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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. Materials and Methods ....................................................................................... 65
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
4.3. Materials and Methods ....................................................................................... 97
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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
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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
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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.,
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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;
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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).
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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.
Post-hoc analysis following a significant interaction between strain and 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.
Post-hoc analysis following a significant interaction between strain and condition
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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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
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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.
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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
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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
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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.
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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
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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,
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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.
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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
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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.
Light F(2,33)=0.31, p=0.74, n.s. F(2,33)=0.89, p=0.42, n.s. F(2,33)=1.97, p=0.11, 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.
Light F(2,33)=2.09, p=0.14, n.s. F(2,33)=1.97, p=0.15, n.s. F(2,33)=4.71, p<0.002
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
Light F(2,33)=1.21, p=0.31, n.s. F(2,33)=1.34, p=0.27, n.s. F(2,33)=4.31, p<0.003
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.
Light F(2,33)=0.04, p=0.96, n.s. F(2,33)=2.77, p=0.07, n.s. F(2,33)=2.15, p=0.08, 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.
Light F(2,33)=0.98, p=0.39, n.s. F(2,33)=0.12, p=0.89, n.s. F(2,33)=0.93, 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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94
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.
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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.
108
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].
111
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.
112
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.
113
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).
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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).
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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.
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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
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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;
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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
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(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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