SEROTONIN: FROM MOUSE MODELS TO HUMAN DISORDERS A ... · Ashley L. Jensen Dissertation Committee: D. Caroline Blanchard, Chairperson Harry Davis IV Kentaro Hayashi Scott Sinnett Lorey

SEROTONIN: FROM MOUSE MODELS TO HUMAN DISORDERS

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE

UNIVERSITY OF HAWAI'I AT MĀNOA IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

PSYCHOLOGY

August 2014

By

Ashley L. Jensen

Dissertation Committee:

D. Caroline Blanchard, Chairperson

Harry Davis IV

Kentaro Hayashi

Scott Sinnett

Lorey Takahashi

David Haymer

Keywords: Serotonin, autism, mouse models, postmortem tissue

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ACKNOWLEDGEMENTS

The work reported in this dissertation was supported by grants awarded by the

National Institute of Mental Health (MH0881845) to Dr. Robert J. Blanchard. The

human tissue used in this research was contributed through the Autism Tissue Program of

Autism Speaks, the Harvard Brain Tissue Resource Center which is supported in part by

a Public Health Services grant R24 (MH 068855), and the NICHD Brain and Tissue

Bank. I am especially grateful to the families for their invaluable donations; without

whom this research would not have been possible.

Special thanks are due to Dr. Michael Corley and Tina Carvalho for their

technical expertise and advice. Some of the immunohistochemical troubleshooting was

performed in the lab of Dr. David Jameson who graciously lent me bench space and

resources. I also benefited greatly from the resources and supplies of Dr. Harry Davis IV

and Dr. Nicholas James, who tried endless variants of immunohistochemistry protocols

with me and encouraged me to not give up.

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ABSTRACT

Autism is an increasingly common neurodevelopmental disorder characterized by

deficits in social interactions, impaired communication, and repetitive and stereotyped

behaviors. While the etiology of autism is unknown, several lines of evidence implicate

dysfunction of the serotonin (5-HT) system in the anatomical and behavioral features of

the disorder. Work reported in this series investigated components of the 5-HT system in

animal models of autism as well as in post-mortem human brain tissue in an attempt to

clarify the role of 5-HT in this disorder. The activity of the central 5-HT system was

investigated in the BTBR T+Itpr3tf/J (BTBR) mouse, a strain that displays behaviors

consistent with all three diagnostic categories for autism. In comparison to the common

C57BL/6J (B6) control strain, BTBR mice showed a pattern of behaviors characterized

by reduced frontal orientations in social interactions and altered cortical and cerebellar

concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). 5-HT

concentrations may be regulated by the serotonin transporter (5-HTT) gene (SLC6A4)

and variants in this gene are implicated in autism. Mice with targeted disruptions of

SLC6A4 displayed increased stereotyped grooming that is consistent with one of the core

symptoms of autism. However, social, aggressive, and communicative behaviors were

not altered in SLC6A4 knockout mice. The research reported here also described altered

structural and molecular composition in the subventricular zone (SVZ) of individuals

with autism. 5-HTT immunoreactivity was significantly increased in the SVZ of autism-

diagnosed (AD) individuals when compared to typically-developing controls.

Furthermore, structural features of the SVZ in AD cases were suggestive of altered

neurogenesis and cellular migration; consistent with the neurodevelopmental nature of

this disorder. Collectively, these results provide strong support for the role of

serotonergic system dysfunction in the anatomical and behavioral traits characteristic of

autism.

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ................................................................................................ ii

ABSTRACT ....................................................................................................................... iii

LIST OF TABLES ............................................................................................................ vii

LIST OF FIGURES ......................................................................................................... viii

LIST OF ABBREVIATIONS ............................................................................................. x

PREFACE ......................................................................................................................... xii

CHAPTER 1. INTRODUCTION ....................................................................................... 1

1.1. Autism Spectrum Disorders ................................................................................. 1

1.2. Etiology of ASD .................................................................................................. 1

1.3. Neuropathology of Autism ....... ..........................................................................2

1.4. Serotonin .............................................................................................................. 5

1.5. Serotonin in Neurodevelopment .......................................................................... 6

1.6. Serotonin in Autism ............................................................................................. 8

1.7. Dissertation Overview........................................................................................ 11

1.8. References .......................................................................................................... 13

CHAPTER 2. NEUROCHEMICAL CHARACTERIZATION OF THE BTBR T+Itpr3

tf/J MOUSE MODEL FOR AUTISM ...................................................... 31

2.1. Abstract .............................................................................................................. 31

2.2. Introduction ........................................................................................................ 33

2.3. Materials and Methods ....................................................................................... 35

2.3.1. Subjects ............................................................................................... 35

2.3.2. Apparatus ............................................................................................ 35

2.3.3. Procedure ............................................................................................ 35

2.3.4. Statistical Analyses ............................................................................. 38

2.4. Results ................................................................................................................ 38

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2.4.1. Experiment 1-Baseline ........................................................................ 38

2.4.2. Experiment 2-Nonsocial and Social Proximity ................................... 41

2.5. Discussion .......................................................................................................... 46

2.6. References .......................................................................................................... 53

CHAPTER 3. BEHAVIORAL CHARACTERIZATION OF THE SEROTONIN TRANSPORTER KNOCK OUT MOUSE MODEL FOR AUTISM .................. 62

3.1. Abstract .............................................................................................................. 62

3.2. Introduction ........................................................................................................ 64


3.3.1. Subjects ............................................................................................... 65

3.3.2. Apparatus ............................................................................................ 66

3.3.3. Procedure ............................................................................................ 67

3.3.4. Statistical Analyses ............................................................................. 71

3.4. Results ................................................................................................................ 71

3.4.1. Visible Burrow System ....................................................................... 71

3.4.2. Three Chamber .................................................................................... 75

3.4.3. Autogrooming ..................................................................................... 77

3.4.4. Social Proximity .................................................................................. 79

3.4.5. Repetitive Novel Object Contact ........................................................ 80

3.4.6. Scent Marking ..................................................................................... 82

3.4.7. Resident Intruder ................................................................................. 83

3.5 Discussion ........................................................................................................... 84

3.6 References ........................................................................................................... 88

CHAPTER 4. CHARACTERIZATION OF THE SEROTONERGIC PROFILE OF THE SUBVENTRICULAR ZONE (SVZ) IN INDIVIDUALS WITH AUTISM AND TYPICALLY DEVELOPING (TD) CONTROLS ..................... 94

4.1. Abstract .............................................................................................................. 94

4.2. Introduction ........................................................................................................ 96


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4.3.1. Postmortem Brain Tissue Samples ..................................................... 97

4.3.2. Procedure .......................................................................................... 100

4.3.3. Statistical Analysis ............................................................................ 106

4.4. Results .............................................................................................................. 107

4.4.1. Clinical Characteristics ..................................................................... 107

4.4.2. Postmortem and Neuropathological Characteristics ......................... 107

4.4.3. Morphology of the SVZ .................................................................... 108

4.4.4. Hypocellular Gap .............................................................................. 109

4.4.5. Localization and Quantification of 5-HTT in the SVZ ..................... 112

4.4.6. Localization and Quantification of FGF-2 in the SVZ ..................... 115

4.4.7. Localization and Quantification of GFAP in the SVZ ..................... 117

4.4.2. Cellular Characterization of the SVZ ............................................... 119

4.5. Discussion ........................................................................................................ 121

4.6. Limitations ....................................................................................................... 125

4.7. Conclusions ...................................................................................................... 126

4.8. References ........................................................................................................ 127

CHAPTER 5. GENERAL DISCUSSION ...................................................................... 134

5.1. Serotonin in Autism ......................................................................................... 134

5.2. Serotonin alterations in the BTBR mouse model for autism ........................... 135

5.3. Enhanced stereotypy in 5-HTT knock-out mice .............................................. 136

5.4. Increased mitogenic factors in the SVZ of young AD cases ........................... 138

5.5. Conclusions, constraints and future directions ................................................ 139

5.6. References ........................................................................................................ 142

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LIST OF TABLES Page

Table 2.1. Regional baseline concentrations of monoamines, metabolites, and

monoamine turnover for BTBR and B6 mice………………………...……….....40

Table 2.2. Regional concentrations of monoamines, metabolites and monoamine turnover

collapsed across nonsocial and social conditions……………………. ...…….…44

Table 3.1. Statistical results for behaviors run in the VBS ……………………..……….73

Table 4.1. Diagnostic characteristics of autism cases.....…………………………….......98

Table 4.2. Antibodies.....................................................……………………………......101

Table 4.3. Postmortem information on autism-diagnosed and typically-developing

controls...........................................................................……………………………......108

viii

LIST OF FIGURES

Page

Figure 1. Distribution of the major serotonergic pathways in the central nervous system ....6

Figure 2.1. BTBR mice in the social proximity test ..............................................................37

Figure 2.2. Frequency of behaviors in the social proximity test ............................................41 44

Figure 2.3. Concentrations of NE in the medial prefrontal cortex in the proximity

conditions ...................................................................................................................45 48

Figure 2.4. Concentrations of MHPG in the cortex in the proximity conditions...................45

Figure 2.5. Concentrations of DOPAC:DA in the hypothalamus in the proximity

conditions ...................................................................................................................45 48

Figure 3.1. Frequency of social behaviors during the light phase of the VBS ......................74

Figure 3.2. Locomotion in the three chamber test .................................................................76 82

Figure 3.3. Time spent in each chamber in the three chamber test ........................................76 82

Figure 3.4. Behaviors displayed in the autogrooming test.....................................................78 84

Figure 3.5. Behaviors displayed in the social proximity test .................................................79 85

Figure 3.6. Behaviors displayed in the repetitive novel object contact test ...........................81

Figure 3.7. Scent marking behavior .......................................................................................82 88

Figure 3.8. Behaviors displayed in the resident intruder test .................................................83 89

Figure 4.1. Tissue acquisition and histology .........................................................................99 104

Figure 4.2. Sample preparation for immunohistochemical staining ......................................103 109

Figure 4.3. Layered cellular organization of the human SVZ ...............................................109 120

Figure 4.4. Hypocellular gap widths in the AD and TD SVZ ...............................................111 122

Figure 4.5. Hypocellular gap density in the AD and TD SVZ ..............................................112 123

Figure 4.6. Area Fraction of 5-HTT in the AD and TD SVZ ................................................114

ix

Figure 4.7. Area fraction of FGF-2 in the AD and TD SVZ .................................................116 127

Figure 4.8. Area fraction of GFAP in the AD and TD SVZ ..................................................118 129

Figure 4.9. NeuN staining of the SVZ ...................................................................................120 131

Figure 4.10. IBA-1 staining of the corpus callosum in autism ..............................................121 132

x

LIST OF ABBREVIATIONS

5-HIAA 5-Hydroxyindoleacetic acid

5-HT Serotonin (5-hydroxytryptamine)

5-HTT/SERT Serotonin transporter

5-HTTLPR Serotonin transporter gene linked polymorphic region

AD Autism-diagnosed

ASD Autism spectrum disorder

B6 C57BL/6J

BTBR BTBR T+Itpr3tf/J

CNS Central nervous system

CSF Cerebrospinal fluid

DA Dopamine

DOPAC 3,4-Dihydroxyphenylacetic acid

FGF-2 Fibroblast growth factor 2

HET Heterozygous

HPLC High performance liquid chromatography

KO Knockout

MHPG 3-Methoxy-4-hydroxyphenylglycol

NE Norepinephrine

NPC Neural precursor cell

NSC Neural stem cell

PC Purkinje cell

PET Positron emission tomography

xi

PMI Postmortem interval

SGZ Subgranular zone

SLC6A4 Serotonin transporter gene

SSRI Selective serotonin reuptake inhibitor

SVZ Subventricular zone

TD Typically-developing

VBS Visible burrow system

WT Wild type

PREFACE

This dissertation has been formatted to allow for the separate publication of

Chapters 2, 3, and 4. Chapter 2 titled "Neurochemical characterization of the BTBR

T+Itpr3tf/J mouse model for autism" and Chapter 3 titled "Behavioral characterization of

the serotonin transporter knockout mouse model for autism" have been prepared for

submission to the journal Behavioral Brain Research.

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CHAPTER 1. INTRODUCTION

1.1. Autism Spectrum Disorders

Autism Spectrum Disorders (ASD) are a group of complex and heterogeneous

neurodevelopmental disorders that includes autism, Asperger's syndrome, Rhett

syndrome, childhood disintegrative disorders, and pervasive developmental disorders not

otherwise specified (American Psychiatric Association, 2013). Disorders within this

spectrum are characterized by social deficits, impaired communication and stereotyped

and repetitive behaviors. In addition to these core features, a number of associated

conditions including enhanced anxiety and responsivity to sensory stimuli (Green and

Ben-Sasson, 2010), as well as gastrointestinal and sleep dysfunction (Buckley et al.,

2010; Mazurek et al., 2013) are frequently observed. These conditions are pervasive,

life-long and carry significant interpersonal, societal and economic costs.

ASDs are reported in all ethnic and socioeconomic groups (Baio, 2012) and are

estimated to afflict 1 in 50 children; making ASDs the most frequently diagnosed

developmental disorders in the United States (Blumberg et al., 2013). Despite improved

awareness and behavioral interventions, prognoses for ASD patients are highly variable.

Significant functional deficits typically persist into adulthood (Howlin et al., 2004;

Billstedt et al., 2005; Farley et al., 2009) and consequently, individuals with an ASD

often require lifetime support (Hume et al., 2009). Families of children with an ASD

report increased stress as well as decreased marital satisfaction and reduced income

compared to families with typically developing children or children with other

developmental disorders (Karst and Van Hecke, 2012). The lifetime costs per capita

associated with autism, the most common ASD, exceed $3 million dollars in the U.S.

alone (Ganz, 2007). The significant personal, societal and economic consequences of

these disorders highlight the need for improved understanding of their causes, prevention

and effective treatment.

1.2. Etiology of ASD

There is no known singular cause of ASD. Rather, these disorders are likely the

consequence of complex interactions between a number of genetic, epigenetic, and

environmental factors (Bale et al., 2010; Lord, 2011). ASDs are among the most

genetically determined of all neuropsychiatric disorders with an estimated heritability of

2

over 90% (Bailey et al., 1995). Concordance rates range from 3%-14% in siblings

(Constantino et al., 2010; Ozonoff et al., 2011) and are highest (75%-95%) among

monozygotic twins (Hallmayer et al., 2011; Ronald and Hoekstra, 2011). Moreover,

higher rates of autism relevant phenotypes have been reported in first-degree relatives of

patients with autism (Piven et al., 1997b; Murphy et al., 2000). The high concordance of

the disorders has been linked to mutations and variations in a number of candidate genes

(International Molecular Genetic Study of Autism Consortium, 2001; Persico and

Bourgeron, 2006; Geschwind and Levitt, 2007; Hussman et al., 2011). The X

chromosome in particular, has received considerable attention given the 4:1 prevalence of

ASDs in males compared to females (Baron-Cohen et al., 2011). However, less than

20% of all autism cases can be accounted for by these known genetic variants and

mutations (Abrahams and Geschwind, 2008). Additionally, whole-genome scans suggest

that interactions between a large number of genes (>10) likely contribute to the disorders

(Muhle et al., 2004).

There is also evidence for environmental contributions to ASD diagnoses (Chaste

and Leboyer, 2012). Several prenatal factors have been associated with these disorders,

including maternal viral infection (Atladóttir et al., 2010; Patterson, 2011), advanced

paternal age (Reichenberg A, 2006; Croen et al., 2007), and maternal medication use

(Croen et al., 2011). Genetic susceptibility and environmental influences may interact to

contribute to the heterogeneity of ASD phenotypes. Promising new research showed that

possessing the low expressing version of the serotonin transporter gene (SLC6A4)

interacted with maternal smoking during pregnancy, increasing problems in social

interaction, and also interacted with low birth weight, increasing rigid behaviors in

offspring (Nijmeijer et al., 2010).

1.3. Neuropathology of Autism

Despite the lack of a clear biological cause, a number of specific brain pathologies

have been described in autism. Clinical, neuroimaging, neurochemical, and

neuropathological studies consistently report findings suggestive of disrupted brain

development and organization; such as accelerated brain growth (Courchesne et al.,

2007), structural abnormalities (Bauman and Kemper, 2005; Amaral et al., 2008), and

atypical structural and functional connectivity between brain regions (Amaral et al.,

3

2008; Ecker et al., 2013b). These dysfunctions are hypothesized to alter information

processing in the brains of autistic individuals and contribute to the social,

communicative, and motor symptoms associated with this disorder (Persico and

Bourgeron, 2006; Hussman et al., 2011; Ecker et al., 2013b).

One of the most consistent findings in autism is altered brain growth. Children

with autism frequently undergo a period of early postnatal brain overgrowth (Courchesne

et al., 2001; Hazlett et al., 2005, 2012; Amaral et al., 2008) and subsequent arrest of

growth (Courchesne et al., 2001, 2011a) coinciding with the onset of behavioral

symptoms. Head circumference (Courchesne et al., 2003), brain volume (Courchesne et

al., 2001; Palmen et al., 2004a) and brain mass (Palmen et al., 2004b; Redcay and

Courchesne, 2005) are larger in 2-5 year-old autistic children when compared to typically

developing controls of the same age. These volume differences diminish after age 5 and

are negligible by adulthood (Courchesne et al., 2007). Larger than average brains have

also been reported in first-degree relatives of autistic individuals (Woodhouse et al.,

1996; Fidler et al., 2000). The molecular and cellular contributions to this overgrowth

are unknown, however, excessive neurogenesis, disturbed migration, abnormal apoptosis,

and disrupted synaptogenesis have been proposed as potential mechanisms (McCaffery

and Deutsch, 2005; Persico and Bourgeron, 2006; Courchesne et al., 2007; Vaccarino et

al., 2009). Consistent with these hypotheses, increased neurons (Courchesne et al.,

2011b), cell density (Casanova et al., 2002, 2006), microglia-neuron clustering (Morgan

et al., 2012), and neurogenesis (Wegiel et al., 2010; Kotagiri et al., 2013; Pearson et al.,

2013) have all been reported in the postmortem brains of autistic individuals.

Structural abnormalities are consistently described for the cortex and cerebellum

in autism; suggesting disturbances in neuronal migration and organization. In some

patients cortical minicolumns are increased in number and narrower in width, with

reduced neuropil space and smaller neuron bodies (Bailey et al., 1998; Casanova et al.,

2002, 2006; Wegiel et al., 2010). These minicolumn disruptions are thought to contribute

to the increased cortical gyrification (Wallace et al., 2013) and thickness (Ecker et al.,

2013a) as well as the attentional and sensory-discrimination deficits in autism (Opris and

Casanova, 2014). The most consistent neuropathologies of the cerebellum are hypoplasia

and reduced cerebellar Purkinje cells (PC) (Piven et al., 1997c; Fatemi et al., 2002;

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Palmen et al., 2004b; Wegiel et al., 2010, 2013). A recent study suggests that cerebellar

dysplasia and PC cell loss are associated with the atypical gaze that is a characteristic

feature of autism (Wegiel et al., 2013). Taken together, these observations suggest that

developmental disturbances in neural migration and organization may contribute to at

least some of the neuropathological and clinical features of autism.

In addition to the reported morphological abnormalities, there is evidence for

altered structural and functional connectivity in the brains of autistic individuals

(Belmonte et al., 2004a, 2004b; Courchesne and Pierce, 2005; Geschwind and Levitt,

2007; Rudie et al., 2012). Neuroimaging studies report structural differences in white-

matter volume (Boddaert et al., 2004; McAlonan et al., 2005), microstructural integrity

(Alexander et al., 2007; Catani et al., 2008), and atypical connectivity between brain

regions (Just et al., 2007; Pugliese et al., 2009). Consistent reports of reduced corpus

callosum volumes (Egaas et al., 1995; Piven et al., 1997a) are indicative of reduced

intrahemispheric connectivity (Anderson et al., 2011) and are suggested to contribute to

behavioral features of autism. For example, smaller corpus callosum volumes have been

associated with lower intelligence quotient scores (Alexander et al., 2007) and greater

symptom severity (Hong et al., 2011) in individuals with autism.

Functional imaging of the brains of autistic individuals during tasks of working

memory (Koshino et al., 2005, 2008), executive function (Just et al., 2007), as well as

language (Just et al., 2004) and emotion processing (Kleinhans et al., 2008) reveal

abnormal patterns of regional activation and synchronization. In particular, dysfunction

of the cerebello-thalamo-cortical circuit is thought to contribute to the cognitive and

affective symptoms characteristic of autism (Hoppenbrouwers et al., 2008). Functional

magnetic resonance imaging (fMRI) of high functioning autistic children revealed

reduced cerebellar activation with increased cortical activation in a simple motor task

when compared to typically developing controls (Mostofsky et al., 2009). An additional

fMRI study found that cerebellar activation was reduced while thalamo-cortical

activation was increased in high functioning autistic individuals during a sensorimotor

control task (Takarae et al., 2007).

Several lines of research suggest that neuronal communication is disturbed in

autism. Genes known to regulate neurotransmitter function (Sutcliffe et al., 2005;

5

Coutinho et al., 2007), axonal and dendritic growth (Gilman et al., 2011), and

synaptogenesis (Toro et al., 2010; Hussman et al., 2011) have been implicated in the

disorder. Biomarker analyses (Cook and Leventhal, 1996), neuroimaging investigations

(Chugani et al., 1997, 1999) and pharmacological manipulations (Volkmar, 2001)

implicate abnormalities in several neurotransmitter systems. Furthermore, in rodent

studies, early disruptions to monoamine neurotransmitter systems, especially serotonin

(5-HT) and dopamine (DA), result in anatomical and behavioral features relevant to

autism (Kahne et al., 2002).

Collectively, these findings highlight the neurodevelopmental nature of autism-

associated pathology. Evidence of altered neuronal development (Wegiel et al., 2010),

irregularities in brain cytoarchitecture (Piven et al., 1997c; Casanova et al., 2002),

disordered interregional connectivity (Egaas et al., 1995; Boersma et al., 2013), and

disturbed neurotransmission (Lam et al., 2006) suggest enduring changes in the

organization, connectivity and communication of the autism brain (Belmonte et al.,

2004b; Pardo and Eberhart, 2007). However, the cellular and molecular mechanisms that

drive these anomalies remain unknown. Therefore, studies of the underlying mechanisms

that contribute to altered neurodevelopment in autism are needed.

1.4. Serotonin

Serotonin (5-HT) is a phylogenetically ancient and highly conserved monoamine

neurotransmitter. 5-HT is found in nearly every organ of the body and in virtually every

living organism (Müller and Jacobs, 2010). In the central nervous system (CNS), 5-HT

synthesizing cells originate in the raphe nucleus of the brainstem and provide a dense

innervation to all regions of the brain (Jacobs and Azmitia, 1992) (Figure 1). 5-HT

neurons also project into the walls of the ventricles, forming a dense supra- and sub-

ependymal plexus (Richards et al., 1981; Jahanshahi et al., 2011). 5-HT affects

numerous biological processes including appetite, circadian rhythms, sensory processing,

cognition, motor system function, emotion, and reward processing (Lucki, 1998).

Disturbed 5-HT function has been implicated in a variety of neuropsychiatric conditions

including depression, anxiety disorders, obsessive compulsive disorders, and autism

(Cook and Leventhal, 1996; Elhwuegi, 2004; Hart et al., 2010).

6

Figure 1. Distribution of the major serotonergic pathways in the central nervous

system (Kruk and Barkus, 1993).

1.5. Serotonin in Neurodevelopment

5-HT is one of the earliest developing and most widely distributed

neurotransmitters in the mammalian CNS (Sodhi and Sanders-Bush, 2004). In the human

brain, neurons expressing 5-HT appear by the fifth week of gestation (Sundström et al.,

1993), grow rapidly until the 10th

week (Shen et al., 1989; Kontur et al., 1993; Levallois

et al., 1993) and assume the typical adult organization in the raphe nuclei by week 20

(Kinney et al., 2007). Neurons expressing 5-HT innervate the cortical subplate around 10

weeks post gestation (Verney et al., 2002), during a highly active period of neural

proliferation and migration (de Graaf-Peters and Hadders-Algra, 2006). 5-HT synthesis,

7

receptors and activity increase significantly during the first two years after birth and then

decline to adult levels after the age of five (Hedner et al., 1986; Tóth and Fekete, 1986;

Chugani et al., 1999); corresponding with peak periods of neural development and

synaptogenesis (Chugani et al., 1987, 1999; Chugani, 1998).

5-HT modulates important neurodevelopmental processes such as: cell

proliferation and apoptosis, as well as neuronal migration, maturation, and

synaptogenesis (Azmitia, 2001; Pino et al., 2004; Sodhi and Sanders-Bush, 2004;

Daubert and Condron, 2010). Collectively, these processes determine brain

cytoarchitecture and connectivity (Lesch and Waider, 2012). Consequently, disturbances

to 5-HT during development may produce enduring changes in brain structure and

function and contribute to the pathology of neurodevelopmental disorders such as autism.

5-HT acts as a mitogen on a number of cell types. Increasing 5-HT during fetal

development stimulates the proliferation of neural precursor cells (NPC) (Peng et al.,

2013) and glial cells (Tajuddin et al., 2003). Excess 5-HT also can also produce

morphological changes similar to those seen in autism, such as increased cortical cell

density and thickness (Altamura et al., 2007). Conversely, early depletion of 5-HT

reduces neurogenesis (Lauder and Krebs, 1976), gliogenesis (Khozhai, 2006), and

embryonic brain size (Holson et al., 1994). Neurogenesis persists in two discrete regions

in the adult brain, the subventricular zone (SVZ) and the hippocampal subgranular zone

(SGZ) (Ming and Song, 2005). 5-HT stimulates neurogenesis in both the SVZ and SGZ

(Banasr et al., 2004) and protects against cell death by reducing apoptosis in the

developing (Persico et al., 2003; Wang et al., 2011) and adult (Liu et al., 2011) brain.

Thus, 5-HT has been suggested to play a role in both the development and life-long

plasticity of the brain.

During later stages of development 5-HT regulates the migration and

differentiation of neurons. High concentrations of 5-HT decrease the migratory speed of

various cortical neurons (Riccio et al., 2008, 2011) and cause malformations of cortical

minicolumns (Janušonis et al., 2004) similar to those reported in autism (Casanova et al.,

2002, 2006). 5-HT activity is also critical for directing axonal development and dendritic

growth; including the outgrowth of 5-HT producing neurons themselves. Excessive

amounts of 5-HT disrupt the development of axons in the primary somatosensory cortex

8

(Cases et al., 1996) and primary visual cortex (Upton et al., 1999, 2002). Excess

extracellular 5-HT, as a consequence of serotonin transporter (5-HTT) ablation, increased

the number of 5-HT projections from the median raphe to the medial prefrontal cortex

and decreased the number of corpus callosum projection neurons (Witteveen et al., 2013).

5-HT activity also regulates the growth (Mazer et al., 1997), complexity (González-

Burgos et al., 1996; Vitalis et al., 2007) and pruning (González et al., 2008) of dendrites.

Specifically, 5-HT has been shown to modulate the development (Oostland et al., 2013)

and arborization (Kondoh et al., 2004) of cerebellar PCs; which are reduced in the brains

of autistic individuals (Palmen et al., 2004b; Bauman and Kemper, 2005; Wegiel et al.,

2010, 2013).

1.6. Serotonin in Autism

Due to its involvement in the regulation of a diverse number of physiological and

emotional functions, dysfunction of 5-HT neurotransmission has been implicated in a

number of psychopathologies, including autism. One of the most consistently reported -

findings in the disorder is a 50-70% increase in platelet concentrations of 5-HT. This

hyperserotonemia is observed in one third of people with autism (Cook and Leventhal,

1996; Lam et al., 2006) and their first degree relatives (Kuperman et al., 1987; Cook Jr. et

al., 1988; Abramson et al., 1989; Leboyer et al., 1999). Urinary concentrations of 5-HT

are also increased in the disorder (Barthelemy et al., 1988; Martineau et al., 1992). The

significance of these peripheral measures however, remains unclear. Attempts to

correlate blood concentrations of 5-HT to autism symptoms have yielded inconsistent

findings (Kuperman et al., 1987; Mulder et al., 2004; Kolevzon et al., 2010).

A number of genes related to 5-HT homeostasis and function have been

implicated in autism. 5-HT homeostasis is maintained by 5-HTT, encoded by the gene

SLC6A4 (Kalueff et al., 2010). Polymorphisms of the promoter region for SLC6A4

result in altered 5-HTT expression and 5-HT concentrations (Lesch et al., 1996) and have

been associated with autism susceptibility (Cook and Leventhal, 1996; Tordjman et al.,

1997; Kim et al., 2002; Devlin et al., 2005; Sutcliffe et al., 2005). Furthermore, these

polymorphisms are associated with increased cortical gray matter volume (Wassink et al.,

2007), increased amygdala activity (Wiggins et al., 2013) more severe communication

deficits (Brune et al., 2006) and hyperserotonemia (Coutinho et al., 2004, 2007) in some

9

individuals with autism. The gene TPH2 encodes for tryptophan hydroxylase-2 the rate

limiting enzyme for 5-HT synthesis in the CNS (Müller and Jacobs, 2010). Single

nucleotide polymorphisms in TPH2 are associated with autism in Caucasian (Coon et al.,

2005) and Korean (Yang et al., 2012) populations. Variants in MAO A, the gene for

monoamine oxidase A, an enzyme responsible for 5-HT metabolism (Müller and Jacobs,

2010), are associated with an increased risk of autism (Yoo et al., 2009; Cheng et al.,

2010; Tassone et al., 2011), as well as more severe symptom expression in individuals

with autism (Roohi et al., 2009; Cohen et al., 2011). A recent study reported reduced

expression of TPH2 and MAO A in cultured cell lines from individuals with autism

(Boccuto et al., 2013). Additionally, several 5-HT receptor genes have also been

associated with autism (Zafeiriou et al., 2009).

Additional evidence for the involvement of the 5-HT system in autism comes

from drug treatment and challenge studies. Drugs that increase 5-HT, such as selective

serotonin reuptake inhibitors (SSRIs) effectively alleviate anxiety, aggression and

stereotypies in adults with autism (Cook and Leventhal, 1996). However the efficacy of

these drugs in the treatment of children is debated (Volkmar, 2001). In adults, reducing

5-HT by tryptophan depletion exacerbates autism symptoms (McDougle et al., 1996).

Prenatal exposure to compounds that increase 5-HT levels such as antidepressants (Croen

et al., 2011; Rai et al., 2013), cocaine (Davis et al., 1992) and alcohol (Nanson, 1992;

Aronson et al., 1997) increase the risk for autism. These compounds can cross the

placenta (Rampono et al., 2009) or be excreted in the breast milk (Sie et al., 2012) and

alter fetal brain development (Mulder et al., 2011) and infant behavior (Casper et al.,

2003; Oberlander et al., 2005).

Neuroimaging studies suggest that 5-HT synthesis, concentrations and binding

activity are reduced in autism. Developmentally, whole brain concentrations of 5-HT

synthesis are altered in autism (Chugani et al., 1999). A positron-emission tomography

(PET) study showed that typically developing children synthesized high concentrations of

5-HT that peaked around age 5 and then declined to adult values. Autistic children did

not show this pattern, but rather had lower 5-HT synthesis capacity which increased

slightly with age. Additional studies by this group showed that a subset of children with

autism had asymmetries in cortical, thalamic, and cerebellar 5-HT synthesis (Chugani et

10

al., 1997; Chandana et al., 2005) that correlated with the degree of language impairment

and handedness (Chandana et al., 2005). 5-HTT binding is also reportedly reduced in

both children (Makkonen et al., 2008) and adults (Nakamura et al., 2010) with autism.

Reduced 5-HT2 binding has been noted in high functioning autistic adults (Murphy et al.,

2006; Beversdorf et al., 2012), and in the parents of children with autism (Goldberg et al.,

2009). Collectively these neuroimaging studies provide strong support for altered

serotonergic neurotransmission in individuals with autism, as well as their family

members.

Postmortem analyses of 5-HT in the autistic brain have only recently been

reported. Contrary to the imaging studies mentioned, investigations of neurons

expressing 5-HT, labeled with 5-HTT antibodies, are reportedly thicker and denser in the

brains from autistic donors compared to those from controls (Azmitia et al., 2011b;

Wegiel et al., 2013). Regions of increased neurons expressing 5-HTT include the

amygdala, hippocampus, cortex (Alzmitia et al., 2011a) and cerebellum (Wegiel et al.,

2013) of autism donors. Reports of increased 5-HTT expression do not necessarily

conflict with reports of reduced 5-HTT binding. Although still reportedly increased,

Azmitia's group noted that starting as young as age 12, the neurons expressing 5-HTT in

the brains of autistic individuals took on a degenerative morphology (2011a, 2011b). The

authors suggest that these dysmorphic 5-HT neurons may reflect degenerating neurons

and consequently a loss of transporter function. Therefore, it is possible that the 5-HT

system imbalance observed in autism reflects developmental over stimulation followed

by degeneration in adolescence.

5-HT plays a critical role in the development of the brain and regulates many of

the processes thought to be altered in ASDs, including, neurogenesis, apoptosis, cell

migration and synaptic plasticity (Pino et al., 2004). It has been suggested that

developmental disruptions to the 5-HT system, such as early exposure to elevated 5-HT

concentrations, may down-regulate central 5-HT circuitry and activity in the developing

brain (Whitaker-Azmitia, 2005) and contribute to the anatomical, behavioral and

emotional disturbances reported in autism (Zafeiriou et al., 2009). Pharmacologically or

genetically increasing neonatal 5-HT in rodents produces morphological and behavioral

pathologies consistent with autism (Kahne et al., 2002; McNamara et al., 2008; Moy et

11

al., 2009). For example, rodents exposed to high concentrations of 5-HT had thinner

corpus callosums (Bortolato et al., 2013; van der Marel et al., 2013), increased dendritic

arborization of prefrontal cortical neurons (Wellman et al., 2007; Bortolato et al., 2013),

and reduced numbers of cerebellar PC cells (Bortolato et al., 2013) when compared to

controls. Behaviorally, rodents exposed to high 5-HT concentrations showed reduced

juvenile play (Homberg et al., 2007; Simpson et al., 2011), reduced distress vocalizations

(Kahne et al., 2002), and reduced social interactions as adults (Overstreet et al., 2000;

Moy et al., 2009; Bortolato et al., 2013). Collectively the available data provides strong

evidence for a role in disruptions to 5-HT homeostasis in the neuropathology and

behavioral deficits of autism.

1.7. Dissertation Overview:

Autism is a disorder of altered brain development. 5-HT is a critical regulator of

brain development and dysfunctional 5-HT signaling is associated with many of the

neuroanatomical and behavioral symptoms of autism. Therefore, the aim of the current

studies was to investigate the role of 5-HT in experimental and clinical models of autism.

Chapter 2 quantifies brain monoamine neurotransmitter concentrations, especially

5-HT, in the BTBR Itpr3tf/J (BTBR) mouse, a strain which displays behaviors resembling

all three diagnostic symptom clusters of autism. In comparison to the common C57BL/6J

(B6) control strain, BTBR mice showed cortical and cerebellar changes in 5-HT and its

metabolite concentrations. BTBR mice had increased concentrations of cortical 5-HT

and 5-hydroxyindoleacetic acid (5-HIAA) as well as reduced cerebellar 5-HT and 5-

HIAA when exposed to novel environments. These findings are consistent with the

hypothesis of impaired cerebellar-cerebral communication in autism, and suggest that

disturbed 5-HT-signaling pathways may contribute to some of the autism relevant

behaviors displayed by the BTBR.

Chapter 3 aims to identify the consequences of genetic modulation of the

serotonergic system in a mouse model of autism. The serotonin transporter (5-HTT) gene

(SLC6A4) regulates 5-HT concentrations and polymorphisms in this gene are associated

with an increased risk for autism (Devlin et al., 2005; Huang and Santangelo, 2008).

Furthermore, reduced 5-HTT function appears to contribute to the anatomical and

behavioral features of the disorder (Sutcliffe et al., 2005; Brune et al., 2006; Wassink et

12

al., 2007). Chapter 3 assesses autism-relevant behaviors of mice deficient in the

serotonin transporter gene (5-HTT KO). 5-HTT KO mice displayed increased

stereotyped grooming when compared to wild-type littermates. These results suggest that

mice lacking 5-HTT display some modest behavioral changes relevant to autism and may

be a useful model for investigating molecular mechanisms which contribute to the

symptoms of ASDs.

Chapters 2 and 3 provide evidence that altered 5-HT neurotransmission may

underlie some of the autism-relevant behaviors displayed by the BTBR and 5-HTT

mutant mouse models. Therefore, Chapter 4 investigates the distribution of 5-HTT in

the postmortem human subventricular zone (SVZ), a brain region involved in

neurodevelopment (Brazel et al., 2003; Ming and Song, 2005). The SVZ tissue from

autism-diagnosed (AD) individuals displayed a number of structural and compositional

differences when compared to typically-developing (TD) controls. 5-HTT and fibroblast

growth factor-2 (FGF-2) immunoreactivity were significantly increased in AD cases.

Coupled with findings of reduced hypocellular gaps and increased cell density, these

findings support reports of aberrant neurogenesis and migration in the brains of AD

individuals.

13

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CHAPTER 2. NEUROCHEMICAL CHARACTERIZATION OF THE BTBR T +

Itpr3tf/J MOUSE MODEL FOR AUTISM.

2.1. Abstract

Rationale

Animal models of autism provide opportunities to evaluate potential disturbances

in biological systems in the disorders. Several lines of research suggest that

abnormalities of neurotransmitter systems, especially serotonergic, occur in autism. The

inbred BTBR T+Itpr3tf/J mouse strain (BTBR) displays several behaviors analogous to

the core symptoms of autism. Thus, the BTBR mouse provides an excellent opportunity

to explore neurotransmitter systems and potential disturbances that may be relevant to

autism.

Objectives

Experiment 1 measured neurotransmitter concentrations of BTBR mice and

C57BL/6J (B6) mice at baseline. Experiment 2 measured neurotransmitter

concentrations of BTBR and B6 mice in a novel environment with a nonsocial stimulus

and in a novel environment with a social stimulus that provided social proximity

conditions. BTBR and B6 behavior was also assessed in the novel nonsocial and social

proximity conditions.

Methods

Brain tissue concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-

HT) and their respective metabolites were measured using high performance liquid

chromatography (HPLC) with electrochemical detection.

Results

In the social proximity test, BTBR mice displayed decreased facial contact and

increased crawl over and crawl under behaviors. Strain differences in brain

concentrations of NE, DA, 5-HT and their metabolites were observed at baseline and in

response to novel environments. Of the regions measured, the cerebellum showed the

most differences in neurotransmitter concentrations between BTBR and B6 mice and the

occurrence and direction of these differences were consistent in experiments 1 and 2.

32

Conclusions

These data provide the first documentation for altered neurotransmitter systems in

BTBR mice. Neurotransmitter disturbances were especially prevalent in the cortex and

cerebellum; consistent with the theory of dysfunctional cerebellar-cortical connectivity

and communication in autism.

33

2.2. Introduction

Autism is a complex neurodevelopmental disorder that appears at an early age and

remains throughout adulthood. There are currently no consistent biological markers or

screening tests (Lord, 2011); therefore, diagnosis relies on the presence of core

behavioral features, which can vary greatly in their severity and manifestation (American

Psychiatric Association, 2013). Despite the lack of consistent biomarkers, growing

evidence suggests altered anatomical and functional connectivity in the autism brain

(Courchesne and Pierce, 2005; Geschwind and Levitt, 2007; Nair et al., 2013). Recent

findings specifically implicate impairments of cerebral-cerebellar connectivity in the

motor and affective symptoms of autism (Rogers et al., 2011; Heck and Howell, 2013).

Additionally, a role for neurochemical dysfunction in autism has been suggested

(McDougle et al., 2005; Lam et al., 2006); especially for the serotonergic (Cook and

Leventhal, 1996; Zafeiriou et al., 2009) and dopaminergic systems (Walker, 2008;

Kałuzna-Czaplinska et al., 2010).

Autism frequently occurs in conjunction with other conditions such as reduced

attention and hyperactivity (Hofvander et al., 2009), hyper-responsivity to sensory stimuli

(Green and Ben-Sasson, 2010), and mood disorders (Mazefsky et al., 2008; van Steensel

et al., 2011). Norepinephrine (NE) mediates arousal and attention (Iversen et al., 2008;

Ettinger, 2010) and has been implicated in the pathophysiology of depression and anxiety

(Ressler and Nemeroff, 2001; Aston-Jones, Gary, 2002). Additionally, drugs that act on

the adrenergic system have provided moderate improvements for hyperactivity and

aggression in individuals with autism (Volkmar, 2001; Lam et al., 2006) . Consequently,

NE has been suggested to play a role in mediating these frequently co-morbid symptoms.

Dopamine (DA) is associated with a variety of social and affiliative behaviors

such as pair bonding, reproduction, maternal care, and aggression (Insel, 2003; Young et

al., 2008). Therefore, the DA system is of particular interest in understanding the deficits

in social interactions observed in people with autism. Interest in DA has also been

stimulated by evidence that DA antagonists such as the antipsychotic haloperidol reduce

motor stereotypies, aggression and hyperactivity (Buitelaar and Willemsen-Swinkels,

2000; McDougle et al., 2005) in people with autism. Hyperactivity and stereotypy,

34

frequently observed in autism, can be induced in animals by agonizing central DA

functioning (Fog, 1969).

5-HT is widely distributed in the CNS and has been implicated in diverse

physiological states and behaviors (Iversen et al., 2008). The 5-HT system is one of the

earliest to develop in all species and is implicated in regulating brain development and

maturation (Whitaker-Azmitia, 2001; Zafeiriou et al., 2009), which may be especially

relevant to developmental disorders such as autism. In rodents, serotonergic system

disruptions produce autism-relevant behavioral phenotypes (Whitaker-Azmitia, 2005;

Borue et al., 2007; Boylan et al., 2007; Martin et al., 2012). Furthermore,

pharmacological interventions that enhance 5-HT activity reduce aggression and

stereotyped movements (Cook and Leventhal, 1996; Tsai, 1999; Buitelaar and

Willemsen-Swinkels, 2000) in autism. Treatments that reduce 5-HT availability, such as

acute tryptophan depletion, exacerbate these symptoms (McDougle et al., 1996). These

findings suggest that at least some symptoms of autism such as stereotypy, repetitive

behaviors, and anxiety may be related to reduced 5-HT function.

Mouse models of autism provide opportunities to evaluate potential disturbances

in biological systems in the disorder. In the absence of a clear biological marker, mouse

models displaying autism-relevant behaviors may be useful for assessing genetic,

environmental and biochemical contributions to the manifestation of symptoms (Bolivar

et al., 2007; Moy et al., 2007; McFarlane et al., 2008; Silverman et al., 2010a). The

BTBR inbred mouse strain presents a robust autism-relevant behavioral phenotype that

includes parallels to all three diagnostic symptom clusters in autism (Bolivar et al., 2007;

McFarlane et al., 2008; Blanchard et al., 2011; Meyza et al., 2012). When compared to

C57BL/6J (B6) mice, previous studies have shown that BTBR mice display low levels of

social interaction in a three-chambered test for social approach (Yang et al., 2007;

McFarlane et al., 2008) and in a visible burrow system providing ethologically relevant

social conditions (Pobbe et al., 2010). Scent marking and ultrasonic vocalizations, two

modes of communication in mice, are also impaired in the BTBR strain (Scattoni et al.,

2008, 2011; Roullet et al., 2011; Wöhr et al., 2011). In addition, BTBR mice display

high levels of spontaneous repetitive self-grooming (Silverman et al., 2010a; Pearson et

al., 2011). Thus, the BTBR mouse provides an excellent opportunity to explore potential

35

neurotransmitter systems and disturbances that may be relevant to autism. In the present

experiment regional neurotransmitter concentrations were measured in BTBR and B6

mice at baseline after single housing, and when exposed to a novel nonsocial stimulus or

to a novel social stimulus in a social proximity condition.

2.3. Materials and Methods

2.3.1. Subjects

Subjects were 60 adult male C57BL/6J (B6) and BTBR T+ Itpr3tf

(BTBR) mice

(64-131 days of age) bred from stock originally obtained from the Jackson Laboratory

(Bar Harbor, ME). At weaning all subjects were housed in same-strain, same-sex groups

of 4 to 6 and maintained on a 12-h light/dark cycle (lights on at 06:00 am). Food and

water were provided ad libitum. All procedures were conducted in accordance with

protocols approved by the University of Hawaii Institutional Animal Care and Use

Committee in accordance with the National Institutes of Health Guidelines.

2.3.2. Apparatus

The social proximity chamber was a clear rectangular acrylic chamber (7 x 14 x

30 cm H).

2.3.3. Procedure

Experiment 1: Baseline

To assess baseline neurotransmitter concentrations, BTBR mice and B6 mice

(n=10/strain), were singly housed 24 hours prior to the experiment. On the day of

testing, subjects were transported to a room separate from the colony room, rapidly

decapitated and their brains were removed for dissection.

Experiment 2: Nonsocial and Social Proximity

To assess neurotransmitter concentrations when in social proximity, subjects were

singly housed 24 hours prior to the experiment and randomly assigned to either the

nonsocial condition or the social condition. Subjects assigned to the nonsocial condition

(n=10/strain) were placed in the proximity chamber with an upright 50 mL conical tube

filled with 35 g of clean bedding to prevent it from falling over onto the mouse. This

nonsocial object was included to provide a novel nonsocial stimulus to investigate and to

restrict the space in the chamber to an extent similar to that in the social condition. In the

nonsocial proximity condition several behaviors were measured including: sniffing,

36

rearing on and contacting the tube, as well as grooming, general rearing/upright and jump

escapes. Subjects assigned to the social condition (n=10/strain) were paired with an

unfamiliar, sex and strain-matched conspecific inside the proximity chamber (Figure 2.1).

In the social proximity condition the frequencies and duration of the following behaviors

were manually quantified:

Nose tip-to-Nose tip (NT): subject's nose tip and or/vibrissae contact the nose

tip and/or vibrissae of the other mouse.

Nose-to-Head (NH): subject's nose tip contacts the head of the stimulus

mouse.

Nose-to-Anogenital (NA): subject's nose tip contacts the base of the tail or

anogenital region of the other mouse.

Crawl Over (CO): subject's forelimbs cross the midline of the dorsal

surface of the other mouse.

Crawl Under (CU): subject's head goes under the ventral surface of the

other mouse to a depth of at least the ears of the

subject animal crossing the midline of the other

mouse's body.

Jump Escape (JE): subject makes a vertical leap with all feet leaving

the ground.

The apparatus was cleaned with 20% ethanol between each exposure session. All

testing was conducted under dim red light during the light phase of the light/dark cycle,

as previously described (Defensor et al., 2011). Social or nonsocial exposures lasted 20

minutes. Immediately following the exposure session mice were moved to a separate

room, rapidly decapitated and their brains were removed.

37

Figure 2.1. BTBR mice in the

social proximity test. The side

walls of the chamber are tinted

a false gray to emphasize the

area of confinement. (Defensor

et al., 2011)

Tissue preparation

Dissections of the medial prefrontal cortex, striatum, hippocampus,

hypothalamus, brain stem, cerebellum, and the remaining cortex were collected for

neurochemical analysis. All tissues were promptly weighed in tared 1.5 mL Eppendorf

tubes and frozen on dry ice. The tissue concentrations of monoamines and metabolites

were quantified by high performance liquid chromatography (HPLC) with

electrochemical detection, using a slight modification of a previously described method

(Dunn, 1993).

HPLC

Brain samples were homogenized by ultrasonic disruption in ice cold 0.1M

HClO4 containing 1 mM EDTA and an internal standard of N-methyldopamine (NMDA).

Samples were centrifuged at 20,000xg for 20 minutes at 4º C. The supernatant was

removed and 100 µL was injected into the chromatographic system at a flow rate of 1.1

ml/min. The HPLC system consisted of a liquid chromatograph pump (LC-20 AD;

Shimadzu Co., Kyoto, Japan), a refrigerated Shimadzu autosampler (20AC), a 5 µm

ODS1 reverse-phase column (Spherosorb 4.6 x 250 mm; Waters Corp., Milford, MA,

USA) maintained at a constant temperature in the range of 35-42º C by a BioAnalytical

Systems (BAS) column heater (LC22A; BioAnalytical Systems, West Lafayette, IN), a

BAS dual channel detector (LC-4B) and collected by a data acquisition system (LC

Solutions, Shimadzu). Electrode potentials were set at 0.78 and 0.95 V with respect to a

38

Ag-AgCl reference electrode. The mobile phase contained 0.1M NaH2PO4, 0.1mM

EDTA (pH 2.8-3.1), 0.3mM 1-octanesulfonic acid and 4% (v/v) acetonitrile.

All tissue samples were analyzed for norepinephrine (NE) and its major

metabolite 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), dopamine (DA) and its

metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), tryptophan (Trp), 5-

hydroxytryptamine (serotonin, 5HT) and its metabolite 5-hydroxyindoleacetic acid

(5HIAA). Concentrations of the monoamines and their metabolites within each sample

were calculated with reference to known standards. Corrections were made relative to

these standards and for brain tissue weight. Standard solutions were injected every day

before each region and after every 5 samples. Monoamine turnover was calculated by

determining the ratio of the metabolite to its monoamine as described previously (Dunn,

1988). Estimates of monoamine turnover are commonly used measures of the rate of

utilization of a neurotransmitter; which are thought to directly reflect the activity of the

neurons that release it (Commissiong, 1985; Jones et al., 1996). Data are expressed as

nanograms of compound per milligram of brain tissue.

2.3.4. Statistical analyses

The neurochemical data from experiment 1 and behavioral data from the social

proximity test of experiment 2 were analyzed using a two-tailed Student’s t-test. The

neurochemical data for experiment 2 was analyzed with separate two-way ANOVAs

(strain x condition) conducted for each brain region and each analyte. Some samples

were lost during HPLC preparation resulting in different degrees of freedom across brain

regions. The critical value for significance was set at p < 0.05 for all analyses. Fisher’s

LSD post hoc tests were performed on statistically significant main effects.

2.4. Results

2.4.1. Experiment 1- Baseline

Strain differences in baseline concentrations of the monoamines, metabolites and

utilization ratios are shown in Table 2.1. BTBR mice had significantly less NE in the

cortex [t(18) = 3.81, p < 0.01]; and significantly more NE in the hippocampus [t(18) =

3.39, p < 0.01] and hypothalamus [t(18) = 2.45, p < 0.05] as compared to B6 mice.

BTBR mice had significantly less of the NE metabolite MHPG in the cerebellum [t(18)=

4.79, p < 0.001] and brainstem [t(18) = 2.16, p < 0.05] compared to B6 mice. BTBR

39

mice had significantly increased MHPG:NE ratios in the cortex [t(18) = 3.17, p < 0.01],

but decreased MHPG:NE ratios in the hippocampus [t(18) = 2.67, p < 0.05], and

brainstem [t(18) = 2.84, p < 0.05].

BTBR mice had significantly more DA in the cortex [t(18) = 2.19, p < 0.05], but

significantly less DA [t(18) = 2.81, p < 0.05] and DOPAC [t(18) = 3.90, p < 0.001] in the

cerebellum compared to B6 mice.

BTBR mice had less 5-HT [t(18) = 4.97, p < 0.001] and 5-HIAA [t(18) = 5.45, p

< 0.001] in the cerebellum when compared to B6 mice. There were no significant

differences in baseline concentrations of tryptophan or 5-HIAA:5-HT ratios.

40

Table 2.1. Regional baseline concentrations of monoamines, metabolites, and monoamine turnover for BTBR and B6 mice.

Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM)

NE B6 0.957 (0.046) 0.651 (0.068) 1.614 (0.283) 2.803 (0.062) 5.529 (0.379) 0.870 (0.256) 1.536 (0.163)

BTBR 1.013 (0.066) 0.361 (0.034) ** 1.432 (0.266) 3.165 (0.087) ** 6.739 (0.317) * 0.686 (0.123) 2.210 (0.672)

MHPG B6 0.288 (0.094) 0.158 (0.010) 0.322 (0.041) 0.070 (0.004) 0.147 (0.012) 0.046 (0.003) 0.077 (0.006)

BTBR 0.174 (0.044) 0.173 (0.018) 0.336 (0.067) 0.062 (0.006) 0.157 (0.028) 0.030 (0.002) *** 0.053 (0.009)

MHPG:NE B6 0.281 (0.082) 0.270 (0.032) 0.290 (0.061) 0.025 (0.001) 0.027 (0.002) 0.381 (0.298) 0.053 (0.005)

BTBR 0.193 (0.057) 0.527 (0.074) ** 0.294 (0.068) 0.020 (0.002) * 0.023 (0.003) 0.063 (0.014) 0.031 (0.006)

DA B6 4.491 (2.698) 3.376 (0.218) 9.210 (1.902) 0.161 (0.035) 1.505 (0.157) 0.074 (0.004) 0.159 (0.005)

BTBR 2.346 (1.146) 4.409 (0.419) * 11.845 (2.450) 0.178 (0.053) 1.346 (0.166) 0.054 (0.006) * 0.160 (0.006)

DOPAC B6 0.375 (0.136) 0.140 (0.008) 0.440 (0.077) 0.036 (0.003) 0.172 (0.026) 0.020 (0.001) 0.034 (0.004)

BTBR 0.190 (0.051) 0.153 (0.011) 0.422 (0.078) 0.017 (0.006) 0.156 (0.020) 0.016 (0.001) *** 0.029 (0.003)

DOPAC:DA B6 0.446 (0.261) 0.043 (0.003) 0.063 (0.011) 0.278 (0.032) 0.113 (0.007) 0.280 (0.013) 0.214 (0.023)

BTBR 0.234 (0.074) 0.037 (0.003) 0.053 (0.017) 0.094 (0.030) 0.118 (0.008) 0.311 (0.025) 0.182 (0.017)

5-HT B6 2.655 (0.325) 2.434 (0.105) 1.185 (0.234) 3.164 (0.180) 7.779 (0.546) 1.083 (0.086) 3.093 (0.228)

BTBR 2.396 (0.146) 2.584 (0.069) 1.700 (0.264) 3.269 (0.180) 7.357 (0.679) 0.588 (0.051) *** 2.763 (0.231)

5-HIAA B6 2.329 (0.353) 1.508 (0.093) 2.040 (0.257) 3.205 (0.165) 7.729 (1.069) 0.998 (0.065) 3.947 (0.538)

BTBR 1.955 (0.175) 1.469 (0.060) 1.900 (0.115) 3.074 (0.147) 6.593 (0.663) 0.614 (0.028) *** 2.952 (0.334)

TRP B6 3.779 (0.539) 1.528 (0.141) 4.657 (0.901) 3.790 (0.353) 6.274 (0.634) 2.298 (0.281) 3.693 (0.531)

BTBR 2.945 (0.170) 1.341 (0.049) 3.873 (0.487) 3.426 (0.108) 5.392 (0.408) 2.137 (0.179) 3.375 (0.076)

5-HIAA:5-HT B6 0.896 (0.091) 0.626 (0.626) 2.438 (0.507) 1.036 (0.074) 0.989 (0.097) 0.957 (0.070) 1.237 (0.105)

BTBR 0.817 (0.056) 0.569 (0.019) 1.433 (0.293) 0.953 (0.045) 0.896 (0.044) 1.082 (0.061) 1.065 (0.082)

Cerebellum Brainstem

Strain

mPFC Cortex Striatum Hippocampus Hypothalamus

Means are presented in ng/mg tissue. mPFC, medial prefrontal cortex; NE, norepinephrine; MHPG, 3-methoxy-4-

hydroxyphenylethyleneglycol; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-

hydroxyindoleacetic acid; TRP, tryptophan. Significant differences between BTBR and B6 mice are noted with *p < 0.05, **p < 0.01,

and ***p < 0.001.

41

2.4.2. Experiment 2 - Nonsocial and Social Proximity

Behaviors in the nonsocial and social proximity conditions

No significant differences between the strains were detected for any of the

behaviors in the nonsocial proximity condition (Figure 2.2A). There were clear strain

differences in the frequency of behaviors observed in the social proximity chamber

(Figure 2.2B). Consistent with the results of Defensor et al. (2011), BTBR mice made

significantly fewer nose tip-to-nose tip investigations [t(18) = 3.64, p < 0.01] and

significantly more nose-to-anus investigations [t(18) = 3.03, p < 0.01]. Additionally,

BTBR mice displayed reliably increased crawl over behaviors [t(18) = 3.94, p < 0.001].

There was also a trend for BTBR mice to demonstrate increased crawl under behaviors,

but this failed to reach statistical significance [t(18) = 1.92, p = 0.07].

Figure 2.2. Frequency of behaviors in the nonsocial (A) and social proximity (B)

conditions. Data are expressed as mean (+ SEM). NT, nose tip-to-nose tip; NH, nose-

to-head; NA, nose-to-anogenital; CO, crawl over, CU, crawl under; JE, jump escape.

Significant differences between BTBR and B6 are indicated by * p < 0.05, ** p < 0.01,

*** p < 0.001.

Neurochemistry

Main effects of strain and condition

Significant main effects of strain indicated differences between BTBR and B6

mice when collapsed across the social and nonsocial conditions. Main effects of strain

for brain NE, MHPG and MHPG:NE ratios are presented in Table 2.2a. BTBR mice had

42

higher concentrations of NE in the medial prefrontal cortex [F(1,33) = 4.58, p < 0.05] and

hippocampus compared to B6 mice [F(1,34) = 20.62, p < 0.001]. BTBR mice had less

MHPG in the hippocampus [F(1,34) = 20.56, p < 0.001] and in the cerebellum [F(1,36) =

42.45, p < 0.001]. BTBR mice also had lower MHPG:NE ratios in the hippocampus

[F(1,34) = 60.94, p < 0.001].

Main effects of strain for brain DA, DOPAC and DOPAC:DA are presented in

Table 2.2b. BTBR mice had more DA [F(1,36) = 7.87, p < 0.01] and DOPAC

[F(1,36) = 15.05, p < 0.001] in the cortex and less DA [F(1,36) = 13.03, p < 0.001] and

DOPAC [F(1,36) = 31.42, p < 0.001] in the cerebellum as compared to B6 mice.

Main effects of strain for brain 5-HT, 5-HIAA and 5-HIAA:5-HT ratios are

presented in Table 2.2c. BTBR mice had more 5-HT [F(1,36) = 7.17, p < 0.05] and 5-

HIAA [F(1,36) = 9.21, p < 0.01] in the cortex, less 5-HT [F(1,36) = 28.80, p < 0.001] and

5-HIAA [F(1,36) = 18.06, p < 0.001] in the cerebellum, and less 5-HT [F(1,36) = 8.28, p

< 0.01] and 5-HIAA [F(1,36) = 8.70, p < 0.01] in the brainstem when compared to B6

mice. BTBR mice had higher 5-HIAA:5-HT ratios [F(1,36) = 19.85, p < 0.001] in the

cerebellum.

A significant main effect of condition indicated a difference between the social

and nonsocial conditions when collapsed across strains. A significant main effect of

condition indicated that the DOPAC:DA ratios in the cerebellum [F(1,36) = 8.79, p <

0.01] were higher in the nonsocial condition when compared to the social proximity

condition.

Interactions between strain and condition

Post-hoc analysis following a significant interaction between strain and condition

for NE in the medial prefrontal cortex [F(1,33) = 5.17, p < 0.05] showed that BTBR mice

in the social condition had more NE in the medial prefrontal cortex than B6 mice in the

social condition (Fig. 2.3). In addition, B6 mice had less NE in the social condition when

compared to B6 mice in the nonsocial condition.


for cortical MHPG concentrations [F(1,36) = 6.06, p < 0.05] showed that BTBR mice in

the social condition had more MHPG in the cortex than B6 mice in the social condition

43

(Fig. 2.4). In addition, B6 mice had less MHPG in the social condition compared to their

nonsocial counterparts.


for DOPAC:DA ratios [F(1,36) = 6.08, p < 0.05] showed that BTBR mice in the social

condition had higher DOPAC:DA ratios in the hypothalamus than B6 mice in the social

condition (Fig. 2.5).

There were no significant strain by condition interactions for 5-HT, 5-HIAA, or

5-HIAA:5-HT ratios.

44

Table 2.2. Regional concentrations of monoamines, metabolites, and monoamine turnover collapsed across nonsocial and

social conditions.

a. Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM)

NE B6 1.081 0.071 0.506 0.057 1.982 0.243 3.205 0.079 5.970 0.272 0.760 0.147 1.633 0.136

BTBR 1.266 0.058 * 0.454 0.038 2.133 0.238 3.690 0.072 *** 5.637 0.299 0.627 0.081 1.732 0.122

MHPG B6 0.188 0.028 0.101 0.008 0.388 0.078 0.079 0.002 0.205 0.014 0.061 0.003 0.097 0.004

BTBR 0.228 0.056 0.103 0.011 0.501 0.096 0.061 0.004 *** 0.214 0.016 0.038 0.001 *** 0.087 0.006

MHPG:NE B6 0.194 0.041 0.250 0.031 0.291 0.079 0.025 0.001 0.035 0.003 0.384 0.180 0.064 0.004

BTBR 0.193 0.051 0.267 0.033 0.358 0.088 0.016 0.001 *** 0.039 0.002 0.071 0.005 0.054 0.005

Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM)

b. DA B6 2.220 0.741 2.489 0.163 6.217 1.248 0.188 0.027 1.654 0.081 0.082 0.004 0.186 0.009

BTBR 3.751 1.158 3.167 0.197 ** 9.503 1.787 0.150 0.026 1.498 0.083 0.064 0.003 *** 0.185 0.007

DOPAC B6 0.164 0.029 0.108 0.005 0.342 0.059 0.048 0.004 0.200 0.013 0.026 0.001 0.054 0.004

BTBR 0.228 0.051 0.140 0.007 *** 0.456 0.056 0.026 0.006 0.189 0.012 0.019 0.001 *** 0.048 0.002

DOPAC:DA B6 0.183 0.038 0.045 0.002 0.067 0.006 0.299 0.035 0.123 0.006 0.322 0.014 0.296 0.021

BTBR 0.160 0.031 0.046 0.002 0.068 0.007 0.196 0.043 0.127 0.005 0.311 0.018 0.261 0.012

c. Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM)

5-HT B6 2.237 0.103 1.551 0.048 1.032 0.143 3.395 0.104 7.732 0.452 1.091 0.106 3.251 0.148

BTBR 2.125 0.121 1.782 0.079 * 1.151 0.168 3.246 0.135 7.049 0.450 0.499 0.023 *** 2.619 0.157 ***

5-HIAA B6 2.521 0.178 1.193 0.028 2.131 0.123 4.026 0.123 8.220 0.516 1.118 0.106 4.702 0.308

BTBR 2.481 0.140 1.337 0.041 ** 1.987 0.133 4.051 0.133 7.332 0.467 0.650 0.021 *** 3.409 0.302 ***

TRP B6 5.243 0.436 1.036 0.085 6.506 0.549 5.233 0.217 8.306 0.502 3.051 0.230 5.289 0.386

BTBR 4.247 0.204 1.137 0.093 6.315 0.663 4.925 0.164 7.986 0.346 2.654 0.113 4.713 0.212

5-HIAA:5-HT B6 1.117 0.049 0.776 0.021 2.793 0.295 1.192 0.030 1.062 0.029 1.049 0.045 1.464 0.094

BTBR 1.176 0.040 0.766 0.026 2.451 0.282 1.259 0.022 1.053 0.039 1.326 0.041 *** 1.276 0.074

Hypothalamus Cerebellum Brainstem

mPFC Cortex Striatum Hippocampus Hypothalamus Cerebellum Brainstem

mPFC Cortex Striatum Hippocampus

Hypothalamus Cerebellum BrainstemmPFC Cortex Striatum Hippocampus

Means are presented as ng/mg tissue. mPFC, medial prefrontal cortex; NE, norepinephrine; MHPG, 3-methoxy-4-

hydroxyphenylethyleneglycol; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-

hydroxyindoleacetic acid; TRP, tryptophan. Significant differences between BTBR and B6 mice are noted with * p <0.05, ** p <0.01,

and *** p <0.001.

45

Figure 2.3. Concentrations of NE

in the mPFC in the proximity

conditions. Data are expressed as

mean (+ SEM). * indicates

difference, p < 0.05.

Figure 2.4. Concentrations of

MHPG in the cortex in the

proximity conditions. Data are

expressed as mean (+ SEM). *

indicates difference, p < 0.05.

Figure 2.5. Concentrations of

DOPAC:DA in the hypothalamus

in the proximity conditions. Data

are expressed as mean (+ SEM). *

indicates difference, p < 0.05.

46

2.5. Discussion

Social Proximity

In the current study BTBR mice showed social avoidance in the social proximity

test. These findings are consistent with a previous report of BTBR social deficits in

social proximity (Defensor et al., 2011). Here BTBR mice showed reduced nose tip-to-

nose tip contacts and increased crawl over behaviors (Figure 2.2B), suggesting active

avoidance of frontal orientations. We have previously suggested a parallel between this

unique behavioral pattern and the gaze aversion frequently observed in autism (Defensor

et al., 2011). In support of this hypothesis, the current results revealed no strain

differences in motor or investigative behaviors in the nonsocial condition (Figure 2.2A).

In addition, no aggressive contact was displayed by either strain in the social proximity

condition. Taken together, this suggests that the observed behavioral differences are not

due to altered motor, exploratory or aggressive behaviors. The current results

compliment previous findings of reduced frontal approaches (Yang et al., 2007; Pobbe et

al., 2010) and nose sniffing (McFarlane et al., 2008) in BTBR mice and are consistent

with an interpretation of active social avoidance in the BTBR in social proximity.

Norepinephrine

The noradrenergic system of the BTBR mouse showed disturbances in more of

the brain regions examined than any other neurotransmitter system measured in this

study. At baseline, BTBR mice had significantly different concentrations of NE, MHPG,

and MHPG:NE ratios in cortical, limbic, and hindbrain regions, suggesting widespread

regional differences in activity of NE systems in BTBR vs. B6 mice. Compared to

exposure to a novel nonsocial situation, the social proximity condition reduced

concentrations of NE in the mPFC and MHPG in the cortex for the B6 strain. Although

turnover rates (MHPG:NE ratios) were not significantly different for either of these areas,

these data suggest that regional activation of NE systems may differ in social vs.

nonsocial conditions for the B6 mice. However, the social vs. nonsocial proximity

situations produced no significant differences for any noradrenergic system parameters in

the BTBR mice; suggesting that BTBR mice fail to show a NE-based differentiation

between social and nonsocial conditions.

47

The central noradrenergic system originates in the brain stem and has diffuse

projections throughout the limbic system and cortex (Iversen et al., 2008; Ettinger, 2010).

Norepinephrine regulates several behaviors including attention, anxiety, and stress

response (Iversen et al., 2008; Ettinger, 2010) which are frequently disturbed in autism.

Drugs that reduce NE neurotransmission reduce aggression and impulsivity in individuals

with autism (Volkmar, 2001; Bauman and Kemper, 2006; Lam et al., 2006), suggesting

that disturbed noradrenergic functioning may contribute to certain behaviors observed in

autism.

Tests of anxiety in response to stress have been inconsistent in the BTBR strain;

with reports of no difference (Moy et al., 2007; Yang et al., 2009; Silverman et al.,

2010b), reduced (McFarlane et al., 2008; Pobbe et al., 2011), or enhanced (Benno et al.,

2009) anxiety; or anxiety that is dependent on context/stimulus (Pobbe et al., 2011).

Here, to novel stimuli, B6 mice showed changes in medial prefrontal cortex, and

remaining cortex consonant with those typically associated with stress, such as reductions

in NE, increased MHPG:NE ratios or increased tryptophan (Bliss et al., 1968; Thierry et

al., 1968; Dunn, 1988; Shanks et al., 1990). BTBR failed to show any of these,

suggesting that these particular novelty situations were not highly salient or stressful for

these mice; a view that is hard to evaluate against previous literature given the great

variability in results of behavioral studies of stress responsivity in this strain. However,

given the critical role of NE in the stress response, it is possible that the variable anxiety

behaviors seen in the BTBR are linked to the numerous area-based disturbances in the

NE system shown here under basal conditions.

Dopamine

At baseline and in response to novel situations, BTBR mice had higher

concentrations of DA in the cortex and lower concentrations of DA and DOPAC in the

cerebellum, as well as higher DOPAC in cortex in the novel situation. Recent findings

indicate that stimulation of the cerebellum evokes DA release in the medial prefrontal

cortex through glutamatergic inputs (Mittleman et al., 2008; Rogers et al., 2011, 2013).

The current findings of differences in both the cerebellum and cortex for BTBR mice are

compatible with a view of an impaired cerebellar-cortical pathway in the BTBR mouse,

when compared to B6 mice.

48

Clinically, dysfunction of cerebello-thalamo-cortical circuits has been implicated

in the symptom expression of autism (Catani et al., 2008; Hoppenbrouwers et al., 2008;

Sivaswamy et al., 2010). Cerebellar abnormalities, especially reduced Purkinje cells,

have been proposed to disrupt cerebello-thalamo-cortical communication through

disinhibition of the deep cerebellar nuclei, resulting in increased excitatory output in the

thalamus and cortex (Belmonte et al., 2004a, 2004b). Disturbances to the cerebellum,

which are commonly observed in autism (Palmen et al., 2004; Wegiel et al., 2013), could

result in a loss of functional connectivity between the cerebellum and cortex, which is

manifested as abnormal DA activity in the cortex (Rogers et al., 2011).

Aberrant DA transmission, in the cerebellum and elsewhere, has been implicated

in stereotypic motor behaviors. Rates of stereotyped behaviors were negatively correlated

with the area of cerebellum vermis lobules VI-VIII in a study of children with autism

(Pierce and Courchesne, 2001), while compounds that antagonize central DA activity,

such as haloperidol, are effective in alleviating stereotypies and hyperactivity (Tsai,

1999; Buitelaar and Willemsen-Swinkels, 2000; Volkmar, 2001). Taken together, these

studies suggest that reduced DA availability and activity in the cerebellum may be

involved in the repetitive behaviors observed in autism, in addition to potentially

contributing to cortical changes associated with the disorder. That such DA changes are

also characteristic of the BTBR mouse adds to a view that these mice may provide a

biologically meaningful animal model of autism.

Serotonin

Results from the current study suggest only moderate changes in regional 5-HT

and 5-HIAA concentrations in the BTBR under baseline conditions, with differences

(reductions) restricted to the cerebellum. However, in response to novel situations and

social stimuli, 5-HT and 5-HIAA show a number of additional differences, with

reductions in the cerebellum and brainstem, and with increases in both, in the cortex.

This pattern, similar to the baseline DA differences in the cerebellum and cortex outlined

above, is in agreement with a view that anterior-posterior connections may be disturbed

in the BTBR mouse. In the forebrain, 5-HT is distributed largely by fibers originating in

the superior raphe nuclei (Jacobs and Azmitia, 1992), while the inferior raphe nuclei

project largely in a caudal direction, distributing 5-HT to the cerebellum and brainstem,

49

suggesting some differential involvement of projections originating in the superior and

inferior raphe, for the BTBR mouse. In support of this hypothesis, recent research

showed that 5-HTT expression during development regulates the guidance of 5-HT

projections from the dorsal raphe to their cortical targets (Witteveen et al., 2013). A lack

of developmental 5-HTT resulted in increased 5-HT projections from the median raphe to

the medial prefrontal cortex. Given the reports of reduced 5-HTT in the BTBR brain

(Gould et al., 2011), it is likely that the characteristics and projection patterns of the raphe

neurons may be altered in the BTBR.

The serotonergic system regulates a variety of complex behaviors and affective

states (Lucki, 1998) and is critical to CNS development and maturation (Whitaker-

Azmitia, 2001). Disturbances to this system during development produce anatomical and

behavioral features similar to those observed in autism (Palmen et al., 2004; Whitaker-

Azmitia, 2005). One well documented feature of autism is reduced cerebellar Purkinje

cells (Palmen et al., 2004; Bauman and Kemper, 2005), and 5-HT may be involved in this

reduction. Serotonin innervates all areas of the cerebellum (Ottersen, 1993), modulates

Purkinje cell firing (Dieudonné, 2001) and regulates the development (Oostland et al.,

2013) and arborization (Kondoh et al., 2004) of Purkinje cells through various receptors.

Taken together, these results suggest that altered cerebellar serotonin may be related to

the reduced Purkinje cells observed in autism.

More general support for a role for 5-HT functioning in autism-relevant behaviors

is evident from both genetic and pharmacological research. Aberrant social behaviors

have been noted in 5-HT3A receptor knockout (Smit-Rigter et al., 2010) and serotonin

transporter (5-HTT) knockout mice (Moy et al., 2009). Reduced 5-HTT binding has been

reported in both children (Makkonen et al., 2008) and adults with autism and was

negatively correlated with repetitive behaviors (Nakamura et al., 2010). Consistent with

the clinical data, Gould et al., (2011) found reduced 5-HTT binding in the BTBR brain.

Additionally, BTBR social deficits can be ameliorated by treatment with 5-HT agonists

such as the selective 5-HT reuptake inhibitor (SSRI) fluoxetine (Chadman, 2011; Gould

et al., 2011) or the 5-HT1A receptor agonist Buspirone (Gould et al., 2011). Several gain-

of-function 5-HTT coding variants have also been reported in children with autism (Glatt

et al., 2001; Sutcliffe et al., 2005). Mice with alterations in one of these, 5-HTT Ala56,

50

show enhanced 5-HT clearance rates and hyperserotonemia, with reduced basal firing of

raphe 5-HT neurons, as well as 5-HT1A and 5-HT2A receptor hypersensitivity. They also

display alterations in social function, communication, and repetitive behavior (Veenstra-

Vanderweele et al., 2012).

These findings suggest that serotonin systems may have multiple roles in the

development of an autism-relevant behavior phenotype. The present findings are

consonant with a view that serotonin systems in BTBR mice show differences from those

of B6 mice that are exacerbated in response to novel situations and social stimuli.

Area Focus

In this study, the cerebellum was the brain region that showed the greatest number

of differences in neurotransmitter concentrations for BTBR vs. B6 mice, and these

differences were consistent across Experiments 1 and 2 (Tables 2.1 and 2.2). While the

absolute values of the monoamines in the cerebellum are low in comparison to other

brain regions, there is considerable evidence of noradrenergic (Gordon Shepherd, 1990;

Ottersen, 1993), dopaminergic (Chrapusta et al., 1994; Delis et al., 2008) and

serotonergic (Gordon Shepherd, 1990; Ottersen, 1993) afferents to various layers of the

cerebellum, and they may modulate the activity of the cerebellum through their

interactions with various cerebellar neurons, including Purkinje cells (Gordon Shepherd,

1990; Ottersen, 1993).

Cerebellar disturbances are frequently reported in autism (Fatemi et al., 2012).

Cerebellar volume differences are reported as either increased proportionally with total

brain volume (Piven et al., 1997; Sparks et al., 2002; Herbert et al., 2003), or when

corrected for total brain volume (Hardan et al., 2001). Reduced numbers of Purkinje

cells are consistently reported in the disorder regardless of age, sex, or cognitive ability

(Ritvo et al., 1986; Courchesne, 1991; Bailey et al., 1998; Fatemi et al., 2002). Recent

investigations revealed cerebellar alteration in a signaling pathway (Ras/Raf/ERK1/2)

involved in apoptosis of neural cells in BTBR mice (Zou et al., 2011) and in the brains of

individuals with autism (Yang et al., 2011).

The cerebellum displays functional relevance to the behaviors that occur in

autism. Gaze behavior, which has been associated with the cerebellum (Brettler et al.,

2003; Fuchs et al., 2010; Kheradmand and Zee, 2011), is frequently abnormal in autism

51

and has been used as a predictor for early detection of the disorder (Baron-Cohen et al.,

1996; Clifford et al., 2007). The BTBR mouse also displays gaze aversion-like behavior

(Defensor et al., 2011). In addition, oculomotor deficits, consistent with disturbances in

the cerebellar vermis, have been detected in autism (Takarae et al., 2004; Nowinski et al.,

2005; Wegiel et al., 2013). The cerebellum plays a critical role in the coordination of

motor functions, which are frequently impaired in patients with the disorder (Fournier et

al., 2010; Behere et al., 2012). In addition, impairments in communication are common

following trauma to the posterior fossa, which includes the cerebellum and brainstem

(Gudrunardottir et al., 2011), and reduced sociability has been reported in patients with

lesions to the cerebellar vermis (Riva and Giorgi, 2000).

The cerebellum receives a fine plexus of serotonergic fibers through which 5-HT

is released via volume transmission (Trouillas and Fuxe, 1993). 5-HT concentrations in

the cerebellum are positively correlated to an organism's level of motor activity (Mendlin

et al., 1996) and disturbances to these concentrations could have a number of deleterious

effects. Reduced 5-HT in the cerebellum could contribute to reduced Purkinje cell

complexity and firing (Oostland and van Hooft, 2013). As the primary cellular output for

the cerebellum, Purkinje cells contribute significantly to cerebellar-thalamo-cortical

communication. Therefore, Purkinje cell dysfunction as a consequence of deficient 5-HT

signaling, may contribute not only to the gaze-aversion like behavior displayed by the

BTBR but may also have long-ranging implications for autism brain connectivity

(Hoppenbrouwers et al., 2008).

Summary

In summary, BTBR differences in regional monoamine concentrations and

utilization, compared to those of B6 mice, suggest disturbances in anterior-posterior

connections related to monoamine distribution. These results support and compliment

findings of disturbed long-range connectivity in autism (Khan et al., 2013). The area

most consistently involved in these differences was the cerebellum, a structure that is

both the recipient of monoaminergic inputs and a modulator of monoaminergic

projections to the forebrain. Numerous similarities between the profiles of BTBR

differences compared to the B6 mouse, and findings from autism-diagnosed individuals,

provide additional support for the BTBR mouse as a model of autism. Future analyses

52

may benefit from the inclusion of additional strains with documented autism relevant

phenotypes and the use of in vivo techniques such as microdialysis or electrophysiology.

53

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62

CHAPTER 3. BEHAVIORAL CHARACTERIZATION OF THE SEROTONIN

TRANSPORTER KNOCKOUT MOUSE MODEL FOR AUTISM.

3.1. Abstract

Rationale

Autism is a behaviorally defined disorder with a strong genetic component.

Behavioral phenotyping of mice with targeted mutations in autism candidate genes are an

important tool for discovering mechanisms that underlie the disorder. The serotonin

transporter (5-HTT) gene (SLC6A4) regulates 5-HT concentrations and has been

implicated in autism susceptibility. 5-HTT knockout (KO, 5-HTT -/-) mice show

changes in 5-HT concentrations, neuroanatomy and behavior; however, they have not

been extensively studied in the context of autism.

Objectives

The purpose of the current study was to examine the consequences of life-long 5-

HTT inactivation on rodent behaviors relevant to the core symptoms of autism.

Methods

Comprehensive behavioral phenotyping was conducted on 5-HTT wild-type (WT,

5-HTT +/+), heterozygous (HET, 5-HTT +/-) and knock-out (KO, 5-HTT -/-) mice on

measures of social interactions, communication, and repetitive behaviors. Social

interactions between genotypes were assessed in the visible burrow system (VBS), three-

chamber social approach and social proximity tests. Communication deficits were

assessed by the quantification of scent marking to a conspecific. Stereotyped and

repetitive behaviors were assessed by analysis of grooming patterns and spontaneous

investigation of novel objects. Additionally, aggression, a potential motivating factor in

social interactions, was examined in the resident intruder test.

Results

Results from the behavioral battery indicated that adult 5-HTT KO mice were

similar to their WT and HET littermates in tests of social interaction and communication.

However, 5-HTT KO mice displayed modest reductions in initial investigatory behaviors

in the VBS. 5-HTT KO mice also displayed increased stereotyped and repetitive

grooming patterns. Contrary to reports of reduced aggression in the 5-HTT KO, in the

63

VBS and resident intruder test, 5-HTT KO mice displayed similar levels of aggression to

their WT littermates.

Conclusions

These data suggest that dysfunctional 5-HTT activity may contribute to the

stereotyped and repetitive behaviors associated with autism.

64

3.2. Introduction

Autism is a behaviorally defined disorder characterized by a broad spectrum of

symptoms with varying degrees of severity. The underlying cause of the disorder is

unknown and there are no consistent biological markers available. However, a range of

findings suggest a role for reduced serotonin transporter (5-HTT) function in autism

(Devlin et al., 2005; Rose’meyer, 2013). Functional variants in the 5-HTT gene

(SLC6A4) that result in reduced 5-HTT expression have been associated with

susceptibility to autism (Cook and Leventhal, 1996; Kim et al., 2002; Devlin et al., 2005;

Sutcliffe et al., 2005). Imaging studies indicate that 5-HTT binding is reduced in children

(Makkonen et al., 2008) and adults (Nakamura et al., 2010; Oblak et al., 2013) with

autism. Furthermore, selective serotonin transporter inhibitors (SSRIs), which bind to 5-

HTT, effectively reduces aggression and stereotyped behaviors in adults with autism

(Cook and Leventhal, 1996; Tsai, 1999; Buitelaar and Willemsen-Swinkels, 2000).

5-HTT acts as a key regulator of 5-HT neurotransmission and homeostasis by

removing 5-HT from the synaptic cleft (Murphy et al., 2008). Thus 5-HTT determines

the magnitude and duration of 5-HT activity on post-synaptic sites (Canli and Lesch,

2007). The transcriptional activity of SLC6A4 is modulated by a length variation in the

promoter region of the gene (5-HTTLPR) (Heils et al., 1996). The short variant of this

polymorphism results in reduced 5-HTT expression and therefore reduced 5-HT uptake

from the synaptic cleft (Lesch et al., 1996; Murphy et al., 2008). The low expressing 5-

HTTLPR short variant has been associated with a number of neuropsychiatric disorders,

including autism (Devlin et al., 2005; Huang and Santangelo, 2008). Autistic individuals

with one or two copies of the short allele reportedly have increased cortical gray matter

volume (Wassink et al., 2007), altered amygdala activity in response to faces (Wiggins et

al., 2013), reduced neurochemical metabolites (Endo et al., 2010), hyperserotonemia

(Coutinho et al., 2004, 2007), and more severe impairments in social and communication

domains (Tordjman et al., 2001; Brune et al., 2006).

To explore the neurobiological implications of reduced 5-HTT, mice with a

partial or complete inactivation of 5-HTT function have been created on a variety of

backgrounds (Bengel et al., 1998). Homozygous knockouts (KO) completely lack 5-HTT

and consequently have increased 5-HT synthesis (Kim et al., 2005), extracellular 5HT

65

concentrations (Mathews et al., 2004), and turnover (Kim et al., 2005). Tissue

concentrations of 5-HT are reduced in these mice (Fox et al., 2008). Heterozygous mice

(HET) have approximately 50% fewer 5-HTT binding sites, elevated extracellular 5-HT

but unchanged 5-HT tissue concentrations (Mathews et al., 2004; Kim et al., 2005).

Behaviorally, the loss of 5-HTT results in a number of phenotypes including increased

anxiety (Holmes et al., 2003; Kalueff et al., 2007), increased behavioral despair (Zhao et

al., 2006; Wellman et al., 2007) and impaired fear extinction (Wellman et al., 2007).

Although these mice have been studied extensively, little is known regarding autism-

relevant phenotypes in this strain. Given the considerable evidence or disturbed 5-HTT

functioning in ASD, comprehensive phenotyping of mice with targeted 5-HTT

disruptions may clarify the mechanisms underlying autism-relevant behaviors.

The purpose of the current study was to examine the consequences of 5-HTT

inactivation on autism-relevant behaviors in adult male mice. Deficits in social

interactions, a core feature of autism, were assessed in a semi-natural visible burrow

system (VBS), three-chamber social approach and social proximity tests. Aggression

was examined in the resident intruder test. Deficits in communication, another core

symptom of autism, were assessed by the analysis of scent marking to a conspecific.

Lastly, the presence of stereotyped and repetitive behaviors was assessed by analysis of

grooming patterns and spontaneous investigation of novel objects.

3.3. Materials and Methods

3.3.1. Subjects

Subjects were male homozygous knockout (KO), heterozygous knockout (HET)

and wild type (WT) littermates (n=12 per genotype) bred in the University of Hawaii

Laboratory Animal facilities. The original breeding stock was obtained from The

Jackson Laboratory (Bar Harbor, ME), where the 5-HTT KO mice were generated and

backcrossed on a on a C57BL/6J (B6) strain. Mouse genotypes were determined using

purified DNA from tail biopsy after weaning at postnatal day 21. Genotypes were

identified by gel electrophoresis of DNA fragments of either 318 bp (WT), 210 bp (KO)

or both (HET). Subjects were marked with commercial hair dye (Jerome Russel hair

bleach) and ear tags for individual identification. Stimulus mice used in the social

behavior tests were adult male C57/BL6J (B6) mice again bred in-house from stock

66

obtained from The Jackson Laboratory (Bar Harbor, ME). Before and after weaning,

mice were maintained on a 12-hour light-dark cycle (lights on at 6:00am) with ambient

temperature at 22+2°C. After weaning, mice were housed with same-sex siblings in

groups of 2-6 in ventilated cages (35.5 W x 20 L x 13 cm H) until behavioral testing.

Food and water were available ad libitum. All procedures were conducted in accordance

with the National Institutes of Health Guide for the Care and Use of Animals and

approved by the University of Hawaii Laboratory Animal Service Institutional Care and

Use Committee.

3.3.2. Apparatus

Visible Burrow System

The visible burrow system (VBS) procedure was conducted in a large,

rectangular, galvanized metal bin (86 x 61 x 26 cm H) lined with sawdust bedding as

previously described (Pobbe et al., 2010). Briefly, a black Plexiglas wall divided the

VBS into an open "surface" area and a "burrow" consisting of three clear Plexiglas

chambers (12 x 7 x 6 cm H). Two of the chambers were connected to each other and to

the surface by clear Plexiglas tubes, while the third chamber was connected only to the

surface by a single clear Plexiglas tube. The tubes were fitted into the black dividing

wall permitting free access between the chambers and the surface. Food and water were

freely available on the surface.

Three-Chamber

The three-chamber apparatus was constructed of black Plexiglas (47 x 70 x 28 cm

H) as previously described (Moy et al., 2004). The enclosure was divided into three

equal sized compartments connected by manually operated doors. The outer

compartments each contained an inverted wire cup (Galaxy Cup, Kitchen plus) weighed

down by an empty glass jar. Clear Plexiglas inserts in the front wall of the apparatus

allowed recording of interactive behaviors.

Autogrooming

Autogrooming behavior was assessed in a rectangular, clear Plexiglas chamber (7

x 14 x 30 cm H). A wire mesh lid on top of the chamber allowed air circulation and

prevented escape.

67

Social Proximity

Social proximity testing was conducted in the same rectangular, clear Plexiglas

chamber (7 x 14 x 30 cm H) used in the Autogrooming test.

Repetitive Novel Object Contact Task

The repetitive novel object contact task was conducted in a clean standard

laboratory mouse cage (35.5 x 20 x 13 cm H) with approximately 1 cm of bedding on the

floor. An inverted Plexiglas mouse cage was placed as a lid for the test chamber which

permitted recording of the subject from above. The lid was divided into four equally

sized quadrants using laboratory tape to measure general locomotion.

Urinary Scent Marking

The scent marking arena was an inverted rat cage (43 x 22 x 20 cm H) bisected by

a wire mesh barrier which allowed the exchange of olfactory, visual, and auditory cues,

while preventing direct physical contact between the subject and stimulus. The arena was

lined with a 45 x 61 cm piece of drawing paper as substrate.

Resident Intruder

The resident intruder test was conducted in the home cage of the subject mouse

(35.5 x 20 x 13 cm H) with the food and water hopper removed.

3.3.3. Procedure

Behavioral Testing

Behavioral testing of subjects took place between postnatal days 70-100. All

subjects were exposed to the same series of behavioral procedures in the following order:

visible burrow system, three-chamber social approach, autogrooming, social proximity,

repetitive novel object contact, urinary scent marking, and resident intruder tests. Tests

were separated by 24 hours, except in the case of scent marking: Immediately after the

repetitive novel object contact test mice were singly housed for 15 days to encourage

territorial scent marking (Arakawa et al., 2009). All behavioral tests were performed

under ambient fluorescent lighting (120 lx) during the light phase of the light/dark cycle,

between 0900h and 1700h. Temperature (22+2°C) and humidity (70%) were controlled

in the experimental room. All experimental and stimulus mice were habituated to the

experimental room at least 30 minutes prior to testing. To eliminate odor cues, each

apparatus was thoroughly cleaned with 20% ethanol and dried with paper towels between

68

animals. All procedures were recorded for subsequent scoring by a trained observer blind

to the genotype of the subjects. Behavior was analyzed using Observer software (Noldus

Information Technology, Wageningen, The Netherlands) and J Watcher software

(Blumstein and Daniel, 2007).

Visible burrow system

Testing for social behaviors in the VBS was conducted as previously described

(Pobbe et al., 2010). Three days prior to colony formation, subjects were briefly

anesthetized and given a unique identifying mark with commercial hair dye (Jerome

Russel hair bleach). Colonies were formed by placing three male, non-littermate mice

from the same genotype into the apparatus at the beginning of the first dark period

(1800h). Colonies were maintained for 3 dark and 3 light periods. Video recordings

were collected for four hours during each light (0600-1100h) and dark (1700h-2200h)

period. The frequencies of behaviors for each mouse were analyzed by time sampling for

30 seconds, every 10 minutes. The following behaviors were counted: approaches to the

front of another mouse, approaches to the back of another mouse, huddling, alone,

allogrooming, self-grooming, chase, flight, follow, and vigorous contact (Arakawa et al.,

2007b). The mean frequencies of each behavior were summarized for each genotype and

for each light and dark phase during the three successive days and nights of the VBS

period.

Three-chamber social approach test

Social approach behavior was assessed as previously described (Moy et al., 2004).

Initially the subject mouse was placed into the center compartment and allowed to

explore the apparatus for 10 minutes. At the end of this habituation period, the subject

was returned to the center chamber, and an unfamiliar, same-sex, B6 stimulus mouse was

placed into one of the wire cups in the outer chambers, while the other wire cup remained

empty. The side containing the stimulus mouse was counterbalanced within groups. The

subject mouse was allowed to explore the entire chamber for an additional 10 minutes.

The frequency and duration of time spent in each of the chambers during the habituation

and testing period were recorded. Additionally, during the test phase, the frequency and

duration of investigative behaviors including: rear, self-grooming, contact with the cup,

69

sniff, stretch-attend, quick-withdraw, and nose-to-nose with the stimulus animal were

recorded.

Autogrooming

Grooming behavior was assessed as previously described (Pearson et al., 2011).

Mice were placed into the grooming apparatus for 20 minutes. The frequency and

duration of grooming episodes involving the paw, head, body, leg, or tail/genital areas

were scored. The frequencies of grooming bouts, interrupted bouts, transitions, incorrect

transitions, correct transitions, the percent of interrupted bouts and of incorrect transitions

were also measured. A grooming bout was an uninterrupted occurrence of grooming

followed by at least 6 seconds of non-grooming. An interrupted bout was a bout of

grooming that was interrupted by less than 6 seconds. The percentage of interrupted

bouts was calculated by dividing the number of interrupted bouts by the total number of

bouts. Transitions were identified as a change from one grooming site to another.

Correct transitions included transitions of grooming that followed the typical rostral-

caudal progression (paw/head/body/tail) (Kalueff and Tuohimaa, 2004). Incorrect

transitions were characterized by changes in grooming sites that did not follow the typical

rostral-caudal pattern. The percentage of incorrect transitions was calculated by dividing

the number of incorrect transitions by the total number of transitions.

Social proximity

Social proximity testing was conducted as previously described (Defensor et al.,

2011). The subject mouse was placed into the apparatus with an age and weight

matched, unfamiliar male B6 mouse for 10 minutes. The frequency and duration of the

following behaviors were quantified:

Nose tip-to-Nose tip (NT): the subject animal’s nose tip and/or vibrissae

contacts the nose tip and/or vibrissae of the stimulus

animal.

Nose-to-Head (NH): the subject animal's nose tip and/or vibrissae

contacts the head of the stimulus animal.

Nose-to-Anogenital (NA): the subject animal's nose tip contacts the

anogenital region of the stimulus animal.

70

Crawl Over (CO): the subject crawls over the stimulus animal,

crossing over at least half of the stimulus animal's

body.

Crawl Under (CU): the subject pushes under or crawls under the

stimulus animal, crossing under at least half of the

stimulus animal.

Allogroom (AG): the subject grooms the stimulus animal.

Self Groom (SG): the subject animal grooms its own body.

Repetitive novel object contact task

Investigation of novel objects was conducted as previously described (Pearson et

al., 2011). Subjects were first habituated to a clean mouse cage containing fresh sawdust

bedding. A clear, Plexiglas mouse cage was inverted and marked with four equal sized

quadrants using laboratory tape. This was then placed on top of the cage to permit video

recording from above. Mice were allowed to explore the apparatus for 20 minutes. The

number of quadrant entries was recorded for baseline locomotor activity. Twenty-four

hours later subjects were again placed into a similar clean cage containing fresh bedding.

A small novel plastic toy object was placed in each of the four corners, approximately 2

cm from the wall. The toys included: a Lego piece, a jack, a die, and a bowling pin. The

mice were allowed to explore the apparatus and the novel toys for 10 minutes. The

number of visits to each toy, the sequence of toy visits, and the frequency of repetitive

contacts with 3, or with 4 toys were scored. In order to determine if there was a genotype

effect on the tendency to prefer specific toys, the frequency of contacts with each of the

toys were ranked in decreasing order from maximum to minimum preference for each

subject, averaged by genotype and compared.

Scent Marking

Testing for scent making was conducted as previously described (Arakawa et al.,

2007a). Following the toy test, subjects were individually housed for 15 days. Twenty-

four hours prior to the scent marking test, subjects were placed in one compartment of the

apparatus for a 20 minute habituation period. At the same time the next day, the subject

was again placed into one side of the chamber and an unfamiliar male B6 mouse was

placed in the opposite compartment, separated by the wire mesh. Following the trial the

71

paper substrate was removed and fixed with a 6% Ninhydrin solution (Fisher) that binds

to proteins in the urine making them visible. After drying for 24 hours the number of

urinary scent marks was manually counted by placing a transparent 1 x 1 cm square grid

sheet on top of the marked sheet and counting the number of squares containing scent

marks.

Resident-Intruder

The resident-intruder test was conducted as previously described (Pearson et al.,

2012). A sex, age and weight matched B6 intruder was placed into the resident's home

cage for 10 minutes. The latency of the resident to attack was recorded. Additionally,

the frequency and duration of investigative behaviors including: sniff face, sniff body,

sniff rear, sniff tail, vigorous allogroom, attack, follow, upright, and tail rattle were

recorded (Pearson et al., 2012).

3.3.4. Statistical Analyses

Each of the behaviors recorded in the VBS was analyzed by two-way repeated

measures analysis of variance (ANOVA), with genotype as the between-subjects factor

and period as the repeated- measures within-subject factor. Separate comparisons were

conducted for the dark and light periods. Data from the three-chamber test were analyzed

by two-tailed Student's t-test to compare the time spent in the empty chamber with time

spent in the stimulus chamber for each genotype. One-way ANOVAs were used to

compare the frequencies and durations of behaviors in the autogrooming task, social

proximity test, and resident-intruder test. Scent marking data were analyzed by two-way

repeated-measures ANOVA, with genotype as the between-subjects factor and condition

(habituation vs. test) as the repeated-measures within-subjects factor. Post-hoc analyses,

when appropriate were conducted using Newman-Keul’s. All statistical analyses were

carried out using Statistica and significance was set at p < 0.05 for all tests.

3.4. Results

3.4.1. Visible Burrow System

Repeated measures ANOVA revealed that the frequency of social investigation

for all genotypes decreased across dark periods, suggesting habituation to the social

environment (Table 3.1). This main effect of dark period was observed for approach

front [F(2,6) = 11.61, p < 0.001], approach back [F(2,66) = 10.04, p < 0.001], vigorous

72

contact [F(2,66) = 6.82, p < 0.01], huddle [F(2,66) = 36.51, p < 0.001] and allogrooming

[F(2,66) = 4.69, p < 0.01]. There was no effect of genotype, or any genotype x period

interactions for any of behaviors measured during the dark periods. A main effect of

genotype on vigorous contact during the dark periods approached but did not reach

significance (p < 0.07). The average number of vigorous behaviors for each genotype

suggests that both WT and KO had more vigorous contacts then HETs (WT =1.16, HET

= 0.25, KO = 0.61). During the light periods, neither a main effect of genotype nor

period was observed for any of the behaviors measured (Table 3.1). Approaches to the

back and vigorous contacts were nearly absent in all genotypes during the light period

and were not able to be analyzed. Several genotype x light period interactions were

significant. For 5-HTT KO mice, following [F(4,66) = 2.80, p < 0.05; Figure 3.1A] was

increased during light period 3 when compared to light periods 1 and 2. Huddling

behavior also increased in the 5-HTT KO mice during light periods 2 and 3 when

compared to light period 1[F(4,66) = 4.71, p < 0.01; Figure 3.1B] . Similarly, the

frequency of being alone decreased in 5-HTT KO mice during light period 2 and 3 when

compared to light period 1[F(4,66) = 4.31, p < 0.01, Figure 3.1C].

73

Table 3.1. Statistical results for behaviors run in the VBS.

Behavior Main Effects

Genotype Period Strain x Period

Approach Front Dark F(2,33)=117.32, p=0.25, n.s. F(2,33)=11.61, p<0.0001 F(2,33)=0.09, p=0.98, n.s.

Light F(2,33)=1.90, p=0.17, n.s. F(2,33)=2.89, p=0.06, n.s. F(2,33)=0.81, p=0.52, n.s.

Approach Back Dark F(2,33)=0.91, p=0.41, n.s. F(2,33)=10.04, p<0.0001 F(2,33)=0.61, p=0.66, n.s.

Light N.A.

Flight Dark F(2,33)=0.54, p=0.58, n.s. F(2,33)=2.08, p=0.13, n.s. F(2,33)=1.75, 0.15, n.s.

Light F(2,33)=0.07, p=0.92, n.s. F(2,33)=0.59, p=0.56, n.s. F(2,33)=1.40, 0.24, n.s.

Chase Dark F(2,33)=0.41, p=0.66, n.s. F(2,33)=1.59, p=0.21, n.s. F(2,33)=1.57, p=0.21, n.s.


Follow Dark F(2,33)=2.03, p=0.15, n.s. F(2,33)=0.27, p=0.77, n.s. F(2,33)=1.42, p=0.24, n.s.

Light F(2,33)=1.91, p=0.16, n.s. F(2,33)=2.49, p=0.09, n.s. F(2,33)=2.80, p<0.03

Vigorous Dark F(2,33)=2.92, p=0.07, n.s. F(2,33)=6.82, p<0.002 F(2,33)=1.52, p=0.21, n.s.

Light N.A.

Huddle Dark F(2,33)=2.58, p=0.09, n.s. F(2,33)=36.51, p<0.0001 F(2,33)=0.77, p=0.55, n.s.


Alone Dark F(2,33)=1.80, p=0.18, n.s. F(2,33)=1.34, p=0.27, n.s. F(2,33)=2.78, p<0.05


Allogroom Dark F(2,33)=1.32, p=0.28, n.s. F(2,33)=4.68, p<0.05 F(2,33)=1.12, p=0.36, n.s.


Selfgroom Dark F(2,33)=2.31, p=0.11, n.s. F(2,33)=0.35, p=0.71, n.s. F(2,33)=0.94, p=0.45, n.s.


N.A. not able to be analyzed.

74

Figure 3.1. Frequency of social behaviors during the light phases in the VBS. Frequency of following (A), huddling (B), and alone (C) behaviors in the VBS. Values

are displayed as mean (+ SEM) for each genotype. # p < 0.05, ## p < 0.01, within-group

comparison, different from light period 1. & p < 0.05, within-group comparison,

different from light period 2.

75

3.4.2. Three Chamber

During the 10 minute habituation phase, no differences in locomotor activity

[F(2,33) = 0.53, p > 0.05, data not shown] were observed between genotypes. Similarly,

no side biases were found during the habituation phase (WT: t(22) = -0.20, p > 0.05;

HET: t(22) = -1.08, p > 0.05; KO: t(22) = -1.23, p > 0.05, data not shown). During the

testing phase, 5-HTT KO mice displayed reduced locomotion when compared to HET

littermates. KOs made fewer total compartment entries [F(2,33) = 3.46, p < 0.05, Figure

3.2A], fewer social compartment entries [F(2,33) = 3.81, p < 0.05] and fewer center

compartment entries [F(2,33) = 3.52, p < 0.05] when compared to HET mice (Figure

3.2B). Figure 3.3 displays the amount of time each genotype spent in each of the

compartments of the apparatus during the testing phase. T-tests indicated that all

genotypes displayed a social preference, indicated by spending more time in the

compartment containing the unfamiliar mouse when compared to the empty compartment

(WT: t(22) = 5.75, p < 0.001; HET: t(22) = 3.76, p < 0.001; KO: t(22) = 2.76, p < 0.05).

The genotypes did not differ in the frequencies of social behaviors recorded during the

testing phase (data not shown).

76

Figure 3.2. Locomotion in the three chamber test. Frequency of total (A) and individual (B) chamber entries in the three chamber test.

Values are displayed as mean + SEM. * p < 0.05, between-group comparison, different

from the same measure in HET mice.

WT HET KO

Dura

tion (

sec)

0

100

200

300

400

Social

Center

Empty

# # ### # #

Figure 3.3. Time spent in each chamber in the three chamber test.

Values are displayed as mean + SEM. # p < 0.05, ## p < 0.001, ### p < 0.001, within-

group comparison, different from empty chamber.

WT HET KO

Fre

qu

en

cy

0

10

20

30

40

50

60

70

A

*

WT HET KO

Fre

quen

cy

0

5

10

15

20

25

30

35

Social

Center

EmptyB

*

*

77

3.4.3. Autogrooming

In the autogrooming test, KO mice had significantly increased frequencies of paw

grooming [F(2,33) = 7.13, p < 0.01; Figure 3.4A] when compared to WT and HET mice.

KO mice also showed increased duration of paw [F(2,33) = 8.35, p < 0.01] and head

[F(2,33) = 4.30, p < 0.05] grooming (Figure 3.4B) compared to WT and HET littermates.

With regards to the pattern of grooming, KO mice had more completed bout sequences

[F(2,33) = 11.60, p < 0.001; Figure 3.4C] and correct transitions [F(2,33) = 6.84, p <

0.01; Figure 4D] compared to WT and HET littermates. No significant differences were

found for the number of bouts, interrupted bouts, proportion of interrupted bouts,

transitions, incorrect transitions, or proportion of incorrect transitions.

78

Figure 3.4. Behaviors displayed in the autogrooming test.

Frequency (A) and duration (B) of body site grooming. Frequency of grooming bouts (C)

and transitions (D). Values are displayed as mean + SEM. * p < 0.05, ** p < 0.01, *** p

< 0.001, compared to WT and HET mice.

79

3.4.4. Social Proximity

In the social proximity chamber, HET mice displayed higher frequencies [F(2,33)

= 5.00, p < 0.05; Figure 3.5A] and longer durations of allogrooming [F(2,33) = 4.06, p <

0.05; Figure 3.5B] when compared to WT and KO mice. No other significant differences

between genotypes were found.

Figure 3.5. Behaviors

displayed in the social

proximity test. Values are

displayed as mean + SEM.

NT, nose-to-nose; NH,

nose-to-head; NA, nose-to-

anogenital; CU, crawl

under; CO, crawl over;

AG, allogroom; SG,

selfgroom. * p < 0.05,

compared to WT and KO

mice.

80

3.4.5. Repetitive Novel Object Contact

During the habituation phase of the repetitive novel object contact test, no

significant differences were found between any of the genotypes in the number of

transitions between quadrants [F(2,33) = 3.01, p > 0.05, Figure 3.6A]. There was a trend

for KO mice to make fewer line crosses during the habituation phase, but this failed to

reach statistical significance (WT = 155, HET = 144, KO = 122, p < 0.06). During

testing, the genotypes did not differ in the frequency of visits to any of the four novel

objects (Dice: [F(2,33) = 0.60, p > 0.05]; jax: [F(2,33) = 0.34, p > 0.05]; Lego: [F(2,33)

= 0.89, p > 0.05]; bowling pin: [F(2,33) = 0.78, p > 0.05]; Figure 3.6B). Similarly, no

effect of genotype was observed for the tendency to display a preference for a particular

object (F(2,33) = 0.56, p > 0.05, Figure 3.6C). There were no differences in the number

of visits to identical sequences of three [F(2,33) = 2.48, p > 0.05] or four [F(2,33) = 2.48,

p > 0.05] objects (Figure 3.6D).

81

Figure 3.6. Behaviors displayed in the repetitive novel object task. Number of transitions during habituation (A). Rate of novel object investigation (B).

Object preference ratings (C). Frequency of sequenced toy visits (D). Values are

displayed as mean + SEM.

82

3.4.6. Scent Marking

Repeated measures ANOVA revealed a significant main effect of genotype for

the number of scent marks deposited. In total, HET mice deposited more scent marks

than both WT and KO mice across both conditions [F(2,33) = 8.69, p < 0.001, Figure

3.7). No effect of condition [F(1,33) = 1.93, p > 0.05] was observed for any of the

genotypes. Further, no significant genotype x condition interactions were found [F(2,33)

= 0.68, p > 0.05].

Figure 3.7. Scent marking behavior. Number of squares containing scent marks during habituation and social conditions.

Values are displayed as mean + SEM. *** p < 0.001; main effect of genotype when

compared to WT and KO mice.

***

83

3.4.7. Resident Intruder

No differences in latency to attack were observed between any of the genotypes

[F(2,33) = 0.56, p > 0.05, Figure 3.8A). There were no differences in the frequency

(Figure 3.8B) or duration (Figure 3.8C) of any of the behaviors measured in the resident

intruder test.

Figure 3.8. Behaviors displayed in the Resident Intruder Test. Latency to attack (A), frequency (B) and duration (C) of aggressive behaviors in the

resident intruder test. Values are expressed as mean + SEM. SF, sniff face; SB, sniff

body; SR, sniff rear; ST, sniff tail; VG, vigorous groom; AT, attack; FW, follow; UP,

upright; TR, tail rattle.

84

3.5. Discussion

Autism is a behaviorally defined disorder with no known biological markers or

screening tests (American Psychiatric Association, 2013). Although the causes of autism

are still unclear, several lines of evidence suggest that the 5-HT system may play an

important role (Cook and Leventhal, 1996). Variants in the 5-HTT gene are associated

with an increased risk for autism (International Molecular Genetic Study of Autism

Consortium, 2001; Devlin et al., 2005). Reduced 5-HTT binding has also been reported

in people with autism (Makkonen et al., 2008; Nakamura et al., 2010; Oblak et al., 2013)

and in rodent models of the disorder (Gould et al., 2011). In laboratory rodents, genetic

and pharmacological inactivation of 5-HTT produces physiological and behavioral

alterations relevant to conditions associated with autism, such as: gastrointestinal

dysfunction (Coates et al., 2006), exaggerated neuroendocrine and sympathoadrenal

responses to stress (Tjurmina et al., 2002; Jiang et al., 2009), and heightened anxiety

(Holmes et al., 2003).

In the current study sociability was not significantly influenced by 5-HTT

genotype. Social behaviors were assessed in three tests: the VBS, three-chamber social

approach, and social proximity. There were no effects of genotype for any of the

behaviors measured during the dark/active periods in the VBS. During the light periods,

following and huddling increased over the course of testing while being alone decreased

only in 5-HTT KO mice. Notably, Lewejohann et al., (2010) also failed to find

reductions in social behaviors in 5-HTT KO mice on a B6 background, housed in

enriched environments with same-genotype conspecifics. 5-HTT deficiency failed to

alter social approach or social interaction in the three-chamber and social proximity tests,

respectively. A previous study reported reduced sociability in these 5-HTT KO mice in

the three-chamber social approach test (Moy et al., 2009). However, both the current

study and the Moy et al., (2009) report, failed to find genotype differences in the amount

of social exploration (sniffing) of the social stimulus. Taken together, these findings

suggest that the very minimal reductions in social interactions in 5-HTT KO mice likely

reflect the anxious and hypoactive phenotype of this strain (Kalueff et al., 2007), rather

than a deficit in social motivation.

85

Aggression is an additional component of many rodent social interactions

(Blanchard and Blanchard, 1989). A previous study reported longer attack latencies and

reduced aggression in 5-HTT KO mice in the resident intruder paradigm (Holmes et al.,

2002). However, in the current study, we found no differences between genotypes in the

latency to attack or in the number of aggressive behaviors displayed in the resident

intruder test. In line with our results, two recent studies reported no differences in

aggression between 5-HTT KO and WT mice confronted with an intruding conspecific in

their own territory (Jansen et al., 2011; Kloke et al., 2011). Given that aggressive

behavior depends significantly on the type of opponent used (Brain et al., 1981; Miczek

et al., 2001), these conflicting results are likely due to differences in the aggressiveness of

the intruder used in each study. Holmes et al. used the more aggressive DBA/2J mouse,

in comparison to the more docile C3H mice used by Kloke et al. and Jansen et al., and the

B6 used in the current study (Jones and Brain, 1987; Guillot and Chapouthier, 1996).

Conspecific aggression may be modulated by a variety of factors including the

behavior of the opponent (Brain et al., 1981; Miczek et al., 2001), sexual experience of

the aggressor (O’Donnell et al., 1981), previous social experiences (Flannelly et al.,

1984), and the territory where it takes place (Teskey and Kavaliers, 1987). In the current

study, all genotypes showed comparable levels of aggression when housed in same-

genotype colonies in the VBS. Similarly, a previous study (Lewejohann et al., 2010)

found that 5-HTT KO mice were able to establish and maintain dominance hierarchies

through aggression when housed in same-genotype groups. However, when the 5-HTT

KO mice were placed in mixed genotype groups, the KOs were less likely to establish a

dominant position and were more likely to be injured by the other mice (Lewejohann et

al., 2010). Jansen and colleagues (2011) suggested that the aggression displayed by the

5-HTT KO mice in the resident-intruder paradigm was influenced by the opponent's

behavior and the environmental situation (own territory, opponent's territory or neutral

area) whereas 5-HTT wildtypes showed similar levels of aggression regardless of the

opponent or environment. Similarly, Kloke et al. (2011) found that previous experiences

as a winner or loser in agnostic encounters influenced aggressive behavior in the 5-HTT

mutant mice, while 5-HTT genotype did not.

86

Scent marking is a primary method of communication for mice and is used to

convey information about social status, territory boundaries, and reproductive abilities

(Arakawa et al., 2007a). In the current study, there were no differences between 5-HTT

WT and KO mice in scent marking to a male stimulus. This is consistent with our

findings of normal territorial aggression in these mice in the resident intruder test.

Stereotyped and repetitive behaviors represent a core feature of autism.

Clinically, mutations in the 5-HTT gene have been associated with repetitive and

compulsive behaviors (Sutcliffe et al., 2005; Voyiaziakis et al., 2011). Here, 5-HTT KO

mice displayed higher frequencies and durations of grooming. Additionally, KO mice

had more correct transitions (transitions following the typical cephalo-caudal

progression) and more completed bouts of grooming (paw/head/body/tail), suggesting

adherence to a more rigid and fixed pattern of grooming. Although stress has been

shown to increase grooming in rodents (Kalueff and Tuohimaa, 2004; Hart et al., 2010),

it is unlikely that the current results reflect a heightened anxiety response in this test.

Stress induced grooming is characterized by more incorrect transitions and interrupted

bouts (Kalueff and Tuohimaa, 2004); neither of which were displayed by the KO in this

test.

No differences were found between genotypes in the novel object contact test,

designed to assess restricted interests. KO and WT mice explored the items with similar

frequency, displayed similar preferences for objects, and did not differ on the number of

sequenced visits to 3 or 4 toys. These data suggest that reduced 5-HTT function may

contribute to stereotyped motor behaviors, but provide no evidence for 5-HTT deficiency

in regulating restricted interest. Our findings are in line with clinical data that report a

negative correlation between 5-HTT binding and repetitive behaviors in adults with

autism (Nakamura et al., 2010).

This study provided a wide ranging evaluation of the effects of life-long 5-HTT

deficiency on rodent behaviors designed to parallel the core symptoms of ASD.

Although variants in the 5-HTT gene have been implicated in autism (Cook and

Leventhal, 1996; Sutcliffe et al., 2005), this extensive phenotyping of the 5-HTT KO

mouse revealed only modest and limited disturbances in sociability and repetitive

behaviors. This relative lack of overt behavioral differences between genotypes may

87

reflect compensatory molecular mechanisms of the 5-HT system in these mice. For

example, gene expression for an organic cation transporter with low affinity for 5-HT is

up-regulated in the brain of 5-HTT KO mice (Schmitt et al., 2003). Additionally, 5-HTT

KO mice also display decreased 5-HT1A autoreceptor mRNA, protein levels and binding

sites (Kalueff et al., 2010). These alterations likely represent mechanisms to limit the

adverse effects of continually elevated extracellular 5-HT concentrations.

Furthermore, 5-HTT deficiency may increase one's susceptibility to

environmental challenges or toxins. Reduced 5-HTT gene expression is associated with

increased susceptibility to stress (Kendler et al., 1999; Canli and Lesch, 2007) and

environmental influences such as stressful life events may further impinge on 5-HTT

expression via transcriptional and post-transcriptional mechanisms to contribute to autism

pathology. Given that autism likely results from a complex interaction between genetic

susceptibility and environmental insults (Trottier et al., 1999), it is possible that

additional environmental or epigenetic factors may be important in the manifestation of

an autism-relevant behavioral phenotype in the 5-HTT deficient mice.

88

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CHAPTER 4. CHARACTERIZATION OF THE SEROTONERGIC PROFILE OF THE

SUBVENTRICULAR ZONE (SVZ) IN INDIVIDUALS WITH AUTISM AND

TYPICALLY-DEVELOPING CONTROLS.

4.1. Abstract

Rationale

Autism is a neurodevelopmental disorder associated with alterations in early brain

growth (Courchesne et al., 2007). The SVZ neurogenic niche is a unique

microenvironment critical for supporting the development and plasticity of the brain

(Brazel et al., 2003; Quiñones-Hinojosa et al., 2006, 2007; Gage et al., 2008; Kazanis et

al., 2008). Neurons expressing 5-HT innervate the SVZ and regulate its activity (Brezun

and Daszuta, 1999; Banasr et al., 2004; Jahanshahi et al., 2011). The 5-HT system is

involved in brain development (Daubert and Condron, 2010) and is implicated in autism

(Zafeiriou et al., 2009). Therefore, disturbances in the serotonergic composition and

regulation of the SVZ could play a critical role in autism pathogenesis. However, no

studies to date have investigated the 5-HT innervation of the SVZ in autism.

Objectives

The purpose of the current experiment was to characterize the cellular

organization and the distribution of 5-HT transporters (5-HTT) in the human SVZ.

Furthermore, a primary goal of these studies was to compare the distribution patterns and

densities of 5-HTT in autism-diagnosed (AD) and typically-developing (TD) individuals

across the lifespan.

Methods

5-HTT protein expression in the SVZ from 4 pairs of age and sex matched AD

and TD cases was confirmed by Western Blot. The cellular organization and

composition of the SVZ was assessed using immunohistochemistry techniques.

Results

The current results confirmed and expanded on findings of altered SVZ structure

and molecular composition in AD and TD cases. The hypocellular gap was significantly

smaller and more densely packed with displaced cells in AD cases when compared to TD

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controls. 5-HTT immunoreactivity was significantly increased in the SVZ of the

youngest AD cases compared to the matched TD control. FGF-2, a growth factor

responsive to 5-HT, was also increased in the younger AD cases. Evidence of occasional

neuronal and microglia staining was observed in all cases.

Conclusions

These results are the first known demonstration of altered 5-HTT density in AD

SVZ tissue. Altered composition of molecular factors in the SVZ may contribute to the

altered brain growth trajectory reported in the disorder. Enhanced understanding of the

composition and activity of this brain neurogenic niche could contribute to improved

treatment for autism and similar neurodevelopmental disorders.

96

4.2. Introduction

Autism is a widespread, heterogeneous developmental disorder that affects 1 in 50

children in the United States (Blumberg et al., 2013). The disorder is characterized by

impairments in sociability and communication and the presence of stereotyped behaviors

(American Psychiatric Association, 2013). Further complicating research efforts is the

fact that no unifying pathology exists (Palmen et al., 2004b) and there are no consistently

effective treatments (Canitano and Scandurra, 2011).

While the neurobiological basis for autism remains largely unknown, evidence

suggests that fundamental neurodevelopmental mechanisms are altered in the disorder

(Persico and Bourgeron, 2006). Developmental changes such as altered cellular growth

and migration, disrupted structural organization and connectivity, as well as altered

cellular communication (Persico and Bourgeron, 2006; Ecker et al., 2013) are reported in

the brains of autistic individuals. These findings underscore the importance of

investigating cellular and molecular processes involved in brain development in autism.

The subventricular zone of the lateral ventricle (SVZ) is one of only two areas in

the human brain, the other being the hippocampus, that generates new neurons

throughout life (Quiñones-Hinojosa et al., 2006, 2007). The SVZ is visible around 7-8

gestational weeks in humans (Zecevic et al., 2005), undergoes extreme prenatal growth

(Brazel et al., 2003), and generates neurons and glia that populate the prefrontal cortex

and various subcortical structures (Brazel et al., 2003; Zecevic et al., 2005; Bayatti et al.,

2008; Sanai et al., 2011). Later in development the SVZ shrinks in size but retains its

neurogenic capabilities, producing new neurons and astrocytes into adulthood (Eriksson

et al., 1998; Sanai et al., 2004, 2011).

The SVZ is composed of specific cell-cell contacts, extracellular matrix

molecules as well as paracrine signals from growth factors and neurotransmitters

(Decimo et al., 2012). This specialized microenvironment or “neurogenic niche” is

critical for supporting and controlling the proliferation, migration, differentiation, and

survival of neural stem cells (NSC) (Scadden, 2006). Neurons expressing 5-HT from the

dorsal raphe provide a dense innervation of the SVZ, referred to as the supra- and sub-

ependymal 5-HT complex (Richards et al., 1981; Moore and Speh, 2004). 5-HT released

from these neurons increases ependymal cell metabolism (Prothmann et al., 2001) and

97

ciliary beat frequency (Nguyen et al., 2001). These neurons expressing 5-HT may serve

as a feedback loop, monitoring and regulating the flow and composition of cerebrospinal

fluid (CSF) near the SVZ as well as throughout the brain (Mikkelsen et al., 1997).

Furthermore, infusion of 5-HT into the lateral ventricles increased NSC proliferation in

the SVZ of rodents (Hitoshi et al., 2007) while 5-HT depletion reduced NSC proliferation

(Brezun and Daszuta, 1999). In animal models, the administration of 5-HT agonists has

been shown to enhance cell proliferation in the SVZ (Banasr et al., 2004; Hitoshi et al.,

2007; Lau et al., 2007). These findings suggest an important role for 5-HT in modulating

the microenvironment of the SVZ niche and the proliferation of NSC across the lifespan.

Neural proliferation is markedly increased in the SVZ of postmortem brains from

autistic individuals (Kotagiri et al., 2013; Pearson et al., 2013). Components of the 5-HT

system are also disturbed in various brain regions in the disorder (Cook and Leventhal,

1996). However, the role of the SVZ 5-HT subependymal plexus in autism has received

little attention. Given the critical role of the SVZ in brain development and

neuroplasticity, disturbed 5-HT release in this region could contribute to dysregulated

neurogenesis, aberrant brain growth, and disturbed cerebral functioning. This study

examined the SVZ in autism-diagnosed (AD) and typically-developing (TD) postmortem

brains at different ages. Improved understanding of the composition and function of SVZ

regulatory signals, such as 5-HT, may clarify the mechanisms underlying the

neurodevelopmental origins of this disorder.

4.3. Materials and methods

4.3.1. Postmortem Brain Tissue Samples

Frozen postmortem tissue samples were obtained from the Harvard Brain Tissue

Resource Center and the National Institute of Child Health and Human Development

(NICHD) Brain and Tissue Bank for Developmental Disorders through the Autism

Tissue Program of the Autism Speaks organization. Brain tissue was obtained from four

male autism cases, confirmed by the Autism Diagnostic Interview Revised (ADI-R), and

four control subjects matched for sex and age (within 1 year).

Postmortem donors' clinical records are available online from the Autism Tissue

Program Data Portal (atpportal.org). All diagnoses of autism were based on responses to

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the ADI-R by a parent or legal guardian of the deceased. An autism diagnosis is

indicated when scores in all three behavioral domains meet or exceed the minimum cut

off score (Lord et al., 1994). All cases met or exceeded the cutoffs for a diagnosis of

autism using the ADI-R scale (Table 4.1). Control cases were determined to be free of

neurological disorders including autism, based on information gathered at the time of

death.

Table 4.1. Diagnostic characteristics of autism cases

Case Age(y) ADI-Social a ADI-Communication b ADI-Restrictive & Repetitive c

AN08873 5 22 14 6

AN00764 20 >10 >8 >3

AN06420 39 20 12 4

AN09714 60 26 14 4 ADI-R, Autism Diagnostic Interview-Revised

a Qualitative abnormalities in reciprocal social interaction (cutoff, 10; maximum, 30).

b Qualitative abnormalities in communication (cutoff, 7; maximum nonverbal communication, 14)

c Restricted, repetitive, and stereotyped patterns of interest (cutoff, 3; maximum, 12).

Donor brains were harvested and processed at either the Harvard or NICHD brain

bank (Figure 4.1). Briefly, one hemisphere was fresh frozen and the other was formalin

fixed. Coronal sections from the frozen hemisphere were used for analyses in these

studies. Blocks of tissue were dissected from each coronal section along the dorsal-

lateral ventricle wall including the anterior horn and body of the lateral ventricle. Blocks

also included portions of the medial caudate nucleus and body of the corpus callosum.

Coronal sections were obtained from three anterior-posterior sections for each case.

Tissue blocks (2.0 cm3) from each case were sectioned using a freezing microtome

(Leica, CM1850UV) perpendicular to and containing the ependymal, subependymal and

parenchymal zones in the coronal plane at 10 μM. Tissue sections (3-5 sections per slide)

were mounted on Poly-L-lysine (1:5 Sigma Aldrich) coated glass microscope slides

(VWR superfrost) and stored at -20˚C until processing. Immunostaining was performed

on 6-10 tissue sections per individual for each of the experiments described below. All

tissue sections were coded and investigators were blind to the diagnosis.

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Figure 4.1. Tissue acquisition and histology. Postmortem tissue processing protocol.

(A) Donor brains were collected and processed at the brain bank. Hemispheres were

either fresh frozen or fixed in formalin. (B) Frozen coronal sections were taken from one

hemisphere. (C) Dissections of the subventricular zone were collected from three

anterior-posterior coronal sections for each case. (D) Blocks of tissue were sectioned in

our laboratory on a freezing microtome.

A. B.

C. D.

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4.3.2. Procedure

Immunofluorescent labeling

Immunostaining of the serotonergic system in human post-mortem tissue is

complicated by the fact that 5-HT is unstable and undergoes rapid degradation during the

postmortem period (Azmitia and Nixon, 2008). For this reason studies of the

morphology of the human 5-HT neurons must utilize antibodies generated against the

more robust 5-HT-receptor proteins (Törk et al., 1992). In the current study, the

membrane bound 5-HTT was used as a marker of serotonergic neurons. 5-HTT is a

membrane bound protein which resides primarily in the axons and terminal buttons of

serotonergic neurons (Azmitia and Nixon, 2008). Antibodies to 5-HTT provide patterns

of staining that appear identical to those obtained with 5-HT antibodies in rodents;

suggesting that 5-HTT is a faithful marker of 5-HT neurons in the brain (Zhou et al.,

1996).

Prior to the study, careful pilot testing was done to determine optimal antibody

concentrations, specificity and cross-reactivity (Table 4.2). The primary antibody

concentrations were calibrated using dilutions of 1:50, 1:100, 1:200, and 1:400. Antibody

specificity was determined by performing the standard incubations in the absence of the

primary or secondary antibody. Tissue sections in which the primary antibody was

omitted exhibited no immunostaining (data not shown). Control experiments were also

conducted to ensure that antibodies did not cross-react with each other. To detect cross

reactivity in double labeling experiments, each primary antibody in the pair was

incubated with both secondary antibodies to be used in the double label. The control

experiments showed that the secondary antibodies do not cross-react with each other

(data not shown).

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Table 4.2. Antibodies

Primary Antibodies Host Type

Dilution

(l) Company Product number Specificity

5-HTT goat polyclonal IgG 1/100 Santa Cruz sc-1458 Directed at the C-terminus of the human 5-HTT

FGF2 goat polyclonal IgG 1/400 Santa Cruz sc-1390 Directed to the N-terminus of human FGF-2

GFAP mouse monoclonal IgG 1/2000 Millipore MAB360 Astrocyte cytoskeleton marker

Neun mouse monoclonal IgG 1/1000 Millipore MAB377 Neuron specific nuclear marker

Secondary Antibodies Host Type

Dilution

(l) Company Product number Excitation/Emission (nm)

AlexaFluor 488 donkey anti-goat IgG 1/400 Invitrogen A-11055 488/519

AlexaFluor 546 donkey anti-goat IgG 1/400 Invitrogen A-11056 556/573

AlexaFluor 546 donkey anti-mouse IgG 1/400 Invitrogen A-10036 556/573

AlexaFluor 546 donkey anti-rabbit IgG 1/400 Invitrogen A-10040 556/573

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The selected tissue sections were processed using a standard

immunohistochemistry staining procedure (Figure 4.2). Randomly selected slides from

each case were fixed in -20˚C acetone for two minutes and rinsed in phosphate buffered

saline (PBS) for 5 minutes (Figure 4.2). Fixation is necessary to preserve tissue structure

and morphology while immobilizing the antigens of interest. Wells were created on each

slide with a hydrophobic barrier pen (Vector Labs, H-4000). In order to detect

intracellular antigens, tissue sections were permeabilized with a 0.5% Triton-X-100

solution for 15 minutes. The sections were then blocked with a PBS/gelatin blocking

solution (0.2%) for 10 min to suppress non-specific binding. Primary antibodies were

incubated for 48 hours at 4˚C. The slides were subsequently rinsed in PBS for 5 min

followed by PBS/gelatin for 10 min and incubated for 40 minutes on a moving platform

at room temperature with species-specific fluorescently labeled secondary antibodies

(Table 4.2). The secondary antibodies were washed off (4 x 5 min) in PBS and once in

water. Lipofuscin is a highly fluorescent pigment produced in aging cells that can result

in background autoflourescence (Terman & Brunk, 2004). To reduce signal interference

from lipofuscin all sections were incubated in a 1% Sudan Black solution for 5 minutes

followed by a second incubation in a 3% Sudan Black solution for an additional 5

minutes. Sections were air-dried, mounted with VECTASHIELD mounting medium

with 4',6-diamidino-2-phenylindole (DAPI) nuclear marker (Vector Labs, H-1200), cover

slipped and sealed.

Double immunofluorescence was performed on 8 tissue sections per sample.

Selected tissues were first stained with either anti-5-HTT or anti-FGF-2 antibodies

followed by incubations with the corresponding fluorescently labeled secondary

antibody. Following incubation with the first set of antibodies the sections were then

stained with anti-GFAP followed by incubations with the appropriate secondary

antibody. Sections were air-dried, mounted with VECTASHIELD mounting medium

with 4',6-diamidino-2-phenylindole (DAPI) nuclear marker (Vector Labs, H-1200), cover

slipped and sealed.

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Acetone PBS PBS

PBS/Gelatin

Triton X-100

Primary Antibody Secondary Antibody Sudan Black

DAPINail Polish Mounted Slides

Wells

A B

ED

H IG

F

C

Figure 4.2. Sample preparation for immunohistochemical staining.

(A) Fixation of tissue in 20% acetone, followed by baths in PBS. (B) Drawing of wells

around the tissue using a hydrophobic pen. (C) Incubation of slides in Triton X-100, PBS,

and PBS/gelatin. (D) Incubation of slides in primary antibody in a moistened chamber.

(E) Incubation of slides in secondary antibody in a moistened chamber. (F) Sudan Black

solution washes for quenching of autofluorescence. (G) Mounting of slides with DAPI+

nuclear stain. (H) Sealing of slides with clear nail polish. (I) Mounted and sealed slides

ready for microscopy.

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Immunoperoxidase labeling

Additional tissue sections were prepared for anti-neuron and anti-microglial

immunostaining using the highly sensitive and long lasting avidin-biotin method.

Sections were fixed in a brief acetone bath (-20˚C, 2 min) and rinsed in PBS. The

sections were treated with a blocking solution (0.3% hydrogen peroxide in 0.3% normal

goat serum, 5 min), incubated in the appropriate primary antibody (Table 4.2, 48 hrs),

and processed using goat anti-mouse biotinylated secondary antibodies (1:200; 6 h), and

the Vectastain ABC Elite reagent kit (1:100 dilution, 1 h; Vector Laboratories). The

immunoperoxidase reaction was visualized by using 0.05% diaminobenzidine (DAB;

Sigma) and 0.015% hydrogen peroxide in PBS, which results in a brown reaction

product. The reaction was terminated by rinsing in cold water. Sections were

counterstained with haematoxylin, dried, mounted with Permount mounting medium

(Fisher), coverslipped and sealed with clear nail polish.

Western Blot

5-HTT protein expression in the SVZ was confirmed by Western Blot.

Approximately 12 tissue slices from each case were scraped off of the glass slides and

collected in 1mL of lysis buffer. The absorbance at 280 nanometers (nm) was used to

quantify the protein content of each sample. From each sample, 80 μg of protein was

loaded into a 10% polyacrylamide gel and run in an electrophoresis chamber for

approximately one hour at 120V at room temperature. The proteins were then transferred

from the gel onto a low fluorescence PVD filter membrane (Bio-Rad) for one hour at

15V using the semi-dry transfer method. Membranes were blocked against nonspecific

binding for one hour in Tris-buffered saline with Tween 20 (TBST) containing 5%

bovine serum albumin (BSA). The membranes were then incubated with the primary

antibody overnight at 4˚C on a rotating platform. The following day the membranes were

washed, blocked and incubated with fluorescently labeled secondary antibodies for one

hour.

Data Acquisition

Images of immunofluorescent staining were obtained on an Olympus Fluoview

FV1000 laser scanning microscope mounted on an Olympus IX81 inverted microscope at

105

the Biological Electron Microscope Facility of the University of Hawaii. DAPI,

AlexaFluor 488 and AlexaFluor 546 were excited at 405 nm, 488 nm, and 546 nm

respectively, with a blue diode, an Ar ion set at 488 nm and a red HeNe lasers. All

sections were scanned using identical instrument settings. Emission from each

fluorescent molecule was recorded through spectral filtering. Images were acquired

using Fluoview Viewer software. The SVZ is composed of four cellular layers

(Quinones-Hinojosa et al., 2006). These layers were located on the immunostained slides

by viewing DAPI staining at low magnification (10x). Slides in which the SVZ was not

visible were excluded from all analyses. On each immunolabeled section at least one

photomicrograph was systematically and randomly captured using a 20x UplanApo

(0.70) optical objective. Microscopy images were exported as digital TIFF image files

(1024 x 1024 pixels). High magnification photomicrographs of GFAP immunoreactivity

were captured using a 20x UplanApo (0.70) optical objective with a 2.0x digital zoom

and were exported as digital TIFF image files (1600 x 1600 pixels). No further

processing was performed on the images before analysis.

Images of immunoperoxidase stained slides were obtained on an Olympus BX-51

upright compound microscope using bright-field illumination. Photomicrographs of each

slide were captured with an Optonics MacroFire digital camera mounted onto the

microscope. Images were captured on each section at 4x, 10x, and 20x zoom. All

images were saved and exported as digital TIFF image files (1200 x 1200 pixels).

Peroxidase stained images were used to determine the location of labeled cells in relation

to the anatomical landmarks under study.

Imaging of the Western Blot was performed using a Typhoon 9410 (Amersham

Pharmacia Biotech) system. Images were converted to TIFF files using the ImageQuant

software (Amersham Pharmacia Biotech) and exported to ImageJ.

Quantification

For immunofluorescence quantification five to ten tissue slices were processed

per case and per antigen of interest. At least one image was acquired from a randomly

selected location on each immunolabeled tissue slice. All images were processed using

Image J/Fiji Software (NIH) (Schindelin et al., 2012). The quantification of fluorescently

106

labeled slides was carried out by measuring the area fraction for each of the molecules of

interest in two regions of interest (ROI). Area fraction is a measure of the number of

pixels of a defined color relative to the total area of the tissue under study (Kaczmarek et

al., 2004; Diniz, 2010) and is commonly used as a method of immunohistochemical

quantification (Pham et al., 2007). The two regions of interest were the SVZ (layers I-

IV) and the hypocellular gap (layer II). The SVZ was defined as the region beginning at

and extending 300μm from the ependymal wall. This standardized region was selected as

it encompasses a range of variable SVZ widths that have been previously described (van

den Berg, et al., 2010). The hypocellular gap was defined as the cell-sparse region

between the ependymal wall and the beginning of the astrocytic ribbon ( uinones-

Hinojosa et al., 2007).

The width of the hypocellular zone was assessed by measuring the distance from

the ependymal wall to the astrocytic ribbon layer at five equidistant points along the SVZ

and averaged. The cell density in the hypocellular gap was calculated by manually

counting the number of DAPI+ cell nuclei in the hypocellular gap and dividing this

number by the hypocellular gap area to determine the number of cells per millimeter. The

width of the astrocytic ribbon was quantified by taking the average of five measures of

the GFAP+ area.

The optical density of the Western Blot bands was quantified using the gel

analysis feature in Image J (http://rsb.info.nih.gov/ij/docs/menus/analyze.html#gels).

4.3.3. Statistical analysis

Data are presented as mean + standard error of the mean. The stained area

fraction for each molecule of interest and the hypocellular gap width were compared with

two-way analyses of variance (ANOVA) with condition (AD vs. TD) and Age as main

factors. When main effects or interactions were found, Bonferroni post hoc comparisons

were performed to assess significant differences between conditions at each age group.

T-tests were used to compare demographic variables between AD and TD cases. The

relationship between 5-HTT immunolabeling and protein expression was determined by

Pearson product-moment correlation analyses. In all studies a p-value of < 0.05 was

considered statistically significant.

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4.4. Results

4.4.1. Clinical Characteristics

All of the autistic cases received for this study met or exceeded the criteria for

autism diagnosis on the ADI-R assessment (Table 4.1). Control cases did not have any

known neurological disorders. All of the individuals in the autism group were the

products of full term, normal pregnancies. Each of the four autism cases revealed a

family history of neurological disorders including attention-deficit-hyperactivity disorder

(ADHD), pervasive developmental disorder (PDD), Manic Depression and Alzheimer's.

The 60 year old in the autism group had a history of seizures.

4.4.2. Postmortem and Neuropathological Characteristics

Donor characteristics are presented in Table 4.3. The average age of the AD

cases at death was 26.53 (range 5-60.1 years). The average age of control cases at death

was 32.23 (range 6.9-60 years). The average postmortem interval for AD cases was 22.4

hours and 20.1 hours for controls. There were no statistical differences between groups

in age at death or postmortem interval (data not shown). Consistent with reports of

enlarged head size in autism, the brain from the 5 year old AD case was approximately

15% larger than the normative brain weight reported in the literature (Redcay and

Courchesne, 2005) and nearly 18% larger than the brain of the 6 year old TD case.

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Table 4.3. Postmortem Information on Autism-Diagnosed and Typically-

Developing Controls.

Autism (ADI-R) Diagnosed

ID Age (years) PMI (h)a Cause of death Brain weight (g)

AN08873 5 25.5 Drowning 1560

AN00764 20 23.7 Auto accident 1144

AN06420 39 13.95 Cardiac tamponade 1520

AN09714 60.11 26.5 Pancreatic cancer 1210

Typically-Developing Control

ID Age (years) PMI (h) Cause of death Brain weight (g)

UMB5408 6.86 16 Drowning 1320

UMB1405 21 13 Drowning 1440

ANO1410 41 27.17 N/A N/A

AN10723 60 24.23 Heart attack 1340

a post mortem interval

4.4.3. Morphology of the SVZ

The SVZ in both cohorts was comprised of an ependymal layer, visualized as a

single layer of tightly packed DAPI+ cells bordering the lateral ventricle (Layer I; Figure

4.3). Immediately adjacent to the ependymal layer was a hypocellular layer largely

devoid of DAPI + cells (Layer II; Figure 4.3). Both AD and TD cases displayed a small

number of displaced DAPI+ cells within this region. Adjacent to the hypocellular gap

was a layer of homogeneously dispersed DAPI+ cells (Layer III; Figure 4.3). Previous

research has described Layer III as a dense ribbon of astrocytes (Quiñones-Hinojosa et

al., 2006). DAPI+ cells were also observed in Layer IV, which has been described as a

transitional zone between the astrocytic ribbon and the brain parencyhma (Quiñones-

Hinojosa et al., 2006).

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Figure 4.3. Layered cellular organization of the human SVZ.

Representative photomicrograph of the major layers of the SVZ. LV = lateral ventricle, I

= ependymal wall, II = hypocellular gap, III = astrocytic ribbon, IV = transitional zone.

4.4.4. Hypocellular gap

The hypocellular gap width was smaller in AD cases than in TD cases [F(1, 231)

= 76.50, p < 0.001; Figure 4.4A]. Across cohorts, the width of the hypocellular gap

changed as a function of age [F(3, 231) = 26.87, p < 0.001; Figure 4.4B]. The youngest

individuals (5-21 year olds) had the smallest hypocellular gaps while the oldest

individuals (39-60 year olds) had the largest [t(237) = -2.03, p < 0.05; Figure 4.4C].

Post-hoc analysis following significant interactions between condition and age

showed that in the AD cases the hypocellular gap remained similar in size between the

young ages (5-20 year olds) but increased in size with increasing age (Figure 4.4B). TD

cases showed the opposite pattern. In TD cases, the hypocellular gap increased in size

between the youngest ages (6-21) followed by a decrease in size at 41, which remained

unchanged at 60. This is likely due to the large hypocellular gap in the 21 year old TD

case.

110

The hypocellular gap contains a small number of displaced ependymal cells

(Sanai et al., 2004; Quiñones-Hinojosa et al., 2006), neuroblasts (Wang et al., 2011) and

astrocytes (Kotagiri et al., 2013). Both TD and AD cases had numerous DAPI+ cells in

the hypocellular gap, however, the AD cases had a significantly greater number of

DAPI+ cells/mm2 when compared to TD controls [F(1,76) = 42.67, p < 0.001]. The

young cases (5-21 year olds) had a significantly greater cell density in layer II than the

old cases (39-60 year olds) [F(1,76) = 23.99, p < 0.001]. Post-hoc analyses following

significant interactions between condition and age showed that the young AD cases had a

greater hypocellular gap density when compared to young TD cases and old AD cases

[F(1,76) = 14.11, p < 0.001; Figure 4.5].

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Figure 4.4. Hypocellular gap widths in the AD and TD SVZ.

(A) Average hypocellular gap width measurements for AD and TD cases. (B)

Hypocellular gap widths for AD and TD cases across ages. (C) Hypocellular gap widths

for young versus old cases. * p < 0.05, ** p < 0.01, *** p < 0.001, between-group

comparisons. # p < 0.05, ## p < 0.01, # p < 0.001, within-group comparisons, different

from all other AD ages. & p < 0.05, && p < 0.01, &&& p < 0.001, within-group

comparison, different from all other TD ages.

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Figure 4.5. Hypocellular gap density in the AD and TD SVZ.

Average hypocellular gap density measurements for AD and TD cases. * p < 0.05, ** p <

0.01, *** p < 0.001, between-group comparisons. # p < 0.05, ## p < 0.01, # p < 0.001,

within-group comparisons, different from all other AD ages.

4.4.5. Localization and quantification of 5-HTT in the SVZ

Western blot analysis of tissue sections containing the SVZ confirmed the

presence of 5-HTT in both AD and TD cases with a band measured at ~75kDa (data not

shown). A comparison between 5-HTT values obtained by Western Blot and area

fraction immunoreactivity revealed a highly significant correlation r(8) = 0.95, p < 0.001.

5-HTT immunoreactivity was observed throughout all layers of the SVZ with the

exception of Layer II (Figure 4.6C). 5-HTT immunoreactivity could be seen as punctate

staining in both AD and TD cases. 5-HTT+ staining was consistently found to be the

most intense in Layer I of the SVZ (Figure 4.6C). Layers III and IV revealed moderate

5-HTT+ labeling that appeared to localize with DAPI+ nuclear staining.

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The area fraction of 5-HTT staining in the SVZ was significantly increased in AD

cases compared with typically-developing controls [F(1,88) = 20.57, p < 0.001, Figure

4.6A]. The area fraction of 5-HTT staining also differed as a function of age across both

conditions [F(3,88) = 16.88, p < 0.001, data not shown]. 5-HTT staining in the SVZ was

higher in the youngest cases (5-6 year olds) when compared to the tissues from the young

adults (20-21years old) and the 60 year olds. These main effects of condition and age are

likely due to the high area fraction of 5-HTT+ staining seen in the young AD (5 year old)

tissue.

Post-hoc analyses following significant interactions between condition and age

revealed different age-related patterns of 5-HTT immunoreactivity between AD and TD

tissues. The 5 year old autism case had the highest levels of 5-HTT+ staining when

compared to every other AD and TD case [F(3,88) = 26.80, p < 0.001; Figure 4.6B]. In

contrast the area fraction of 5-HTT+ staining did not differ across ages in the TD tissue.

114

Figure 4.6. Area fraction of 5-HTT in the AD and TD SVZ.

(A) Average area fraction of 5-HTT for AD and TD cases. (B) Area fraction of 5-HTT

for AD and TD cases across ages. (C) 5-HTT immunoreactivity in the SVZ of the human

brain. * p < 0.05, ** p < 0.01, *** p < 0.001, between-group comparisons. # p < 0.05,

## p < 0.01, ### p < 0.001, within-group comparisons, different from all other AD cases.

115

4.4.6. Localization and quantification of FGF-2 in the SVZ

FGF-2 immunoreactivity was found almost exclusively in the hypocellular gap

(Layer II) of both AD and TD cases. FGF-2+ staining was only observed outside of this

region in connection with large blood vessels and capillaries. FGF-2 immunoreactivity

was observed as patchy staining in the hypocellular gap of both TD and AD cases (Figure

4.7C).

The area fraction of FGF-2+ staining in the hypocellular gap was significantly

increased in AD cases when compared to TD controls [F(1,128) = 46.23, p < 0.001;

Figure 4.7A]. FGF-2 immunoreactivity changed as a function of age across both

conditions [F(3,128) = 25.86, p < 0.001; data not shown]. The youngest individuals (5-6

year olds) had the greatest area fraction of FGF-2+ staining compared to every other age

group.

Post-hoc analyses following significant interactions between condition and age

showed that the area fraction of FGF-2+ staining was significantly increased in the 5 and

20 year old AD cases when compared to their respective TD controls [F(3,128) = 10.60,

p < 0.001; Figure 4.5B]. Additionally, FGF-2 immunoreactivity was significantly

increased in the 5 and 20 year old AD cases when compared to the 39 and 60 year old

AD cases (Figure 4.5B). In TD tissue FGF-2+ staining was significantly increased in the

6 year old only when compared to the 21 year old (Figure 4.5B).

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Figure 4.7. Area fraction of FGF-2 in the AD and TD SVZ.

(A) Average area fraction of FGF-2 for AD and TD cases. (B) Area fraction of FGF-2 for

AD and TD cases across ages. (C) FGF-2 immunoreactivity in the SVZ of the human

brain. * p < 0.05, ** p < 0.01, *** p < 0.001, between group comparison. + p < 0.05, ++

p < 0.01, +++ p < 0.001 within-group comparison, different from the young (5 year old)

AD case. ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001 within-group comparison, different from

the young adult (20 year old) AD case. & p < 0.05, && p < 0.01, &&& p < 0.001 within-

group comparison, different from the young (6 year old) TD case.

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4.4.7. Localization and quantification of GFAP in the SVZ

Both TD and AD cases contained a highly dense network of GFAP positive fibers

(Figure 4.8B). Consistent with previously reported descriptions (Sanai et al., 2004) there

was a particularly strong band of immunoreactivity in Layer III directly adjacent to the

hypocellular gap. Diffuse and fibrous GFAP+ projections could also be seen innervating

all layers of the SVZ. The width of the immunoreactive band, which averaged between

240-600 μm, did not differ between AD and TD cases. The area fraction of GFAP+

immunoreactivity did not differ between AD and TD controls (Figure 4.8A). There were

no significant age by condition interactions in GFAP+ staining (data not shown).

Double stained slides for 5-HTT/GFAP revealed occasional cell processes that

exhibited both 5-HTT and GFAP immunoreactivity (data not shown). The few processes

found positive for 5-HTT and GFAP were localized to Layer III of the SVZ. Contrary to

previous reports (Gómez-Pinilla et al., 1992), slides double-stained for FGF-2/GFAP

immunoreactivity did not display co-localization (data not shown). The lack of clear and

robust co-localization, which has been reported in other studies, was likely a result of the

sequential staining method used in the current study (Christensen and Winther, 2009).

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Figure 4.8. Area fraction of GFAP in the AD and TD SVZ.

(A) Average area fraction measurements of GFAP+ staining in AD and TD cases.

(B) Representative GFAP immunoreactivity in the SVZ of the human brain (20x zoom).

119

4.4.8. Cellular characterization of the SVZ

In order to determine additional cell types present in the SVZ, several slides were

stained with either an anti- neuronal (NeuN) or microglial (IBA-1) marker. Anti-NeuN is

a widely used and reliable antibody against postmitotic neurons. Neun immunostaining

was observed in both AD and TD cases. Neun immunoreactivity could be seen as darkly

stained, small, round or tear-drop shaped somata (~15 μm in diameter; Figure 4.9A). At

higher magnification (40x) Neun staining also revealed darkly stained proximal processes

(Figure 4.9B). Neun staining was primarily seen in layer IV of the SVZ (Figure 4.9A).

SVZ neuronal staining was considerably more diffuse in comparison to the adjoining

parenchyma (Figure 4.9B).

IBA-1 is a protein specifically expressed by brain microglia. In all cases a few

lightly stained cells were detected in layers II, III and IV. In the 60 year old autism

tissue, IBA-1 immunostaining revealed a dense population of darkly stained somata in

the corpus callosum (Figure 4.10).

120

Figure 4.9. NeuN staining of the SVZ.

(A) Sparse NeuN immunoreactivity in the SVZ of the human brain at 20x magnification.

(B) Denser immunoreactivity for NeuN in the adjacent parenchyma at 40x magnification.

121

Figure 4.10. IBA-1 staining of the corpus callosum in autism.

IBA-1 immunoreactivity in the corpus callosum and SVZ of the brain of the 60 year old

autistic individual at 20x magnification.

4.5. Discussion

The current experiments were aimed at investigating the structural and molecular

components of the SVZ in AD and TD brains. In addition to expanding on previous

reports of SVZ architecture and cellular composition in neuropsychiatric disorders

(Wegiel et al., 2010; Kotagiri et al., 2013; Pearson et al., 2013), this is the first study to

describe the expression of 5-HTT in the SVZ in autism. Consistent with the existing

literature on the neurodevelopmental nature of autism (DiCicco-Bloom et al., 2006), our

results revealed a number of differences in the tissue from the young AD case.

Morphological examination of the SVZ confirmed that in both TD and AD cases

the SVZ is organized into four distinct layers (Figure 4.3). The SVZ was separated from

the underlying ventricular CSF by a single layer of ependymal cells (I). A hypocellular

region (II) devoid of cell bodies was observed between the ependymal layer (I) and the

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ribbon of GFAP+ astrocytes (III). Astrocytic processes could be observed extending into

this gap region. In addition, the occasional DAPI+ cell were present. The exact function

of this gap is unknown; however, it is thought to reflect a void left over from migrating

stem cells during development (Quiñones-Hinojosa et al., 2006).

Consistent with previous reports, the hypocellular gap width increased as a

function of age in both groups (Figure 4.4C) (Sanai et al., 2004). The width of the

hypocellular gap was significantly smaller in AD cases when compared to TD controls

(Figure 4.4A), possibly reflecting increased cellular proliferation in this region. This

hypothesis supports findings of increased neurogenesis in the SVZ of post-mortem brains

from autistic individuals (Kotagiri et al., 2013; Pearson et al., 2013). Furthermore, a

number of displaced astrocytes and ependymal cells have also been observed in this gap

(Quiñones-Hinojosa et al., 2006) and here we report increased hypocellular density in the

young AD cases compared to controls (Figure 4.5). Taken together, the current findings

of reduced hypocellular gap widths and increased cell density lend further support to

findings of increased cellular proliferation, accelerated brain growth and disturbed

cellular migration characteristic of this disorder (Wegiel et al., 2010).

Alternatively, ventricular expansion could augment the structure and size of the

SVZ. Enlarged cerebral ventricles are frequently seen in the brains of autistic patients

(Piven et al., 1995, 1996; Hardan et al., 2001) and ventricular enlargement in low-birth

weight infants reportedly increases autism risk nearly seven fold (Movsas et al., 2013).

The structural abnormalities found here could reflect damage to this region as a

consequence of autism-related ventricular enlargement. However, we cannot confirm

ventricular enlargement in these individuals as only a small region of the SVZ was

received for analysis.

This study confirmed the existence of 5-HTT+ positive staining in the SVZ of

human brains (Figure 4.6C); documenting increased 5-HTT immunostaining in the SVZ

of young individuals with autism (Figure 4.6B). These findings compliment and expand

on previous reports of increased 5-HTT immunonreactivity in the cortex (Azmitia et al.,

2011a, 2011b) and cerebellum (Wegiel et al., 2013) of autism cases. Collectively these

123

findings provide strong evidence for altered 5-HT function in the autistic brain which

may contribute to the disturbances in neurodevelopment characteristic of the disorder.

Here we report intense 5-HTT staining in the ependymal wall, particularly in the

young autism case. Ependymal cells regulate the flow and molecular composition of the

CSF, and serve as a critical barrier between the CSF and adjacent parenchyma (Bruni,

1998). The activity of ependymal cells is modulated by the release of 5-HT from the

supra- and subependymal plexus. 5-HT increases ependymal cell metabolism

(Prothmann et al., 2001) and ciliary beat frequency (Nguyen et al., 2001). High

concentrations of 5-HT are likely to occur given the extensive plexus of 5-HTT

expression in the SVZ (Porlan et al., 2013). Given that signals generated in distant

locations may diffuse to the SVZ via the CSF, and vice versa, excessive 5-HT release in

this region may have long ranging consequences for overall brain development and

function.

Preclinical studies in rodents exposed to high concentrations of 5-HT during

development show striking parallels in the physiological and behavioral symptoms of

children with autism. Elevated central 5-HT concentrations during development produce

increased cortical thickness (Altamura et al., 2007), disrupted cortical minicolumns

(Janušonis et al., 2004) and deficits in the somatosensory cortex connections (Kinast et

al., 2013). Additionally, increased central 5-HT concentrations permanently reduce

novelty investigation behavior, increase anxiety and depression like behaviors, and

induce sleep abnormalities (Ansorge et al., 2004, 2008; Popa et al., 2008). In the current

study, the presence of dense 5-HTT+ staining in this neurogenic region and the well

described role of 5-HT release on neurogenesis suggests that enhanced 5-HT release in

this region may contribute to the reports of increased cellular proliferation in the SVZ of

AD brains (Kotagiri et al., 2013; Pearson et al., 2013).

One mechanism by which excess 5-HT could alter SVZ activity and consequently

brain development in autism is through the stimulation of growth factors. FGF-2 is a

well documented neurotrophin that regulates the proliferation, self renewal and

differentiation of neural precursors in the SVZ (Wagner et al., 1999; García-González et

al., 2010; Werry et al., 2010). Disturbances to FGF-2 activity produce a range of

124

developmental defects (Itoh and Ornitz, 2004). Over expression of FGF-2 results in

increased cortical size (McCaffery and Deutsch, 2005) and cortical gyrification (Rash et

al., 2013). Similarly, increased brain size (Palmen et al., 2004a) and gyrification

(Wallace et al., 2013) have been reported in autism.

Antidepressants that increase the synaptic concentration of 5-HT increase the

expression of FGF-2 (Mallei et al., 2002; Maragnoli et al., 2004). In turn, FGF-2 may act

upon FGF receptors located on multipotent stem cells (Type B-cells) and proliferating

transient amplifying cells (Type C-cells) resulting in increased NSC proliferation (Mudò

et al., 2007). In the current study the young autism cases had increased 5-HTT and FGF-

2 immunostaining in the SVZ. This suggests that increased 5-HT release in the SVZ by

5-HTT+ neurons may increase FGF-2 concentrations in the developing brain and

contribute to the well documented brain overgrowth reported in autism (Palmen et al.,

2004a). Support for this hypothesis comes from additional studies that report an increase

in BDNF, a well documented neurotrophin and 5-HT tropic factor (Ricci et al., 2013)

coupled with increased neurogenesis (Kotagiri et al., 2013; Pearson et al., 2013) in

autism.

This study confirmed previous reports of GFAP immunoreactivity in Layer III of

the SVZ. GFAP is an intermediate filament protein that is expressed by mature

astrocytes (Waldvogel et al., 2006). Astrocytes are involved in immune function, which is

suspected to be involved in autism pathology (Patterson, 2011). Reportedly, GFAP

immunoreactivity (Vargas et al., 2005) and protein content (Laurence and Fatemi, 2005)

are increased in the cortex and cerebellum of individuals with autism. However, in the

current study there were no differences in GFAP immunoreactivity in the SVZ between

AD and TD cases. Our results are consistent with a report that failed to find GFAP

changes in schizophrenic patients versus controls (Fatemi et al., 2004).

The lack of differences in gross astrocyte morphology in this sample does not

preclude disturbances in astrocyte function in this disorder. Astrocytes provide

structural, metabolic and tropic support for the components of the SVZ (Lim and

Alvarez-Buylla, 1999). For example, astrocytes help regulate synaptic 5-HT content by

uptake via 5-HTT (Kubota et al., 2001) and express FGF-2 and their respective receptors

125

(Porlan et al., 2013). Thus, although quantification of GFAP+ staining was not different

between the groups, astrocytic regulation of 5-HT and/or FGF-2 could be impaired in this

disorder. Astrocytes contribute to synaptic 5-HT content by taking up, releasing and

responding to 5-HT and 5-HT modulating drugs (Inazu et al., 2001). Glutamate has

recently been shown to block the ability of astrocytes to respond to 5-HT (Schipke et al.,

2011). Glutamate is increased in autism (Purcell et al., 2001; Shinohe et al., 2006), and

may therefore, impair astrocytic function in autism by inhibiting regulation of 5-HT.

The tissue from the 60 year old autism case in the current study displayed dense

IBA-1 staining especially in the corpus callosum. This observation supports other studies

that suggest a role for microglial pathology in autism (Morgan et al., 2012) and in the

BTBR animal model of the disorder (Heo et al., 2011). In the current study IBA-1

labeling was most clearly observed in the 60 year old autism sample containing the

corpus callosum. The limited number of samples with adjoining corpus callosum

sections precluded any group comparisons in this study. However, given the consistent

reports of corpus callosum abnormalities in autism, the current findings present an

interesting avenue of investigation for future research.

4.6. Limitations:

Immunohistochemical examination of post-mortem tissue is a valuable tool for

studying the chemical and structural anatomy of the brain in healthy and diseased states.

However, these techniques are not without limitations. In general post-mortem human

brain research is restricted by the availability of appropriate tissue samples. Pediatric

samples, which are critical for neurodevelopmental disorder research, are especially rare

(Abbott, 2011). The relatively small sample size in the current study reflected all of the

available young autism samples at the time and was consequently unavoidable.

Replication with a larger number of samples in addition to parallel investigations of these

systems in animal models may lend support to the current findings.

An additional limitation of human post-mortem tissue research relates to the

unpredictability of immunolabeling when compared to animal tissues. Various pre- and

post-mortem factors that may interfere with molecular preservation can be rigorously

126

controlled in animal studies but will vary considerably in human samples. In the current

study a consorted effort was made to match samples with regard to age, gender and post-

mortem interval. Possible effects of agonal state, mode of death, or time in storage were

not examined. However, the confirmation of 5-HTT protein expression in the current

study by multiple methods (Western Blot and immunostaining) strengthens the validity of

these findings. Given the scarcity of autistic brain tissue, the agreement of the current

results with other published studies (Azmitia et al., 2011a, 2011b; Wegiel et al., 2013)

suggest that these findings may provide valuable and valid biomarkers of this condition.

4.7. Conclusions

The studies described here were undertaken with the goal of determining

structural and molecular differences in the SVZ of AD brains. Improved understanding

of the composition, activity and functions of the SVZ may clarify the mechanisms

underlying autism neuropathology and provide novel therapeutic targets. Although post-

mortem brain research is hampered by the scarcity of appropriately-preserved tissue as

well as an array of technical challenges associated with such tissues, we have shown here

that serotonergic and growth factor proteins have expression patterns that parallel known

cellular aberrations in autism. The increased 5-HTT and FGF-2 density in the autism

SVZ are consistent with the neurodevelopmental overgrowth hypothesis of autism. Our

results provide further evidence that serotonin plays a role in the pathogenesis of autism

and provide strong support for assessing 5-HT specific marker expression in the SVZ as a

biomarker for autism.

127

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CHAPTER 5. GENERAL DISCUSSION

5.1. Autism & Serotonin

Autism is a behaviorally defined neurodevelopmental disorder associated with

pathological disturbances in neural growth and organization (Pardo and Eberhart, 2007).

While the etiology of autism remains poorly defined, a combination of clinical,

neuroimaging, neurochemical and neuropathological studies implicate the serotonergic

system in the disorder (Zafeiriou et al., 2009). Hyperserotonemia is among the most

consistently documented candidate biomarkers for the disorder (Cook and Leventhal,

1996). Furthermore, 5-HT regulates many of the behaviors that are altered in autism

(Lucki, 1998) and increasing 5-HT activity often improves the behavioral deficits (Tsai,

1999), while reducing 5-HT can worsen these behaviors (McDougle et al., 1996). Finally,

a number of genes that regulate 5-HT function have been implicated in autism (Zafeiriou

et al., 2009).

5-HT is a critical developmental signal (Whitaker-Azmitia, 2001) and disruptions

to this system produce physiological and behavioral symptoms associated with autism

(Whitaker-Azmitia, 2005; Borue et al., 2007; Croen et al., 2011; Rai et al., 2013).

Furthermore, neuroimaging studies suggest that the development of the 5-HT system is

blunted in autism (Chugani et al., 1997, 1999). Given the critical role of 5-HT in neural

growth and behavioral regulation, abnormalities of 5-HT development, activity, and the

subsequent effects on downstream circuitry may influence the manifestation of autistic

symptoms.

The studies described here were undertaken to investigate the role of serotonergic

disruptions in autism using a multidimensional approach. Chapter 2 used an

endophenotype method to investigate suspected 5-HT disturbances in an animal model

that displays an autism-relevant phenotype. Chapter 3 used a reverse-genetics approach

to assess the effects of a targeted 5-HTT mutation on mouse behaviors relevant to the

disorder. Chapter 4 was designed as a neuropathological study aimed at identifying

potential serotonergic disruptions that may directly or indirectly contribute to autism

neuropathology. The rodent models reviewed in Chapter 2 and 3 parallel commonly

reported clinical findings such as reduced 5-HT activity, and suggest additional candidate

pathways for further research. Chapter 4 supports findings of increased 5-HTT

135

expressing neurons in post-mortem brains from autistic individuals and provides a

potential novel biomarker and therapeutic target for the disorder.

5.2. Serotonin alterations in the BTBR mouse model for autism

The experiments described in Chapter 2 extend upon previous findings of reduced

serotonergic functioning in the BTBR mouse (Gould et al., 2011) by showing reduced

concentrations of 5-HT in the BTBR cerebellum; a structure consistently implicated in

autism (Allen, 2005). In addition, the behavioral aspects of the experiments confirm

previous reports that BTBR mice display an autism-relevant phenotype, including a

mouse analog of gaze aversion, when confined with another mouse in social proximity

(Defensor et al., 2011). Interestingly, the cerebellar flocculus, which is involved in eye

gaze behavior and is reported to be disturbed in autism (Wegiel et al., 2013), is located in

a 5-HT impaired brain region in this strain expressing gaze aversion. The cerebellum

receives a fine plexus of serotonergic fibers through which 5-HT is released via volume

transmission (Trouillas and Fuxe, 1993). 5-HT concentrations in the cerebellum are

positively correlated to the level of motor activity (Mendlin et al., 1996). Taken together,

the current results suggest that reduced 5-HT activity in the BTBR cerebellum may be

related to the gaze aversion-like behavior displayed by this strain in the social proximity

test.

Reduced concentrations of cerebellar 5-HT may contribute to disturbed

oculomotor activity in the BTBR through the loss of Purkinje Cells (PC). PCs are the

primary output cells of the cerebellum (Llinas and Walton, 1990) and are involved in

oculomotor control (Wegiel et al., 2013). 5-HT activity in the cerebellum modulates PC

development (Oostland et al., 2013), arborization (Kondoh et al., 2004) and firing

(Dieudonné, 2001). Namely, a reduction in 5-HT activity in the cerebellum leads to a

loss of PC complexity and firing (Oostland and van Hooft, 2013). One of the most

consistent cerebellar abnormalities reported in autism is reduced numbers of PCs (Fatemi

et al., 2002). Therefore, the reduced cerebellar 5-HT observed here in the BTBR could

result in reduced PC activity and consequently, contribute to the gaze-like aversion

displayed by this strain.

In addition to oculomotor control, the cerebellum is important for cognitive

function, language, emotion regulation and social interactions (Riva and Giorgi, 2000;

136

Schmahmann, 2004). Dysfunction of the cerebello-thalamo-cortical circuit is thought to

underlie the cognitive and affective symptoms associated with autism (Hoppenbrouwers

et al., 2008). Reduced cerebellar 5-HT activity and consequent reductions in PC firing

could result in a disinhibition of the deep cerebellar nuclei which would in turn lead to

increased excitatory output to the thalamus and subsequently the cortex. Consistent with

these hypotheses, imaging studies revealed altered cerebellar activation (Allen, 2005)

and increased thalamo-cortical output in autism (Mizuno et al., 2006). Similarly, in

Chapter 2 we report reduced 5-HT and 5-HIAA in the BTBR cerebellum as well as

increased concentrations of 5-HT, 5-HIAA, DA and DOPAC in the BTBR cortex when

exposed to novel environments. These neurochemical findings in the BTBR are

consistent with the hypothesis of altered cerebral-cortical feedback in autism, which may

be modulated through 5-HT signaling.

5.3. Enhanced stereotypy in 5-HTT knock-out mice

The experiments described in Chapter 3 aimed to understand the behavioral

consequences of life-long 5-HTT deficiency. 5-HTT is a modulator of circulating 5-HT

concentrations (Jacobs and Azmitia, 1992) and 5-HTT gene mutations have been

implicated in autism (Zafeiriou et al., 2009). In the current study, the lack of 5-HTT

produced modest enhancement of stereotyped and repetitive behaviors but did not alter

social, aggressive, or communicative behaviors. Given the multifaceted etiology of the

disorder and the diversity in symptom expression, it is not surprising that a single genetic

knock-out failed to display the entire spectrum of behaviors typical of the disorder.

Nevertheless, animal models are critical for investigating important aspects of behavior,

physiology, and anatomy, and will continue to further clarify the underlying

neurobiology of autism.

In addition to the enhanced stereotypy described here, additional studies of 5-HTT

KO rodents indicate many of the physiological and behavioral features associated with

autism. 5-HTT KO rodents show a reduction in corpus callosum connectivity (van der

Marel et al., 2013), consistent with findings of reduced corpus callosum density in autism

(Hong et al., 2011). In line with reports of gastrointestinal dysfunction (Mazurek et al.,

2013) and rapid eye movement (REM) sleep deficiencies in autistic patients (Buckley et

al., 2010), 5-HTT KO rodents also have increased gut motility and disturbed REM sleep

137

(Murphy and Lesch, 2008). Additionally, 5-HTT KO rodents show increased anxiety

(Holmes et al., 2003; Kalueff et al., 2007) and depression-related behaviors (Murphy and

Lesch, 2008; Kinast et al., 2013), which are frequently co-morbid with autism (van

Steensel et al., 2011). 5-HTT regulates nearly every physiological and behavior domain

that is disturbed in autism. Therefore, the 5-HTT KO mouse may continue to be a useful

model to study the functional interactions between molecular and neurochemical systems

that may contribute to the associated symptoms of autism.

The 5-HTT KO mouse has no detectable 5-HTT protein (Bengel et al., 1998)

while the BTBR mouse described in Chapter 2 has 20-30% fewer 5-HTT binding sites

compared to B6 mice (Gould et al., 2011). Both strains display autism-associated

pathophysiology such as reduced tissue concentrations of 5-HT (Bengel et al., 1998; Kim

et al., 2005), reduced sensory function reflected by blunted responses to mildly painful

thermal stimuli (Vogel et al., 2003; Silverman et al., 2010) in addition to mild insulin

resistance and obesity (Flowers et al., 2007; Murphy and Lesch, 2008). These findings

are consistent with reports of reduced 5-HTT binding (Makkonen et al., 2008), hypo-and

hyper- sensory responses (Horder et al., 2013) and increased prevalence of obesity

(Curtin et al., 2010) in individuals with autism. Both strains also display autism-relevant

phenotypes such as increased self-grooming (Kalueff et al., 2007; McFarlane et al., 2008;

Pearson et al., 2011) and enhanced responsivity to stress (Tjurmina et al., 2002; Holmes

et al., 2003; Benno et al., 2009; Pobbe et al., 2011). However, the two strains differed

significantly in their manifestation of the three core autism features. BTBR mice display

behaviors relevant to each of the three core symptom domains (Meyza et al., 2012), while

the 5-HTT KO mouse only showed increased stereotypy. Taken together, the results

from the current studies suggest a contributory role for 5-HTT function in the anatomical

and behavioral features of autism. However, given the complex and diverse symptom

expression in autism, other molecular and environmental factors likely contribute to the

manifestation of the disorder.

Autism is recognized as a multi-factorial disorder with both genetic and

environmental contributions (Persico and Bourgeron, 2006). Therefore, variations in 5-

HTT genotype in conjunction with additional genetic and environmental factors could

interact to contribute to autism pathology. For instance, reduced expression of the 5-HTT

138

gene contributes to increased stress sensitivity (Bartolomucci et al., 2010; Caspi et al.,

2010) and a number of species show increased anxiety and depression behaviors after

stressful life events in those individuals with reduced 5-HTT expression (Caspi et al.,

2010). Further support for a 5-HTT gene x environment interaction in autism comes

from a recent study that found that reduced expression of the 5-HTT gene interacted with

maternal smoking during pregnancy and low birth weight to increase autism-relevant

symptoms such as social interaction deficits and rigid behaviors in children with attention

deficit hyperactivity disorder (ADHD) (Nijmeijer et al., 2010). Furthermore, many of the

environmental risk factors associated with autism risk also interact and/or interfere with

the serotonin system. Prenatal factors such as the use of serotonergic agonists during

pregnancy (Croen et al., 2011), stress (Ronald et al., 2010), infection (Patterson, 2011),

and a history of maternal depression (Rai et al., 2013) are recognized as risk factors for

autism and are known to modulate serotonergic activity (Dunn et al., 1999). On the basis

of these observations, it is possible that a combination of genetic vulnerability and an

accumulation of environmental factors could interact to further disrupt the 5-HT system

and increase the risk for autism. Overall, the findings from the two animal models

described here support the general hypothesis that 5-HTT deficiency underlies complex

and diverse phenotypes with implications for a number of psychiatric disorders, including

autism.

5.4. Increased mitogenic factors in the SVZ of young autism brains

In contrast to the animal models described thus far, the final study revealed

significantly increased 5-HTT immunostaining in the SVZ tissue from the 5 year old

autism donor. These results compliment and extend on reports of increased 5-HTT

immunoreactivity in the cerebellum (Wegiel et al., 2013), median forebrain bundle, ansa

lenticularis, stria terminalis, and superior temporal cortex (Azmitia et al., 2011, 2011) in

post-mortem brains of autistic individuals. Collectively these results suggest a global

increase in all serotonergic pathways in the brains of autistic individuals when compared

to TD controls.

5-HT is a well documented modulator of brain development (Whitaker-Azmitia,

2001) and the SVZ is critically involved in neural development and life-long plasticity

(Brazel et al., 2003). Increased 5-HTT immunostaining in the SVZ likely reflects

139

increased 5-HT release in this region (Porlan et al., 2013). Increased concentrations of 5-

HT in the SVZ could produce a number of neurodevelopmental effects including

increased neurogenesis (Banasr et al., 2004), reduced apoptosis (Persico et al., 2003),

inhibition of cellular differentiation (Menegola et al., 2004) and decreased cellular

migration (Riccio et al., 2008). These cellular disruptions are consistent with reports of

autism pathology (Palmen et al., 2004), suggesting a critical role for 5-HT signaling in

the SVZ as a mediator of altered neurodevelopment in this disorder.

Excessive 5-HT signaling in this region may act through an increase in the

secretion of growth factors. Compounds that increase 5-HT concentrations also increase

FGF-2 expression (Maragnoli et al., 2004; Bachis et al., 2008). Moreover, astrocytes,

which are abundant in this region, express 5-HTT (Kubota et al., 2001) and can secrete

FGF-2 (Gómez-Pinilla et al., 1992); providing a possible mechanism for 5-HT action.

Here we show increased 5-HTT and FGF-2 immunoreactivity in the brains of young AD

individuals. FGF-2 promotes neurogenesis and neural migration (García-González et al.,

2010), and is responsible for regulating cerebral cortex size, neuron number and

gyrification (Vaccarino et al., 2009; Rash et al., 2013). The increased brain weight

observed in the 5 year old autism case could reflect seroteonergic stimulation of FGF-2

signaling. Taken together, the findings of increased 5-HTT and FGF-2 immunoreactivity

in the young autism samples support findings of accelerated brain growth in young

autism individuals (Courchesne et al., 2001). Furthermore, these findings propose a

potential mechanism for this brain overgrowth through 5-HT regulation of SVZ activity.

5.5. Conclusions, constraints & future directions

The studies described here represent an attempt to systematically examine the 5-

HT system in autism using a variety of neurochemical, behavioral, and neuropathological

techniques. The animal models described in Chapter 2 and 3 investigated neurochemical

and genetic contributions to autism-relevant behaviors. In each of the mouse models,

reduced brain tissue concentrations of 5-HT were associated with behaviors relevant to

the symptoms of autism. In contrast, the neuropathological findings of Chapter 4

suggest increased 5-HT activity in the brain from the young autistic individual, as

revealed by increased 5-HTT staining in the SVZ. Increased 5-HT in this mitogenic

region may contribute to the altered brain growth trajectories observed in autism. One

140

implication of the current findings is that optimal 5-HT activity may be confined to a

narrow range, such that reduced or elevated function may contribute to autism

vulnerability and to the wide range of behavioral phenotypes observed in the disorder

(Veenstra-VanderWeele et al., 2009).

The current studies support and extend on the existing literature describing altered

serotonergic function in autism (Cook and Leventhal, 1996) and suggest that 5-HT

changes may correlate with the anatomical and behavioral features of the disorder. The

use of animal models has many advantages for studying the suspected neurobiological

underpinnings of this disorder. Thanks in large part to the mapping of the mouse

genome, mice with genetic mutations relevant to human disorders can be easily created

(Guénet, 2005). In comparison to humans, mice have a relatively short life-span (2-3

years), allowing for quick and convenient longitudinal studies. Furthermore, a number of

experimental factors can be easily controlled and/or manipulated with animal models,

which is not possible in studies with humans. However, as autism is a uniquely human

disorder, animal models do not recapitulate all of the behavioral, physiological and

neuroanatomical features of the disorder. Nevertheless, large-scale efforts are underway

to improve behavioral phenotyping assays (Defensor et al., 2011; Pearson et al., 2011;

Pobbe et al., 2011; Wöhr and Scattoni, 2013) and create gene x environment models

(Moy and Nadler, 2007) to better examine the neurobiological, genetic, endophenotypic

and pathogenic factors contributing to autism.

Given the uniquely human nature of autism, examination of the affected brain

tissue is critical to understanding the underlying biological mechanisms that cannot be

modeled through animal models or cell lines. Tissue studies are limited by the

availability of donated samples. Brain samples from young autistic children are

especially rare (Abbott, 2011), likely due to the fact that autism is a non fatal disorder.

Individuals with autism often live into adulthood and while the adult brain may retain

remnants of the neurodevelopmental disturbances of autism, investigation of brains at the

age of symptom expression (2-3 years old) may yield more clues as to the causative

factors of the disorder. Similarly, post-mortem tissue allows us to examine an individual

only at a single time point; precluding longitudinal studies in autistic individuals. As

with other research using human subjects, postmortem tissue research is confounded by

141

the inherent genetic, environmental and behavioral heterogeneity of human populations.

Additionally, a number of factors that have no relationship to the disease pathology may

produce variability in protein-expression analyses. Factors such as postmortem interval,

length of tissue storage and agonal state are known to modulate gene and protein

expression analyses of postmortem tissue (González-Maeso et al., 2002; Lewis, 2002).

Autism research will undoubtedly progress more quickly with the adoption of a

multi-disciplinary approach that utilizes parallel investigations in animal models and

human subjects. Furthermore, additional efforts should be made to publicly highlight the

importance of brain tissue donation. The devastating loss of over 50 autism brain

samples in 2012 (Weintraub, 2013), underscores the need for efficient allocation of

samples to programs using varied and technologically advanced methods including high

resolution microscopy as well as microarray and methylation expression analyses.

Improved identification of biomarkers may further contribute to the therapeutic and

preventative progress made through animal modeling.

The studies described here suggest a fine-tuning mechanism for 5-HT activity in

regulating autism relevant pathology. Reduced 5-HTT expression and 5-HT tissue

concentrations in the BTBR and 5-HTT KO mouse models produced autism relevant

analogs of gaze aversion and stereotypy, respectively. In contrast, increased 5-HTT

immunostaining, suggestive of increased 5-HT activity (Azmitia et al., 2011; Porlan et

al., 2013), was seen in the SVZ of the young individual with autism. Given the diverse

range of physiological and behavioral features mediated by 5-HT, these studies suggest

that alterations in 5-HT activity, in either direction, may result in anatomical and

behavioral features associated with autism. Continued research with the addition of more

cases, autism-relevant brain regions, and complimentary methodologies may clarify the

mechanisms that prompt 5-HT dysfunction in autism and lead to developments in the

disorder's diagnosis, treatment, and prevention.

142

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SEROTONIN: FROM MOUSE MODELS TO HUMAN DISORDERS A ... · Ashley L. Jensen Dissertation Committee: D. Caroline Blanchard, Chairperson Harry Davis IV Kentaro Hayashi Scott Sinnett Lorey