaviour in mouse pups (Horii-Hayashi et al., 2012). However,
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maternal separation in our study lasted only for 4 minutes. The maternal separation trial used in
this study is long enough to assess anxiety using a stress-inducing paradigm but not long enough
to be considered maternal deprivation, which avoids other unaccounted for effects. Furthermore,
the pups were sacrificed immediately after the behavioral test. Therefore, the differences that
were observed are likely due to genotype and/or treatment and not to the testing paradigm.
Alterations in USV patterns in a gene-dose-dependent manner have previously been shown in
mice with a mutation in the reelin gene, which is involved in the plasticity of dendritic spines and
synaptic transmission. Null mutant mice and heterozygous mice were generated, and both
produced fewer USVs than WT controls, but heterozygous mice produced an intermediate level
of USVs. However, the decrease in USVs was also associated with other factors, such as
decreases in body weight, and was reversed by epigenetic factors (Laviola et al., 2006). In our
study, environmental factors such as temperature, noise, and smell were controlled for. In
addition, there was no effect of either body weight or litter on the number of USVs. Therefore,
the Gtf2i gene-dosage-dependent alterations in the number of USVs that we reported in our
previous work (Mervis et al., 2012), and the Gtf2i gene-dosage-dependent changes in USVs
following injection of either escitalopram, 8-OH-DPAT, or MK-801, are likely dependent on the
Gtf2i gene copy number and the drug and not on environmental and/or other confounding
factors.
3.3.2.6 Stress- and Pain- Induced Elevations in Brain c-fos Expression with
a Change in Gtf2i Gene Copy Number
c-fos is an immediate early gene that has been widely used to map neuronal activation. This gene
is responsive to many stimuli including stress induced by maternal separation (Shin et al., 2010).
The expression level of c-fos in mice with different Gtf2i gene copy number was altered
following the maternal separation-induced USVs test (p<0.05). An increase in c-fos expression,
normalized to housekeeping gene, Sdha, was observed in wild-type mice and those with a
heterozygous and homozygous duplication of Gtf2i (Gtf2i +/+
, Gtf2i +/dup
, and Gtf2i dup/dup
respectively) but not in mice with a deletion of Gtf2i (Gtf2i +/del
). This may be due to the
difference in separation anxiety between these groups. Gtf2i +/del
mice show a tendency towards
reduced separation anxiety, as shown by the lower number of maternal separation-induced USVs
110
emitted (previously reported in Mervis et al., 2012). As Gtf2i gene copy number increases, the
total number of USVs also increases, which suggests elevated separation anxiety. Since c-fos is
responsive to extracellular signals such as stress induced by maternal separation, it is expected
that higher c-fos levels would be present in mice that emit an increased number of USVs because
they perceive the maternal separation as more stressful. As a corollary, mice that emit a
decreased number of USVs, such as the Gtf2i +/del
mice, would have lower levels of c-fos
expression, which is supported by our findings. Although a Gtf2i gene-dosage dependent effect
on USVs was observed (previously reported in Mervis et al., 2012), a clear Gtf2i gene-dosage
effect on brain c-fos expression levels is not evident in pups that only underwent the USVs trial.
Perhaps Gtf2i +/+
, Gtf2i +/dup
, and Gtf2i dup/dup
mice show similar levels of c-fos, albeit higher than
Gtf2i +/del
mice because additional copies of Gtf2i do not provide any added benefits in terms of
maternal separation-induced c-fos activation.
In comparison to control mice that did not receive an injection, mice that received a saline
injection prior to the maternal separation test exhibited a different pattern of c-fos expression as
Gtf2i gene copy number increased. Gtf2i +/del
mice showed similar quantities of c-fos expression
as mice of same genotype that did not receive an injection. However, Gtf2i +/+
, Gtf2i +/dup
, and
Gtf2i dup/dup
mice show higher levels of c-fos expression than mice of same genotype that did not
receive an injection. Furthermore, a difference between these three groups was observed as Gtf2i
gene copy number changes. In mice with a heterozygous and homozygous duplication of Gtf2i
(Gtf2i +/dup
and Gtf2i dup/dup
respectively), higher expression levels were found in comparison to
wild-type mice (Gtf2i +/+
). This may be due to the pain associated with the subcutaneous
injection and differences in pain sensitivity in our Gtf2i mouse models. c-fos expression can be
induced by a number of stimuli, which include stress associated with maternal separation as well
as noxious stimuli (Shin et al., 2010). Pain has been previously shown to induce c-fos
expression, such as in the spinal cord neurons following mechanical or thermal noxious
stimulation (Wang et al., 2012). Therefore, both the maternal separation paradigm and the pain
associated with the injection induce an increase in brain c-fos expression. However, because of
the hypothesized changes in pain sensitivity with a change in Gtf2i gene copy number, we expect
pain to induce c-fos expression at a different extent depending on Gtf2i copy number. For
example, since higher pain sensitivity is believed to characterize Gtf2i +/del
mice, the pain due to
111
the injection is expected to induce c-fos expression. On the other hand, reduced pain sensitivity
may be a phenotype of mice with a duplication of Gtf2i (i.e. Gtf2i +/dup
and Gtf2i dup/dup
) and
therefore it is expected that the injection induces c-fos expression minimally, if at all. This
however is the opposite of what we report here. One potential explanation comes from
considering the functions of TFII-I. TFII-I, upon phosphorylation by kinases, will promote c-fos
activation together with other critical promoter-binding proteins by binding to and activating the
c-fos promoter (Kim et al., 1998). Therefore, heightened TFII-I activity in mice with increased
Gtf2i copy number could induce c-fos expression, counteracting the pain induced expression of
c-fos in the brain. Therefore, results of mice receiving a saline injection prior to the maternal
separation-induced USVs trial need to undergo several considerations when examining changes
in c-fos expression corresponding to changes in Gtf2i gene copy number: 1) the stress inducing
maternal separation trial 2) the pain associated with the subcutaneous injection and 3) TFII-I
activation of the c-fos promoter.
3.3.2.7 Anxiolytics Induced Changes in Brain c-fos Expression with a
Change in Gtf2i Gene Copy Number
Anxiolytics have been shown to reduce c-fos activity that was initially brought on by a stress-
inducing paradigm (Troakes et al., 2009). However, increased levels of c-fos expression
following anxiolytic delivery have also been reported in specific brain regions such as the
amygdala (Troakes et al., 2009). When looking at the effects of the two anxiolytics,
allopregnanolone and MK-801, on c-fos expression, different results are observed.
Allopregnanolone reduced c-fos expression to similar levels as seen in control mice that did not
receive an injection. Furthermore, the pattern of c-fos expression levels between mice with
altered Gtf2i gene copy number was comparable to the pattern seen in control mice that did not
receive an injection. These findings suggest that allopregnanolone may be an effective anxiolytic
in the Gtf2i mouse models. In contrast, MK-801 slightly increased c-fos levels across all
genotypes. However, when comparing relative c-fos expression between mice of different Gtf2i
genotypes, a similar pattern of expression was observed in mice that received a saline injection.
This suggests that MK-801 is an ineffective anxiolytic with respect to its effects on brain c-fos
expression. The efficacy of allopregnanolone, as compared to MK-801, may be due to the
potency of allopregnanolone, a GABA-A positive allosteric modulator (Reddy, 2004), to
112
increase inhibitory GABAergic transmission in the brain. This may reduce the hyperexcitability
assumed to be present in an anxious mouse, and as a result increase c-fos expression . On the
other hand, MK-801 is an mGluR2/3 receptor antagonist and reduces the excitatory
glutamatergic transmission in the brain, which leads to an overall reduction in brain
hyperexcitability (Takahashi et al., 2009). Our findings of increased c-fos expression levels
suggest that MK-801 was not sufficiently potent as an anxiolytic to reduce this hyperexcitability.
3.3.2.8 TFII-I Activation of the c-fos Promoter versus Anxiolytic Mediated
Reduction of C-fos Expression.
TFII-I plays a role in activating the c-fos promoter (Kim et al., 1998). TFII-I, upon
phosphorylation by kinases, will promote c-fos activation together with other critical promoter-
binding proteins (Kim et al., 1998). Therefore, a higher number of Gtf2i gene copies should lead
to higher c-fos activation. In control mice that did not receive a subcutaneous injection but
underwent the maternal separation trial, lower levels of c-fos expression were found in mice with
one copy of Gtf2i (Gtf2i +/del
) compared to mice with increased Gtf2i gene-dosage. However, an
increase in Gtf2i copy number (i.e. Gtf2i +/+
, Gtf2i +/dup
, and Gtf2i dup/dup
respectively) did not
elevate c-fos expression levels. We therefore hypothesize that additional Gtf2i copies do not
further increase activation of the c-fos promoter when pups are only exposed to the maternal
separation-induced USV paradigm. However, in mice that received a saline injection prior to the
maternal separation trial, a difference in c-fos expression levels was observed in all of the Gtf2i
mice (Gtf2i +/del
, Gtf2i +/dup
, and Gtf2i dup/dup
) compared to wild-type pups with two copies of
Gtf2i (Gtf2i +/+
). Furthermore, all of the mice that received a saline injection showed higher
levels of c-fos expression than those that did not. Therefore, the addition of another stimulus, the
subcutaneous injection, to the maternal separation trial led to an increased activation of the c-fos
promoter. This additional injection stimulus also exposes the potential Gtf2i gene-dosage-
dependent effect on c-fos expression. This may be due to the pain associated with the
subcutaneous injection, which might be a stronger stimulus than the stress associated with
maternal separation in promoting Gtf2i activation of the c-fos promoter. In conclusion, two
opposing forces may be present in controlling c-fos expression levels. Anxiolytics tend to reduce
c-fos activity, but TFII-I provides an opposing effect by promoting c-fos activity. The resulting
c-fos expression level may be determined by the factor that succeeds in overshadowing the other.
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Chapter 4 Conclusions and Future Directions
4.1 Summary
4.1.1 Overview
Deletion of a 1.5 million base pair segment of human chromosome 7q11.23 causes Williams-
Beuren syndrome (WBS), a neurodevelopmental disorder characterized by intellectual disability,
deficits in visuospatial construction, relative strength in concrete vocabulary, lack of stranger
anxiety, social disinhibition, and non-social anxiety (Mervis et al., 2010). Duplication of this
region results in a contrasting syndrome, Dup7q11.23, associated with speech delay and/or
language impairment and separation anxiety (Somerville et al., 2005, Mervis and Velleman,
2012). Deletion and duplications of the 7q11.23 region are both associated with anxiety. This
suggests that a gene (or genes) within the region is responsible for the anxiety phenotype in a
dose dependent manner (Osborne, 2007). Gtf2i is one of 26 genes commonly deleted in WBS
and duplicated in Dup7q11.23. Studies of individuals with atypical deletions have implicated
Gtf2i in the neurocognitive profile of WBS (Antonell et al., 2010) and we previously linked
duplication of Gtf2i to separation anxiety in both mice and humans (Mervis et al., 2012).
Behavioral and molecular studies of mice with altered Gtf2i gene copy number were undertaken
to assess the effectiveness of drugs acting on the glutamatergic, GABAergic, and serotonergic
systems. Drugs acting on the glutamate receptors were most effective at attenuating USVs across
genotypes, compared to GABAergic and serotonergic modulators. Furthermore, an effect of
subcutaneous injection in a Gtf2i dose dependent way was reported on both USVs and plasma
corticosterone levels. Brain c-fos expression immediately after maternal separation was reduced
by modulators of GABA but not glutamate neurotransmission. Collectively, these results
highlight the important role of the glutamatergic and GABAergic systems in anxiety phenotypes
in the genomic disorders involving copy number variation of GTF2I. A potential difference in
pain sensitivity based on Gtf2i copy number was hypothesized based on the effect of
subcutaneous injections on USV production and plasma corticosterone levels, but needs to be
further assessed, as discussed below.
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4.1.2 Stronger Anxiolytic Effects of Drugs Targeting the Glutamate and
GABA Systems than Serotonin System
Drugs targeting the glutamate neurotransmitter system, MK-801, and the GABA
neurotransmitter, allopregnanolone, were most effective at attenuating total number of USVs
emitted during maternal separation across all genotypes. MK-801 showed a Gtf2i gene-dosage
dependent effect whereby it was most effective in attenuating USVs in Gtf2i+/-
pups, and less
effective as Gtf2i gene copy number increased. Mixed results were observed with drugs targeting
the GABA system whereby allopregnanolone was effective at reducing USVs across all
genotypes, but chlordiazepoxide was not. Serotonin targeting drugs were least effective across all
genotypes. Although both escitalopram and 8-OH-DPAT reduced USVs in wild-type pups, the
drugs were less and less effective as Gtf2i gene copy number increased. This resistance in pups
with duplication of Gtf2i of the anxiolytic effects of drugs targeting the serotonin
neurotransmitter we suggest could be due to an inhibitory role of Gtf2i on serotonergic
transmission. These results suggest a higher involvement of the glutamate and GABA
neurotransmitter systems in the mechanism responsible for separation anxiety assessed by
maternal separation-induced USVs. These results also highlight the utility of drugs targeting
these neurotransmitters as therapeutic interventions for anxiety, and for separation anxiety
particularly.
4.1.3 Hypothesized Changes in Pain Sensitivity with Change in Gtf2i Gene
Copy Number
Subcutaneous injection of saline affected the total number of USVs emitted during maternal
separation as well as plasma corticosterone levels in a Gtf2i gene-dosage dependent manner. We
observed a decrease in USVs as Gtf2i gene copy number increased in PND8 pups. This is in
contrast to our previous work where we showed an increase in total number of USVs as Gtf2i
gene copy number increased (Mervis et al., 2012). Furthermore, this change in USVs with a
change in Gtf2i gene copy number paralleled changes in plasma corticosterone levels for both
groups of PND8 pups: those that received and those that did not receive a subcutaneous injection
115
of saline. We hypothesize that differences in pain sensitivity between mice with altered Gtf2i
gene copy number underlie the differences observed in PND8 mice, dependent on whether they
received a subcutaneous injection or not. Specifically, we propose that hypersensitivity to pain
characterizes mice with a deletion of Gtf2i and hyposensitivity to pain those with a duplication of
Gtf2i. This is in line with previous reports of increased sensitivity to painful stimuli in mice with
a deletion of half the WBS syntenic region, encompassing Gtf2i (Li et al., 2009). These results
suggest a role of Gtf2i in pain sensitivity that needs to be formally addressed in both individuals
with WBS and Dup7q11.23 and in the Gtf2i mouse models.
Pain is associated with intense recruitment of the HPA axis and downstream corticosterone
release and c-fos expression, which could profoundly interfere with the behaviour of the animal
and the mechanism of action of the injected drugs (Aubrun et al., 2004, reviewed in Benedetti et
al., 2012, and Wang et al., 2012). Furthermore, the pain condition must be clear since some
mechanisms underlying spontaneous pain likely differ from those mechanisms associated with
hyper- or hypo-sensitivity pain states (Mogil and Crager, 2004). In addition, clinical pain has
been shown to be altered by anxiety (Seminowicz et al., 2004). Specifically, stress associated
with anxiety may affect nociception by altering the concentration of endogenous opioids or by
inducing changes in neurotransmitter systems, such as the serotonergic, noradrenergic and
adenosinergic systems (reviewed in Benedetti et al., 2012). Therefore, a clear distinction must be
made between the pain- and anxiety- associated phenotypes. Future studies should assess pain
sensitivity in the Gtf2i mouse models and in individuals with WBS and Dup7q11.23 syndrome.
If our hypothesis regarding changes in pain sensitivity in these two disorders holds true, the
current paradigms used to assess anxiolytic efficacy in these two disorders must be revised to at
least account for differences due to pain sensitivity changes and potentially avoid inducing pain
in the subject.
4.1.4 Stress- and Pain- Induced Elevations of Brain c-fos Expression with
a Change in Gtf2i Gene Copy Number
Both stress and presumed pain from injection were found to induce brain c-fos expression in a
somewhat Gtf2i gene-dosage dependent way. A slight increase in c-fos expression was observed
116
for wild-type mice and those with a duplication of Gtf2i after undergoing the test of maternal
separation. A much higher increase was noted in mice of same genotypes but that received a
subcutaneous injection of saline prior to the test of maternal separation, presumably due to the
added stress associated with the injection itself. Furthermore, in mice that received a
subcutaneous injection, c-fos was expressed at higher levels in mice with a heterozygous and
homozygous duplication of Gtf2i than wild-type mice. This elevation could be due to the
additional Gtf2i gene copy number leading to higher activation of the c-fos promoter (Kim et al.,
1998). Therefore, both stress induced by maternal separation and pain induced by subcutaneous
injection are believed to induce brain c-fos expression in a Gtf2i gene-dosage dependent way for
mice with increased copy number of Gtf2i.
4.1.5 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
Allopregnanolone, a GABA-A agonist, reduced c-fos expression whereas MK-801, a glutamate
antagonist, did not. These findings suggest that allopregnanolone is a better drug at reducing
stress- and/or pain- induced c-fos expression. These results also suggest that GABA
neurotransmission might be more relevant to anxiety mechanism of action when considering
changes in immediate early-gene c-fos expression.
GTFII-I is a transcription factor with a long list of roles one of which includes the activation of
the promoter of c-fos immediate early gene (Kim et al., 1998). TFII-I, upon phosphorylation by
kinases, will promote c-fos activation together with other critical promoter-binding proteins
(Kim et al., 1998). Therefore, intuitively, a higher number of Gtf2i gene copies should lead to
higher c-fos activation. However, GTFII-I activation of the c-fos promoter could be hindered by
the efforts of anxiolytics in reducing c-fos levels. Therefore, final c-fos expression levels may
likely be determined by the factor that succeeds in overcoming the other.
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4.2 Future Directions
4.2.1 Assessment of Pain Sensitivity in WBS and Dup7q11.23
Gtf2i appears to be a likely candidate in altering pain sensitivity. The 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 assess pain sensitivity. The hot
plate test is one method that can be used to assess changes in pain sensitivity in these mice. It
provides insight into pain sensitivity by using a thermal stimulus to assess thermal threshold
(Hargreaves et al., 1988). The latency to respond to the painful stimuli of high temperature, by
licking the hindpaw, is measured. A shorter latency to respond indicates an accelerated response
to thermal pain and suggests hypersensitivity to pain (Bonin et al., 2011). Future studies of the
Gtf2i mouse models should therefore assess pain sensitivity using the hot plate test.
To date, no clinical studies have assessed sensitivity to pain in individuals with WBS or with
Dup7q11.23 syndrome. However, high pain thresholds in individuals with Dup7q11.23 have
been inferred through anecdotal reports (personal communication by C. Mervis and C. Morris).
Therefore, it would be of high importance to formally assess pain sensitivity in individuals with
WBS and Dup7q11.23. Reported changes in pain sensitivity in individuals with
neurodevelopmental and other disorders have often been based in anecdotal observations and
parent questionnaires (reviewed in Breau et al., 2002). NCCPC, FPS, and OSBD are some of the
questionnaires commonly used to assess pain sensitivity in children with cognitive impairments
(Breau et al., 2002 and Nader et al., 2004). Specifically, these tests have been previously used to
assess the pain and distress in children with autism experiencing a venipuncture. Therefore,
measures of pain sensitivity using similar methods as the ones described for individuals with
autism need to be employed for individuals with WBS and Dup7q11.23. The Gtf2i mouse
models could then be used to elucidate the molecular mechanism of this phenotype and the
genetic contributions of Gtf2i gene to this mechanism.
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4.2.2 Additional Measures During and After Maternal Separation-Induced
USVs Test
The drugs of choice and their respective doses were selected after careful review of the literature
reporting maximum anxiolytic effects on USV numbers accompanied by negligible side effects
for the selected doses (Fish et al., 200, 2003, and Takahashi et al., 2009). In our study, we tested
for maternal separation-induced USVs and did not include measures of potential drug side
effects, such as the effects that they might have on locomotion. Therefore, we might be omitting
important information by excluding locomotion measurements in our study. Manipulation of
each of the systems – serotonergic, GABAergic and glutamatergic – in young animals could
interfere with the role of these systems in ongoing neuronal development. (Azmitia, 2001).
Despite this point of caution, our study involved a single, acute delivery of the drug and
immediate sacrificing of animals after the behavioral test ensured that it is highly unlikely that
any developmental changes occurred within that short time period.
A number of studies looking at ultrasonic vocalizations have pointed at the physical interaction
between mother and pup as being an important player in the number of calls emitted by pups
independent of maternal genotype itself (Wohr et al., 2008). It is possible that by excluding
assessment of maternal behavior in this study, whether during the separation period and/or after
retrieval of the pup, we might be missing important data that will help us better interpret
maternal separation-induced USVs. Future studies should therefore address this issue by
assessing maternal behavior during interaction with the pups.
Changes in the ventral hippocampus during the early postnatal period have been linked to the
development of anxiety and have been shown in the 5-HT1A receptor knock-out mouse model of
anxiety. These changes were reported to be caused by either the maternal or offspring 5-HT1A
receptor genotype, or both (Bannerman et al., 2003, Gleason et al., 2011). Therefore, future
studies of the Gtf2i mouse models need to assess potential changes in the brain development of
pups due to both offspring and maternal genotype.
119
Mouse pups may be differentially sensitive to anxiolytic drugs than adult mice because they are
still young and developing, and therefore differences in pharmacokinetics and the number of
receptors may exist. Although the effects of anxiolytics in maternal separation-induced
vocalizations was compared to littermate controls of the same age in this study, extending this
work to adult mice requires careful consideration of potential developmental changes. Lastly,
despite the validation of the maternal separation-induced USVs test as a measure of the efficacy
of anxiolytics (Hodgson, 2008), such drugs must be examined in other animal models of anxiety
as well to determine the extent to which these results are dependent on genetic background and
age.
The maternal Gtf2i genotype effects were mainly elucidated in the Gtf2i duplication mouse
models. However, it would be equally important to study maternal genotype effects in the Gtf2i
deletion mouse model since it would further assess maternal Gtf2i genotype effects on maternal
separation-induced USVs and also examine potential Gtf2i gene-dosage dependent effects on
maternal care.
4.2.3 Assessment of c-fos Changes in Specific Brain Regions
Changes in c-fos expression have previously been measured in animals subjected to a test of
anxiety with and without previous delivery of an anxiolytic. Animals subjected to a test of
anxiety, such as the elevated plus maze, showed a characteristic increase in c-fos expression in
several regions of the hippocampus, including CA1, CA3, dentate gyrus, and the central nucleus
of the amygdala (Linden et al., 2004). LY354740, an mGlu2/3 agonist, showed anxiolytic
properties in mice using the elevated plus maze, as well as elevated c-fos in the central nucleus
of the amygdala (Linden et al., 2004). Other anxiolytics, such as benzodiazepines, also induce
neuronal activity in the central nucleus of the amygdala (Beck et al., 1995). Although it may
initially seem more reasonable for anxiolytics to reduce c-fos expression induced by the anxiety-
measuring tests, the opposite might also be valid when specific brain regions and neuronal
populations are considered. It has been suggested that inhibitory GABAergic neurons in the
central nucleus of the amygdala regulate the excitatory output from the medial amygdala (Sun et
al., 1993 & 1994, and Veinante et al., 1998). Therefore, brain-region-specific changes in c-fos
expression have been reported in some studies, which were not attempted in our study. One
120
hypothesis regarding the changes that anxiety induces on brain activation is that anxiety has a
paradoxical effect whereby it suppresses activity in the hippocampus but enhances activity in the
amygdala (Cortese et al., 2005). Therefore, by studying expression at the whole-brain level, we
may be missing important information on the effects that maternal separation-induced USVs,
injection, and anxiolytics may have on c-fos expression in specific brain regions. This critical
information would be beneficial in the process of elucidating a mechanism of action. Future
studies should therefore employ Fos immunohistochemistry techniques to assess changes in c-fos
expression in specific brain regions.
4.3 Conclusion
This study investigated the mechanisms underlying the role of Gtf2i in separation anxiety, using
mice with altered Gtf2i gene dosage. The glutamate and GABA neurotransmitter systems appear
to be more important than serotonin system in the anxiety phenotype of mice with altered Gtf2i
gene copy number. Changes in plasma corticosterone levels paralleled behavioral measures of
anxiety and were also affected in a Gtf2i gene-dosage dependent manner. Brain c-fos expression
levels were controlled by inhibitory actions of anxiolytics. Lastly, we may have uncovered an
unexpected role for Gtf2i in pain sensitivity. Further assessment of the Gtf2i mouse models with
an array of behavioral and molecular approaches can continue to be used to dissect genotype-
phenotype correlations in WBS and Dup7q11.23.
121
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