University of Southern California Dissertations and Theses

Genotype and sex effects on anxiety behavior and functional activation in corticolimbic circuits in serotonin transporter knockout mice (5-HITT KO)

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Genotype and sex effects on anxiety behavior and functional activation in corticolimbic circuits in serotonin transporter knockout mice (5-HITT KO)

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GENOTYPE AND SEX EFFECTS ON ANXIETY BEHAVIOR AND FUNCTIONAL
ACTIVATION IN CORTICOLIMBIC CIRCUITS IN SEROTONIN TRANSPORTER
KNOCKOUT MICE (5-HTT KO)

by

Raina D Pang

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)

August 2012

Copyright 2012 Raina D Pang

ii

ACKNOWLEDGEMENTS

I would like to thank my doctoral adviser Dr. Daniel Holshneider for giving me
the opportunity to work on a project that I felt truly inspired by. Dr. Holschneider was
always there to help me with technical problems, as well as provide professional
guidance for me along the way. In addition to my primary mentor, I would like to thank
my dissertation committee members for their support academically and in graduate
student affairs. My committee members always showed great enthusiasm for my work
and asked questions that broadened my scientific outlook. The generosity of their time
and support allowed me to maintain a foreword path in my scientific career.
I would also like to acknowledge and thank the following for their contributions
to the work presented here: David Herman for completing the whisker study experiments
presented in Chapter one and Tansu Celikel for his guidance on these studies. Dr. Zhuo
Wang provided technical training in the imaging methods and functional connectivity
analysis shown in Chapters two and three. Yumei Guo provided invaluable technical
assistance and general laboratory support.
I was fortunate to work with very talented young scientists and graduate students.
Marta Vukavic was extremely supportive and someone to look up to. Marta provided me
with a steady supply of advice, encouragement, and was instrumental in keeping me on
the correct timetable towards completing the requirements for the neuroscience program.
I will always be grateful for the warmth and open ears of Letisha Wyatt. Letisha was
always there to be my voice of reason and the calm throughout the storm. William

iii

Fowler kept me grounded throughout my dissertation studies. Anna Kamitakihara, Vilay
Khandelwhal, Martha Hernandez, Jenny McGrady, Kelly Kent and Elizabeth Zuniga
provided me kind words and encouragement that allowed me to stay on the path to
graduation.
I am grateful for the opportunity to work with talented undergraduates. Lauren
Klosinski helped me get my project up and running. She provided invaluable support
during the early phase of my graduate career. Hilary Lazarus was extremely helpful
during the later part of my graduate career. In particular her editorial skills helped firm
up my writing.
Funding for the studies presented in this dissertation came from a young
investigator grant from the brain and behavior research foundation awarded to Dr.
Holschneider.

iv

TABLE OF CONTENTS

Acknowledgements ii

List of Figures

v

Abstract

vi

Introduction
Mood and anxiety disorders
Anatomy and function of circuits involved in emotional regulation
Serotonergic system
5-HT role in mood and anxiety disorders
5-HTTLPR effects central processing
5-HTT KO mice display increased anxiety and mood behavior and
alterations in emotional and sensory circuits
Translational models of mood and anxiety disorders
Cerebral blood flow (CBF) as a measure of neuronal activation

1
2
3
4
5
6
9

12
14

Chapter One: Sex and genotype effects on anxiety-like behavior

19

Chapter Two: Conditioning, genotype, and sex effects on functional
activation in corticolimbic circuits
Table 1: Significant changes in rCBF in the cortex and subcortex
in the left and right hemispheres

40

57

Chapter Three: Genotype and conditioning effects on functional
connectivity in corticolimbic circuitry

65

Conclusions

81

References

87

Appendices
Appendix A. Genotype effects on marble burying
Appendix B. Genotype effects on circadian rhythmicity in male 5-
HTT KO mice

111
113

v

LIST OF FIGURES

Figure 1: Known structural effects of developmental blockage of 5-HTT
in corticolimbic and sensory structures
10

Figure 2: Fear Conditioning and Recall

27

Figure 3: Open Field

29

Figure 4: Light/Dark exploration

31

Figure 5: Impaired whisker function in male 5-HTT KO mice

34

Figure 6: Sample 3D reconstruction of a mouse brain

46

Figure 7: Factorial analysis examining a main effect of genotype, sex or
conditioning

48

Figure 8: Factorial analysis examining interactions between genotype, sex
and conditioning

51

Figure 9: Effects of genotype on functional activity in response to a tone

53

Figure 10: Effects of sex on functional activity in response to a tone

55

Figure 11: Interregional correlation matrix in WT-CON mice

70

Figure 12: Interregional correlation matrix in WT-CF mice

72

Figure 13: Interregional correlation matrix in KO-CON mice

74

Figure 14: Interregional correlation matrix in KO-CF mice

76

Figure 15: Marble Burying

112

Figure 16: Sample actograms

118

vi

ABSTRACT

In humans, the serotonin transporter linked polymorphic region (5-HTTLPR)
results in altered transcriptional efficiency of the transporter gene, as well as uptake of
serotonin (5-HT) by neurons. The low expressing ‘s’ allele of this polymorphism has
been linked to increased susceptibility to stress and altered connectivity of corticolimbic
neural circuits, the disruption of which is widely invoked to explain emotional
dysregulation in anxiety and mood disorders.
Serotonin transporter (5-HTT) knockout (KO) mice offer a promising model for
psychiatric research as close parallels exist between the human polymorphism and the
mouse model at the level of serotonergic profile, behavior, physiological function, and
stress hormone response. Although much work has gone into characterizing 5-HTT KO
mice, little work to date has examined functional activation patterns and connectivity of
circuits involved in emotional regulation in these mice. The experiments outlined in this
dissertation address this gap in knowledge by examining sex and genotypic differences in
the underlying functional activation of corticolimbic circuits. The inclusion of females in
this study provides important information about how sex differences in serotonergic
function alter brain function. Functional connectivity analysis is also undertaken in a
combined male/female groups to determine genotypic effects on a network level.
Results presented showed robust increases in freezing behavior during fear-
conditioned recall in 5-HTT KO mice. This genotypic effect was preserved but not
accentuated in females. In contrast to the robust genotypic effects found during fear

vii

conditioning, anxiety like behavior was mainly limited to reduced exploration in 5-HTT
KO mice in ‘naturalistic’ anxiety tests. Because exploratory-based anxiety tests rely
heavily on motor and sensory systems, additional home cage locomotor and whisker
function tests were performed to determine genotypic effects on motor and sensory
systems. Home cage locomotion did not significantly differ between genotypes
suggesting that hypolocomotion seen in 5-HTT KO mice was limited to novel
environments. 5-HTT KO mice did show deficits in whisker function in a gap cross test.
The results from these studies show that genotypic effects on anxiety like behavior are
most apparent during conditioned anxiety and less apparent in ‘naturalistic’ tests reliant
on exploration. Females explored more than males, but this increase in exploration was
mainly attributed to female WT mice.
To determine the effects of genotype on functional activation and connectivity,
functional brain mapping using [
14
C]-iodoantipyrine was performed during recall of a
fear-conditioned tone. Regional cerebral blood flow (rCBF) was analyzed by statistical
parametric mapping (SPM) from autoradiographs of three-dimensionally reconstructed
brains. Results of these studies showed robust main effects of conditioning, genotype
and sex in corticolimbic circuitry implicated in fear behavior. Specifically, conditioning
showed main effects in the conditioned fear circuit including the prelimbic, cingulate,
retrosplenial, amygdala, dorsal hippocampus, and sensory regions. Genotype
significantly altered functional activation in the fear circuit including main effects in the
cingulate, retrosplenial, insula, motor, sensory, amygdala, and hippocampus. These
results provide further evidence that 5-HTT related 5-HT availability plays a role in the

viii

function of corticolimbic circuitry, which may contribute to abnormalities in fear and
anxiety like behavior. Although no sex differences were seen in freezing behavior, sex
showed main effects in fear circuitry including the prelimbic, cingulate, insula, motor
cortex, sensory cortex, hippocampus and cortical amygdala. Interestingly, the effects of
sex were most apparent in the prelimbic and limited in the amygdala. This suggests that
sex may exert its effects on fear behavior more through emotional regulation rather than
reactivity.
Correlational connectivity analyses were applied to further explore genotypic
effects on corticolimbic circuitry on a network level. Differences in reorganization of
functional connectivity were especially apparent in control animals. Specifically, KO
control animals compared to WT control animals showed significant reorganization of
functional connectivity within motor, sensory and insular cortex and between these
regions and the striatum, dorsal hippocampus and raphe. The prefrontal cortex showed
increased connectivity with the raphe and decreased connectivity to the amygdala, motor
and sensory cortex, visual and auditory cortex, and the insula. These results suggest that
even in the presence of a non-threatening auditory cue genotype significantly alters the
organization of circuits implicated in stress.
Overall, the experiments contained in this dissertation contribute novel
information about the role of sex and 5-HTT genotype in anxiety like behavior and
function of corticolimbic circuits. Specifically, this is the first study using whole brain
functional perfusion mapping in the 5-HTT KO mouse model. Furthermore, these studies
support sex differences in functional regulation of the prefrontal-amygdala network in a

ix

fear-conditioned paradigm. These studies establish the translational value of functional
brain mapping endpoints and suggest greater specificity than the behavioral endpoints.
Future studies could address the underlying molecular and structural changes
accompanying functional changes seen in 5-HTT KO mice.

1

INTRODUCTION

The goal of the studies comprising this dissertation was to further our
understanding of the effects of loss of the serotonin transporter (5-HTT) and sex on
function of neural circuits involved in emotional regulation and how these changes relate
to anxiety and mood behavior. The first chapter focuses on genotype and sex effects on
anxiety behavior in 5-HTT knockout (KO) mouse model. Specifically, we test the
hypothesis that 5-HTT KO mice would show increased anxiety behavior in fear-
conditioned and exploratory-based anxiety tests. Furthermore, we expect females to
show an exaggerated effect of genotype. Additional experiments testing motor and
sensory function in 5-HTT KO mice serve to address non-anxious genotypic differences
that can contribute to differences in anxiety tests. The second chapter focuses on
conditioning, genotype and sex effects on functional brain activation in corticolimbic and
motor and sensory regions. Specifically, we test the hypothesis that genotype and sex
will alter patterns of activation in corticolimbic circuitry. The third chapter focuses on
genotypic effects on functional connectivity of circuits involved in the processing of
emotional stimuli. Specifically, we hypothesize that KO mice will have altered
functional connectivity in corticolimbic circuitry. Completion of the studies outlined in
this thesis furthers our understanding of the effects 5-HTT and sex on central processing
of corticolimbic circuits. This knowledge will play an important role in our
understanding of how genetic alterations in 5-HTT availability and sex lead to an
increased vulnerability for the development of anxiety and mood disorders.

2

1.1 Mood and Anxiety Disorders
Mood and anxiety disorders are a prevalent (Greenberg et al., 2003; Kessler et al.,
2005; Lepine, 2002) and costly (DuPont et al., 1996; Lecrubier, 2001; Lepine, 2002)
problem in our society. Mood and anxiety disorders arise from complex interactions
between genes and the environment; thus in these disorders the relationship between
genes and behavior is indirect. Despite intense study, the underlying neurobiology of
these disorders is still not thoroughly understood. A major limiting factor in our
understanding of these disorders is the symptom-based classification system of
psychiatric disorders (Association, 2000). Because of the variability in the presentation
of these disorders, this symptom based classification system limits the ability to model
these disorders in animals (Gould and Gottesman, 2006). Breaking down these complex
behaviors into endophenotypes, i.e. quantifiable components along the genes-to-behavior
pathway, increases our ability to detect relevant genetic variants and our understanding of
how these genetic variants contribute to mental disease (Gould and Gottesman, 2006).
Neuroimaging has been proposed as a potential endophenotypic measure that
could bridge genetic research with cognitive phenotypes. This method provides
quantitative data that is generally more sensitive than behavioral measures (Glahn et al.,
2007). Neuroimaging studies are providing evidence that function of the circuitry
involved in emotional regulation responds abnormally in patients with mood and anxiety
disorders.

3

1.2 Anatomy and function of circuits involved in emotional regulation
Neural circuits underlying normal emotional behavior include: limbic-cortical-
striatal-pallidal-thalamic circuits (LCSPT), orbital prefrontal network and the medial
prefrontal network (Drevets et al., 2008). LCSPT includes connections between orbital
and medial prefrontal cortex, amygdala, hippocampal subiculum, ventral medial striatum,
mediodorsal thalamic nuclei, midline thalamic nuclei and ventral pallidum (Drevets et al.,
2008). This circuit is believed to play an important role in the expression of emotional
behavior via its anatomical connections to visceral control structures (Drevets et al.,
2008).
The orbital prefrontal network involves cortical connections between the central
and lateral orbital cortex with sensory association areas (somatic sensory areas, olfactory
and taste regions) (Drevets et al., 2008). This network seems important for integrating
multi-modal sensory information, assessing the relative value of the stimuli, reward and
aversion (Drevets et al., 2008; Price and Drevets, 2010).
The medial prefrontal network is a component of the default network seen during
the “resting” state of imaging (Drevets et al., 2008; Price and Drevets, 2010). This
network includes dorsomedial and dorsal anterolateral prefrontal cortex, mid and
posterior cingulate, a section of anterior superior temporal gyrus and sulcus, entorhinal
and posterior parahippocampal cortex (Drevets et al., 2008). The medial prefrontal
network has prominent limbic and visceral control connections; thus positioning it to play
an important role in introspection and visceral reaction to emotional stimuli (Drevets et
al., 2008; Price and Drevets, 2010).

4

Disorders of anxiety and mood are thought to result from abnormal perception,
response and storage of emotional stimuli. Patients with mood and anxiety disorders
have been shown to have both structural and functional abnormalities in areas involved in
the regulation of normal emotions (Drevets et al., 2008; Drevets et al., 1992).
Dysfunction in these circuits may result in cognitive and emotional dysregulation seen in
mood and anxiety disorders. Genes have also been shown to affect these circuits, which
could result in increased susceptibility to disorders of anxiety and mood. In particular,
genes affecting the serotonergic system have been investigated for their role in regulating
emotional circuitry and susceptibility to mood and anxiety disorders.

1.3 The serotonergic system
After being synthesized from tryptophan, the vesicular monoamine transporter-2
packages 5-HT into vesicles. Once released into the extracellular space, 5-HT can
interact with 14 known receptors (Barnes and Sharp, 1999; Borue et al., 2007), each of
which exerts a specific effect depending on its location on the cell and the cell’s location
in the brain (Barnes and Sharp, 1999). Removal of 5-HT from the synaptic cleft
terminates serotonergic signaling. The main route of removal occurs via the serotonin
transporter (5-HTT); thus positioning 5-HTT to play a critical role in the regulation of
serotonergic signaling. In addition to 5-HTT, 5-HT is removed from the synapse with
low efficiency (low affinity and selectivity) by dopamine transporters (Daws, 2009; Zhou
et al., 2002) and organic cation transporters (Baganz et al., 2008; Chen et al., 2001;

5

Daws, 2009). After being removed from the synaptic cleft, 5-HT is broken down by
monoamine oxidase. 5-HT that is not broken down is repackaged for re-release.
5-HT cell bodies are located at the midline of the brainstem, mainly the raphe
nuclei (Frazer and Hensler, 1999; Kandel et al., 2000; Paxinos, 2004). These neurons
develop early supporting the notion that 5-HT plays an important role in
neurodevelopment (Paxinos, 2004). As 5-HT projects vastly throughout the brain, 5-HT
is thought to play an important modulatory role in a wide variety of behaviors including,
but not limited to, food intake, maternal behavior, sex, appetite, circadian rhythms, and
mood and anxiety symptoms. Most relevant to this thesis is the role of 5-HT in
regulating neural circuits involved in emotional regulation and how this relates to its role
in mood and anxiety behavior.

1.4 5-HT's role in mood and anxiety behavior
The serotonergic system is commonly studied in psychiatric research. Although
complex emotional states seen in mood and anxiety disorders most likely involve
imbalances in more than one neurotransmitter system, it is generally accepted that
abnormal serotonergic signaling contributes to mood and anxiety disorders. The
presumed importance of 5-HT in mood and anxiety disorders is reflected in
pharmacological treatment of these disorders, with selective serotonin reuptake inhibitors
(SSRIs) being the most commonly prescribed medication for these disorders (Vaswani et
al., 2003).

6

Research investigating the role of 5-HT signaling in mood and anxiety disorders
dates back to the 1960s and has shown that depressed individuals show low 5-HT binding
and 5-HT metabolites in cerebrospinal fluid (Bryer et al., 1992; Gao et al., 2008; Owens
and Nemeroff, 1994; Parsey et al., 2006). Tryptophan depletion studies provide further
evidence that maintaining 5-HT levels is important in depression prone individuals
(Altman et al., 2010; Feder et al., 2011). Because 5-HT plays such an integral role in
mood and anxiety behavior, genetics that alter serotonergic homeostasis have been
investigated for their role in mood and anxiety disorders. Particularly, the serotonin
linked polymorphic region (5-HTTLPR) and animal models of this polymorphism have
been investigated for their role in mood and anxiety disorders (Caspi et al., 2010; Murphy
and Lesch, 2008).

1.5 5-HTTLPR effects central processing
In humans, 5-HTTLPR results in altered transcriptional efficiency of 5-HTT and
the uptake of 5-HT by neurons. Individuals with the 's' allele of 5-HTTLPR have reduced
transcription of 5-HTT and reduced 5-HT uptake (Greenberg et al., 1999; Lesch et al.,
1996). Having the ‘s’ allele has been associated with increased susceptibility to mood
disorders in the face of environmental stressors (Caspi et al., 2010; Caspi et al., 2003) and
anxiety symptoms (Canli et al., 2005; Lesch et al., 1996), but not all studies find an effect
(Gillespie et al., 2005; Lang et al., 2004; Munafo et al., 2009). The inconsistent
behavioral response may result from differences in testing protocols (Caspi et al., 2010)
or definitions of stressors (Caspi et al., 2010), the role of additional polymorphisms of the

7

SLC6A4 gene (Lazary et al., 2008), interaction with other genes, sex differences
(Brummett et al., 2008; Sjoberg et al., 2006), or a relatively small effect of this gene.
Since genes play a role at the circuitry level, which then may or may not affect
overt behavior, neuroimaging studies provide new insight about the effect of 5-HTTLPR
polymorphism (Hariri and Weinberger, 2003). It has been suggested that 5-HTT-related
availability of 5-HT in the developing brain is related to the development of key regions
of the limbic system, in particular the prefrontal cortex and infragenual cingulate cortex
(Caspi et al., 2003; Holmes, 2008). Consistent with the central role of 5-HT in
neurodevelopment, evidence of a robust 5-HTTLPR influence on brain anatomy has
emerged. In healthy individuals, the low expressing ‘s’ allele has been associated with a
12-30% reduction in gray matter volume in limbic regions critical for processing of
negative emotion, including the anterior cingulate cortex and amygdala (Frodl et al.,
2008; Pezawas et al., 2005; Scherk et al., 2009), as well as the dorsolateral prefrontal
cortex and hippocampus (O'Hara et al., 2007; Pezawas et al., 2005).
On a functional level, several neuroimaging studies have found that healthy
individuals carrying a ‘s’ allele display amygdala hyper activation (Drabant et al., 2012;
Hariri et al., 2005; Hariri et al., 2006; Hariri et al., 2002; Heinz et al., 2005; Lau et al.,
2009; Rao et al., 2007) and altered activity in prefrontal regions (Drabant et al., 2012;
Heinz et al., 2005; Pezawas et al., 2005). Functional and structural connectivity has also
been shown to be modulated by 5-HTTLPR; thus amygdala hyper activation could arise
from poor modulatory control from cortical regions (Heinz et al., 2005; Pezawas et al.,
2005). A circuit of particular interest is the amygdala-perigenual anterior cingulate

8

(pACC) circuit. This circuit includes two functionally distinct connections from the
amygdala to the pACC (a positive functional connection to the subgenual cingulate and a
negative connection to the supragenual cingulate), which appear to be positively
connected to each other (Pezawas et al., 2005). The extent of functional connectivity in
this circuit has been shown to account for 30% of variance in personality trait scores
linked to anxiety traits (Pezawas et al., 2005). Healthy carriers of the 's' allele have
reduced functional connectivity between the amygdala and pACC (Pezawas et al., 2005).
Differences have also been shown in 5-HTTLPR in the default network during a non-
emotional task suggesting general cognitive effects of this gene on brain function
(Wiggins et al., 2012). Individuals with the 's' allele also show reductions in the uncinate
fasciculus, the white matter tract connecting the amygdala to the orbital and medial
prefrontal cortex with the amygdala (Pacheco et al., 2009).
Overall, evidence from neuroimaging studies support the notion that 5-HTTLPR
modulates neural circuitry involved in emotional regulation and reactivity. These
changes may predispose individuals to be hyperresponsive to emotional stimuli, which
may increase susceptibility to stress reactivity disorders (Pezawas et al., 2005; Rao et al.,
2007). Because of limitations to human research, animal models have also been
investigated to provide further insight into how alterations in 5-HTT lead to mood and
anxiety behavior.

9

1.6 5-HTT KO mice display increased anxiety and mood behavior and
alterations in emotional and sensory circuits
Recently, there has been interest in the role that 5-HT plays in neurodevelopment
before it assumes its role as a neurotransmitter (Gaspar et al., 2003). Serotonergic
neurons develop relatively early; thus putting them in a position to modulate
developmental processes of the brain. Evidence has implicated 5-HT in numerous
developmental processes, such as modulation of neuronal proliferation of a variety of cell
types (Chubakov et al., 1986; Chubakov et al., 1993; Pronina et al., 2003; Schmitt et al.,
2007), migration (Chubakov et al., 1986; Chubakov et al., 1993; Pronina et al., 2003;
Riccio et al., 2009), differentiation (Chen et al., 2007; Lavdas et al., 1997; Menegola et
al., 2004; Pronina et al., 2003) and prevention of cell death (Chen et al., 2007; Menegola
et al., 2004; Persico et al., 2003; Stankovski et al., 2007). Because 5-HTT (Hansson et
al., 1998; Lebrand et al., 1998; Narboux-Neme et al., 2008) and VMAT2 (Lebrand et al.,
1998) are developmentally expressed by non-serotonergic neurons, these neurons are able
to transiently express 5-HT through its uptake and storage (Lebrand et al., 1996;
Narboux-Neme et al., 2008); thus serotonergic tone has an effect on the development of
both serotonergic and non serotonergic systems. In fact, systems expressing 5-HT solely
during development may be most affected by alterations in prenatal and early postnatal
serotonergic homeostasis because they rely exclusively on extra-cellular concentrations
of 5-HT.
5-HTT knockout mice (KO) offer a promising model for psychiatric research as
parallels exist between the human polymorphism and the mouse model at the levels of

10

serotonergic profile, behavior, physiological function, and stress hormone response
(Carroll et al., 2007; Holmes et al., 2003a; Lira et al., 2003; Tjurmina et al., 2002;
Wellman et al., 2007)(for review see (Murphy and Lesch, 2008). Though 5-HTT KO
animals lack high-affinity cellular uptake of 5-HT, 5-HT can be transported
intracellularly with low efficiency (low affinity and selectivity) by the dopamine
transporter (Zhou et al., 2002) and polyspecific organic cation transporters (Baganz et al.,
2008), the latter of which have been shown to be upregulated in 5-HTT KO mice (Baganz
et al., 2008). Thus, 5-HTT KO mice have reduced, but not absent 5-HT clearance, an
observation similar, though not analogous, to findings in the human 5-HTTLPR
polymorphism.

Figure 1. (Homberg et al., 2010) Known structural effects of developmental blockage of 5-HTT
in corticolimbic and sensory structures.

11

Studies in the 5-HTT KO mice have provided evidence for structural alterations in
emotional and sensory circuitry (Figure 1). Circuits involved in emotions appear to be
affected by altered serotonergic homeostasis during development (Holmes, 2008).
Morphologically, abnormalities in 5-HTT KO mice have been observed in the basolateral
amygdala (Nietzer et al., 2011; Wellman et al., 2007). In the infralimbic cortex, both an
increase and decrease in dendritic length has been observed in 5-HTT KO mice (Nietzer
et al., 2011; Wellman et al., 2007). Furthermore, functional anatomy of this system also
seems to be perturbed in 5-HTT KO mice, with a bias from the prefrontal cortex to areas
involved in the reward circuit (Bearer et al., 2009). These alterations in emotional
circuitry may contribute to the increased mood and anxiety behaviors seen in 5-HTT KO
mice (Carroll et al., 2007; Kalueff et al., 2006; Wellman et al., 2007). Sensory circuits
have also been shown to be disturbed in 5-HTT KO mice. It is thought that excess 5-HT
exerts its effects on somatosensory projections via activation of 5-HT1B receptors, which
have been implicated in playing a role in alterations of barrel field formation and
segregation of retinal projections (Salichon et al., 2001). In 5-HTT KO mice, barrel
formation does not occur correctly in the face of excess 5-HT (Salichon et al., 2001).
Cortical density also appears affected in 5-HTT KO mice. Increased extra cellular 5-HT
during development alters cortical thickness and neuronal density, but how it alters it
depends heavily on an epistatic interaction of the 5-HTT gene with other genetic variants
(Altamura et al., 2007).

12

1.7 Translational models of mood and anxiety disorders
Mood and anxiety disorders are complex emotional states that arise from a
mixture of emotion, cognition, environmental, and genetic history. To understand how
these complex disorders come about a reductionistic approach is often applied. Because
the anxiety response has an evolutionarily advantage, the anxious response is highly
conserved across species with mouse models displaying similar behavioral and
autonomic responses to anxiety provoking stimuli (Hohoff, 2009). Furthermore, the
brain structures and neurotransmitter systems believed to modulate fear behavior is
highly conserved across species (Hohoff, 2009; Lu et al., 2012). While behavioral
studies in animals cannot fully capture the complexity of human disorders, they can
validate specific aspects of human studies while providing underlying mechanisms and/or
treatment approaches (Hohoff, 2009).
Anxiety behavioral tests for mice split into two main classes: conditioned and
unconditioned. Conditioned based anxiety tests investigate abnormalities in the ability to
learn and recall cues signaling safety and threat, which is considered a core feature of
anxiety (Casey et al., 2011). During the training phase of fear conditioning, neutral
stimuli such as a tone are paired with an aversive stimulus such as a footshock. After
conditioning, the conditioned stimuli itself elicits the unconditioned response of
behavioral immobility ("freezing"). In cued conditioning recall, the animal is placed in a
novel context and presented with the cued stimuli. The amount of freezing in response to
the cued stimuli serves as a measures of the fear response. Conditioned based anxiety
tests require motivation, sensory inputs, and learning and memory. This means that

13

deciphering the exact mechanism behind a change in behavior (i.e. differences in anxiety
or differences in learning) can be complicated. Since humans readily undergo fear
conditioning (Delgado et al., 2006), fear conditioning is particularly suited as a
translational model. Many of the findings in animals have been replicated in human
studies and provide novel hypotheses to be tested in humans. Studies in humans, on the
other hand, have the benefit of providing information about affective regulatory
strategies, which can then promote new hypotheses to be tested in animals (Delgado et
al., 2006). Such translational and reverse translational studies have played an important
role in understanding the underlying circuitry involved in the acquisition, expression and
extinction of fear.
Unconditioned anxiety models, such as the light-dark exploration and the open
field, are innate models that pit the desire to explore novel environments versus the
aversion to the novel environments because of threat assessment (Hohoff, 2009). These
tests provide information about spontaneous and innate anxiety reactions (Hohoff, 2009).
The open field is one of the most commonly used behavioral paradigm and assesses
mouse exploratory behavior and motor activity in an unfamiliar environment (Hart et al.,
2010). Exploration of the environment and time spent in the center of the arena are used
to access anxious behavior and animals that explore less and spend less time in the center
of the arena (thigmotaxis) are considered to be more anxious (Hart et al., 2010). The
light-dark exploration box also examines exploratory behavior, and animals that spend
less exploratory time (time spent, vertical exploration, etc.) in the light portion of the box
are considered more anxious (Hart et al., 2010). The major advantage of exploratory

14

based tests of anxiety is their ethological relevance and lack of application of external
noxious stimuli (Trullas and Skolnick, 1993). These tests, however, are limited by
behavioral reliance on non-anxious behaviors (i.e. locomotor, sensory and cognitive
abilities). While some suggest that fear avoidance behaviors in exploratory based tests
can be dissociated from general ambulatory effects (Trullas and Skolnick, 1993), this is
debated (Milner and Crabbe, 2008); thus when conducting these tests it is important to
account for variability in these domains before determining whether behavioral
differences actually relate to changes in anxiety.

1.8 Cerebral blood flow (CBF) as a measure of neuronal activation
The studies conducted in the following chapters include an autoradiographic
measure of CBF using a classical perfusion tracer [
14
C]-iodoantipyrine. When discussing
functional brain activation as measured by CBF, it is important to understand how CBF
relates back to neural activation, and this is discussed below. Unique about the
application of the [
14
C]-iodoantipyrine method is that it allows evaluation of changes in
functional brain activation across the entire brain. Perfusion mapping using
autoradiographic methods (Holschneider and Maarek, 2004; Holschneider and Maarek,
2008; Nguyen et al., 2004) fills a gap in the current armamentarium of imaging tools in
that it can deliver a 3-D assessment of functional activation in the awake, nonrestrained
animal, with a temporal and spatial resolution of around 5-10 seconds and 100 microns
respectively. This distinguishes it from other histologic methods such as c-fos or
cytochrome oxidase which integrate brain responses over a duration of hours to days,

15

electrophysiological recordings which typically target only very limited regions of the
brain and fMRI or microPET, which provide whole brain analysis, but require sedation of
the animal.
Neural activation is the basis of communication of neurons in the brain. This
communication is the electrical signaling of neurons and the resulting transmission of
information via an electrical signal (electrical synapse) or a flux of neurotransmitters
(chemical synapse). Action potentials are the result of changes in the flow of ions
through voltage-gated channels, which results in changes of the membrane potential
(Kandel et al., 2000). Electrical synapses rely on ion current to transmit information
(Kandel et al., 2000). At chemical synapses, a chemical transmitter is released as a result
of influx of calcium via the opening of voltage-gated calcium channels by the action
potential (Kandel et al., 2000). Furthermore, different discharge patterns (tonic or regular
spiking, phasic or bursting, fast spiking, and thin spiking) represent different
electrophysiological properties of different neurons. Because neural activation is tied to
changes in membrane potential, ion flow, neurotransmitter flow, and spiking patterns,
these changes provide direct measurement of changes in neural activation.
Functional neuroimaging measures are the attempt to elucidate the function of
brain structures through creation of a map of task related neural activation. Although it is
often implied that functional imaging techniques directly measure changes in neural
activation, in reality imaging signals usually only measure physiological changes that
occur during neural activation: energy metabolism (glucose consumption and oxygen
consumption) and hemodynamics (blood flow, blood oxygenation, and blood volume).

16

It has long been hypothesized that blood flow, neuroenergetics, and neural activity are
coupled (Roy and Sherrington, 1890), but only recently have studies investigated how
imaging signals of energy consumption and hemodynamics actually relate back to neural
activation.
CBF has been linked to two indices of neuronal activity: neuroenergetics, an
indirect measure of neural activity, and neurotransmitter related signaling, which directly
relates to neural activity. One hypothesis for increased CBF into a “neurally activated”
area is based around neuroenergetics. Neural processing of the brain is extremely
demanding metabolically (Shulman et al., 2004). Since the brain contains few energy
stores (Clarke and Sokoloff, 1999), most of the energy required for brain activity must be
constantly produced. Increased CBF, which provides glucose and oxygen, helps provide
for the increased energy demand required by neural activity. If this hypothesis is correct
then one can relate perfusion-based imaging signals to neuronal activity via
neuroenergetics, which in turn has been shown to measure neural activity (Hyder et al.,
2002; Patel et al., 2004; Shulman et al., 2001; Sibson et al., 1998; Smith et al., 2002);
thus CBF relates to measures of neural activity. This is supported by evidence linking
changes in CBF with changes in neuroenergetics (CMRO
2
) in a linear fashion (Uludag et
al., 2004).
Although changes in CBF and neuroenergetics do correlate (Hyder, 2009),
evidence suggests that changes in energy demand are not solely responsible for changes
in CBF (Mintun et al., 2001), which occurs over a larger area (Attwell and Iadecola,
2002). Another hypothesis about changes in CBF as a result of neural activity posits

17

neurotransmitter release as the driving force for these changes (Attwell and Iadecola,
2002). CBF increases have been correlated with neurotransmitter release (Attwell and
Iadecola, 2002; Drake and Iadecola, 2007). Glutamate and astrocytes are thought to play
an important role in this. Astrocytes, which are proximal to both neurons and blood
vessels, are in an ideal location to couple synaptic activity with hemodynamic responses
(Iadecola and Nedergaard, 2007). Chaigneau et al., 2007 showed a correlation between
CBF, postsynaptic glutamate activation and increases in dendritic calcium levels.
Increased intracellular astrocytic calcium levels have been shown to dilate arteries
(Iadecola and Nedergaard, 2007). Furthermore, (Petzold et al., 2008) found that CBF
changes were mediated by glutamatergic uptake by astrocytes; thus these changes can be
seen as a representation of incoming neural activity. In addition to a role for glutamate in
CBF changes, dopamine (DA), noradrenaline, and 5-HT have been shown to be localized
in neurons innervating microvasculature involved in vasoconstriction and cholinergic
axons have been shown to connect to arterioles and capillaries involved in vasodilatation
(Attwell and Iadecola, 2002). Overall, the data on CBF suggests that it is driven by
incoming synaptic inputs, rather than output signals (Buxton et al., 2004).
CBF correlates with measures of neural activity and with energy metabolism.
However, limitations to viewing CBF as a measure of neural activation still exist. The
temporal relationship between neural activity, changes in calcium levels and CBF
response is still unclear (Iadecola and Nedergaard, 2007). Furthermore, factors other
than synaptic activity such as neuromodulators and hormones may result in vasculature
signals not related to neural activity (Iadecola and Nedergaard, 2007). Additionally,

18

there is the possibility that certain neurotransmitters such as 5-HT can alter CBF
response, which could be important in imaging of genetic alterations in the serotonergic
system.

19

CHAPTER ONE: SEX AND GENOTYPE EFFECTS ON
ANXIETY-LIKE BEHAVIOR

INTRODUCTION
Recently, emphasis has been placed on rectifying the once common notion that
data collected in males could be applied to females (Cahill, 2010). The understanding of
sex differences in behavior and brain function may be particularly important in disorders
that show sexual dimorphism such as disorders of mood and anxiety. Although females
are at a greater risk to experience disorders of anxiety and mood (Zender and Olshansky,
2009), the majority of studies on pharmacological assessment of selective serotonin
transporter inhibitors (SSRIs) and in 5-HTT KO mice have been done in male animals.
The growing literature suggesting that serotonergic function varies by sex makes the
omission of females in serotonergic research particularly problematic. Human studies
have suggested that females may be more responsive to alterations in serotonergic
function (Booij et al., 2002; Khan et al., 2005; Kornstein et al., 2000; Martenyi et al.,
2001; Nishizawa et al., 1997; Young et al., 2009). In rodents, differences have been
noted in both the basal response of 5-HT (Carlsson and Carlsson, 1988; Drossopoulou et
al., 2004), as well as in response to exposure to behavioral models of depression (Dalla et
al., 2010; Drossopoulou et al., 2004). The handful of studies in 5-HTT KO mice that
included females suggest female 5-HTT KOs are more sensitive to stress (Joeyen-
Waldorf et al., 2009; van den Hove et al., 2010), but not all studies find an effect of sex
(Carroll et al., 2007). Discrepancies in the effects of sex on anxiety warrants further

20

investigation into genotype and sex effects on anxiety behavior.
The current study investigated the effects of loss of 5-HTT on conditioned and
exploratory (light/dark, open field) measures of anxiety behavior. Specifically, I tested
the hypothesis that 5-HTT KO mice would show increased anxiety like behavior and
reduced exploration of a novel environment and that these differences would be
accentuated in female mice. Home cage locomotor activity and experiments on whisker
function were performed in male animals to test whether differences in exploratory-based
anxiety tests might be confounded by deficits in motor and/or sensory systems.

METHODS
Animals:
Male and female mice were bred at the university vivarium from pairs obtained
from Taconic (Taconic, Hudson, NY). Mice had been backcrossed onto a C57BL/6
background for greater than 15 generations from an original mixed background
[129/P1ReJ (ES cells), C57BL/6J and CD-1] (Bengel et al., 1998; Salichon et al., 2001).
Mice were weaned at 3 weeks, housed in groups of 3-4 on a 12-hour light/dark cycle
(lights on at 0600) with direct contact bedding and free access to rodent chow (NIH
#31M diet) and water. At the start of behavioral testing, animals were individually
housed. Genotyping of the homozygous knockout and wildtype animals were performed
by Transnetyx, Inc. (Cordova, TN) from tail snips obtained post mortem with primer
sequences obtained from Taconic (m5-HTT-C: 5’ TGA ATT CTC AGA AAG TGC
TGT C 3’, m5-HTT-D: 5’ CTT TTT GCT GAC TGG AGT ACA G 3’, neo3a: 5’ CAG

21

CGC ATC GCC TTC TAT C 3’). All behavioral testing was conducted during the light
phase of the light/dark cycle (0930 to 1430).

Cued fear conditioning:
Conditioned fear training phase. Fear conditioning experiments (Wehner and
Radcliffe, 2004) were conducted three days post surgery. Animals were habituated to the
experimental room for thirty minutes in the home cage. Thereafter, mice were placed in
a Plexiglas box (22.5 cm x 21cm x 18cm) with a floor of stainless steel rods. The
chamber was illuminated with indirect ambient fluorescent light from a ceiling panel (930
lx) and was subjected to background ambient sound (65 dB). After a two-minute
baseline, the animals were presented a tone six times (30 s, 70dB, 1000 Hz/8000 Hz
continuous, alternating sequence of 250 ms pulses). Each tone was separated by a one-
minute quiet period. In the conditioned fear (CF) groups (MKO-CF: body weight = 27g
± 0.6g, age =12.4 wks ± 0.3 wks, n = 12; MWT-CF: body weight = 26g ± 0.5g, age =12.8
wks ± 0.3 wks, n =13; FKO-CF: body weight = 23.8 g ± 0.5g, age =13.0 wks ± 0.2 wks,
n =13; FWT-CF: body weight = 21.2g ± 0.5g, age = 12.1 wks ± 0.2 wks, n = 13) each
tone was immediately followed by a foot shock (0.5 mA, 1 s). Control (CON) animals
(MKO-CON: body weight = 27g ± 0.4g, age =12.4 wks ± 0.2 wks, n =13; MWT-CON:
body weight = 26g ± 0.3g, age =12.4 wks ± 0.2 wks, n =11; FKO-CON: body weight =
23.6g ± 0.4g, age = 13.0 wks ± 0.3 wks, n = 12; FWT-CON: body weight = 21.7g ± 0.6g,
age = 12.5 wks ± 0.2 wks, n = 13) received identical exposure to the tone but without the
foot shock. One minute following the final tone, mice were returned to their home cages.

22

Conditioned fear recall. Twenty-four hours after the training session, animals were
placed in the experimental room for one hour in their home cage. Thereafter, the animal's
percutaneous cannula was connected to a tethered catheter containing the perfusion
radiotracer ([
14
C]-iodoantipyrine, 325 µCi/kg in 0.180 mL of 0.9% saline, American
Radiolabelled Chemicals, St. Louis, MO) and a syringe containing a euthanasia solution
(50 mg/kg pentobarbital, 3M KCl). Animals rested in a transit cage for ten minutes prior
to exposure to the behavioral cage (a cylindrical, dimly lit (300 lx), Plexiglas cage with a
flat, Plexiglas floor). CF and CON animals received a two-minute exposure to the
behavioral cage context followed by a one-minute continuous exposure to the
conditioned tone.

Behavioral analysis of conditioned fear. Behaviors were recorded using Windows
Movie Makes (Microsoft) by a camera placed in front of the cage. The duration of the
animal's freezing response, defined as the absence of all visible movements of the body
and vibrissae aside from respiratory movement, served as the behavioral measure of
conditioned fear memory. Behaviors were analyzed in a blinded fashion using the
Observer 8.0 (Noldus Inc., Leesburg, VA). The freezing data were transformed to a
percentage of time spent freezing. Statistical comparison was performed with a two
factor repeated measure analysis of variance (ANOVA) using "genotype", "sex" and
"conditioning" as between subject factors. The repeated measure was "time" (time

23

intervals during training were 90 s, i.e. 30s tone followed by a 1 minute quiet period, time
intervals during recall were baseline and tone).

Exploratory-based measures of anxiety:
Open field. Mice (MKO n = 28, FKO n =27, MWT n = 25, FWT n = 23) were
habituated for 30 minutes to the behavioral room. They were then placed in the bottom
portion of a test chamber (a novel circular arena, diameter 42.5 cm, height 11.5 cm),
which was illuminated from the ambient fluorescent light from the ceiling (558 lx), and
allowed to freely explore for ten minutes. Latency to enter the center zone (diameter 16.5
cm), time spent in the center zone, and frequency of entries into the center zone was
assessed for each animal from the digitized video recordings using EthoVision 3.1
(Noldus, Inc., Leesburg, VA). Path length traveled in each one-minute interval in the
arena was calculated for each animal. ANOVA was performed on path length using
"genotype" and “sex” as between subject factors. Group averages were compared using a
t-test (two tailed, p<0.05).

Light-dark exploration. Mice (MKO n = 28, FKO n =20, MWT n = 25, FWT n = 32)
were placed in the dark portion (10 cm x 20 cm x 13 cm; ~0 lx) of the light/dark
exploration box and allowed to explore for ten minutes. Latency to enter the light portion
(25 cm x 20 cm x 13 cm ~23,000 lx), transitions between compartments (4 paws in), time
spent in dark, and number of rears in light portion was scored off video recordings using
the Observer (Version 8.0, Noldus, Inc., Leesburg, VA). ANOVA was performed on

24

path length using "genotype" and “sex” as between subject factors and "time" as a within
subject factor. Group averages were compared using a t-test (two tailed, p<0.05).

Homecage locomotor activity (male mice only):
Home cage activity was recorded during a standard 12 hour light, 12 hour dark
(LD 12:12) in male 5-HTT KO (n =11; average age = 14.5±0.5 weeks) and WT mice (n =
14; average age = 15.7 ± 0.6 weeks). Mice were placed within frames containing three
infrared photo beams spaced 8.8 cm apart (San Diego Instruments, San Diego, CA,
USA). Locomotion indicated by photo beam breaks was recorded electronically using
PAS software (San Diego Instruments, San Diego, CA, USA). Mice had one week of
habituation to the room before the start of recording. Locomotion indicated by photo
beam breaks was recorded electronically using PAS software (San Diego Instruments,
San Diego, CA, USA). Group differences in total activity counts were analyzed using a
t-test (two tailed, p < 0.05).

Testing of whisker deficits (male mice only):
Spontaneous gap crossing (sGC). The apparatus and training procedures have been
described before (Voigts et al., 2008). In short, after initial habituation to the
experimenter and the apparatus, individual animals (n=4/group) were placed on one of
the two elevated platforms separated from each other with randomly varying gap-distance
(range: 3-8 cm, step-size: 0.5 cm) and their probability of successful object localization
across gap-distances was quantified. The training was performed under infrared light and

25

white noise; the platforms were cleaned using 70% isopropanol between sessions.
Animal mobility on the platforms was quantified using custom made infrared motion
sensors placed at the two ends and the middle of each platform. Trial duration, duration
of sensory exploration at the gap, number of attempts prior to successful gap-crossing,
and duration of mobility were quantified and genotypes were compared using Student’s t-
test. Animals had ad libitum access to the food and water at all times, except when they
were performing the task (1 session/day for 7 days; session duration: 30 min). Animals
were not baited for successful task execution.

Gap cross training (GCt). GCt (Celikel and Sakmann, 2007) was similar to the sGC
with the exception that the animals were food deprived (to ~90% of their free-feeding
rate) throughout the training period and were rewarded (1 pellet, 14 mg/pellet, BioServ,
product #F05684) for successful gap crossing on the task. Unlike in the sGC, with
repeated GCt animals increase their probability of successful object localization. The
training apparatus and quantification of the variables were as described above. Each
animal (n=4/group) received 3 weeks of training on the apparatus (1 session/day; 7
sessions/week; session duration: 30 min). Tactile exploration of the animal onto the
target platform was recorded using a high-speed camera (Allied Vision Technologies,
Model: Pike) at 300 fps and a human observer confirmed that animals performed the task
using their whiskers.

26

RESULTS
Cued fear conditioning:
During the training phase (day 1) conditioning significantly increased percent
time freezing (conditioning: F
1, 95
= 320.0; p < 0.001) in a time dependent manner (time x
conditioning: F
4, 419
= 140.2; p < 0.001). There was a significant genotypic difference in
percent time freezing (genotype: F
1, 95
= 9.0, p < 0.05), but no significant interaction
between genotype and conditioning (genotype x conditioning: F
1, 95
= 0.7, p > 0.05)
(Figure 2a). Sex affected freezing in a time dependent manner (time x sex: F
4, 419
= 2.6; p
< 0.05).
During recall testing (day 2), mice that were conditioned to the tone (CF) froze
significantly more than control (CON) mice (conditioning: F
1, 94
= 266, p < 0.001) in a
time dependent manner (time x conditioning: F
1, 94
= 284.2, p < 0.001). 5-HTT KO mice
froze significantly more than WT mice (genotype: F
1, 94
= 79, p < 0.001), with a
significant interaction between genotype and conditioning (genotype x conditioning: F
1, 94

= 5.9, p < 0.05) (Figure 2b). Genotypic differences in freezing response were
significantly increased during tone exposure compared to the baseline condition
(genotype x conditioning x time: F
1,94
= 3.9, p < 0.05) (Figure 2b). Sex did not
significantly affect freezing behavior (sex: F
1, 94
= 0.001, p > 0.05), nor interact with
genotype (sex x genotype: F
1, 94
= 0.005, p > 0.05) or conditioning (sex x conditioning:
F
1, 94
= 0.1, p > 0.05) (Figure 2b).

27

a)

b)
Figure 2. Fear Conditioning and Recall
a) Percent freezing (mean ± SEM) during training during which tone-footshock was delivered
during each 30s interval (T1-6). b) Percent freezing during recall of the tone.

28

Exploratory-based measures of anxiety:
Open field. Over time locomotor activity in the open field decreased (time: F
1, 92
= 74.9,
p < 0.001). There was a main effect of genotype with 5-HTT KO mice moving around
the open field arena significantly less than WT mice (genotype: F
1, 92
= 55.9, p < 0.001,
Figure 3a). Sex also affected distance traveled in the arena of the open field with females
traveling significantly more than males throughout the arena (sex: F
1, 92
= 18.4, p < 0.001,
Figure 3a), which was mainly attributed to increased exploratory activity in female WT
mice (genotype x sex: F
1, 92
= 3.9, p = 0.05, Figure 3a). 5-HTT KO mice displayed an
increased latency to enter the center zone of the arena (genotype: F
1, 92
= 9.8, p < 0.01,
Figure 3b). 5-HTT KO mice entered the center zone less frequently (genotype: F
1, 92
=
8.2, p < 0.01, Figure 3d) and showed decreased locomotor activity in the center zone
(genotype: F
1, 90
= 4.4, p < 0.05, Figure 3c). Female mice moved significantly more in
the center zone of the open field (sex: F
1, 90
= 12.3, p < 0.01, Figure 3c).

29

a)

b)

c)

d)

Figure 3. Open field Sex and genotype alter exploratory behavior in a novel open field. a)
Female WT mice moved throughout the arena significantly more than WT males, KO males and
KO females (t-test with Bonferroni correction, *p < 0.001). b) KO mice took significantly longer
to first entry into the center zone of a novel arena. Sex did not effect time to first entry into the
center zone c) KO mice moved within the center zone less then WT mice and male mice moved
within the center zone less then female mice d) Female WT mice entered the center significantly
more than WT males, KO males and KO females (t-test with Bonferroni correction, *p < 0.05).
Shown are group means ± SEM.

Light-dark exploration. In the light-dark box, KO mice compared to WT mice had
significantly decreased latency to enter the light portion of the light-dark box (genotype:
F
1, 93
= 4.0, p < 0.05, Figure 4a). Latency to enter the light did not show an effect of sex
(sex: F
1, 93
= 1.5, p > 0.05, Figure 4a) or an interaction between sex and genotype (sex x
Latency to Center Zone
0
10
20
30
40
50
60
70
80
Time to first enter center zone (s)
WT KO
Male
Female
Distance Center Zone
0
100
200
300
400
500
600
700
800
Distince center zone (cm)
WT
KO
Distance in Arena
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Distance Traveled (cm)
Male Female
WT KO
*
Frequency to Center Zone
0
10
20
30
40
50
60
# Entries center zone
Male
Female
*

30

genotype: F
1, 93
= 0.02, p > 0.05, Figure 4a). There was no effect of genotype (genotype:
F
1, 93
= 3.4, p > 0.05), sex (sex: F
1, 93
= 0.5, p > 0.05), or an interaction (sex x genotype:
F
1, 93
= 0.36, p > 0.05) on percent time spent in the dark (Figure 4b). Females had a
significantly increased number of transitions between compartments (sex: F
1, 93
= 6.34, p
< 0.05, Figure 4d). Genotype (genotype: F
1, 93
= 1.00, p < 0.05, Figure 4d) did not affect
number of transitions, and there was no interaction between genotype and sex (sex x
genotype: F
1, 93
= 0.34, p < 0.05, Figure 4d). In the light portion of a light dark box,
knockout mice compared to WT mice had significantly reduced vertical exploration
(rears, genotype: F
1, 93
= 69.01, p < 0.001, Figure 4c). Sex did not significantly affect
rearing behavior (sex: F
1, 93
= 3.1, p > 0.05, Figure 4c), and there was no significant
interaction between sex and genotype (sex x genotype: F
1, 93
= 0.00, p > 0.05, Figure 4c).

31

a) b)
c) d)

Figure 4. Genotype and sex alter exploratory behavior in the light dark exploration task a) 5-HTT
KO show decreased latency to enter the light portion of the light dark box b) Sex and genotype
did not significantly change amount of time spent in the dark portion of the light dark box c) 5-
HTT KO mice reared significantly less in the light portion of the light dark box d) Females
crossed compartments significantly more often than males.

Latency to Light
0
20
40
60
80
100
120
Time to light (s)
WT
KO
Male Female
*
*
% Time Dark
0
10
20
30
40
50
60
70
80
90
100
% Time Dark
Male Female
Rearing
0
5
10
15
20
25
30
35
40
45
Male Female
# of Rears
*
*
Transitions between Compartments
0
2
4
6
8
10
12
14
16
18
20
# Transitions
Male
Female
WT KO
* *

32

Homecage locomotor activity:
No significant differences in total activity counts (KO = 1010 ± 42; WT = 1141 ±
79) were seen between male WT and male KO mice during the standard 12:12 light-dark
cycle (p > 0.05).

Testing of whisker deficits:
Spontaneous gap crossing (sGC). 5-HTT deletion impaired whisker sensation. On the
spontaneous gap-crossing task (sGC), KO mice failed to locate the target object when the
object was placed at whisker distances, 4.5-7.5 cm (Figure 5a). Moreover at shorter
distances (3 cm < x < 4.5 cm) KO animals failed significantly more often than WT mice
(WT 0.66 ± 0.13, KO 0.19 ± 0.11, values are mean ± SEM, p < 0.01). This impairment
was not due to lack of sensory exploration (Figure 5c) although KO mice explored the
gap for shorter periods than WT mice independent from whether they ultimately located
the target (p < 0.01) or failed to do so (p <0.001). Longer duration of sensory exploration
in those trials that KO mice failed to locate the target, compared to successful trials,
suggest that duration of exploration was not the cause of the failures. The sensory deficit
was not due to lack of motivation as KO mice spent more time performing the task
(Figure 5d; all trials combined, WT 20.2 ± 1.0 seconds, n: 2674, KO 79.0 ± 2.2, n: 2036,
p < 0.001) and made as many attempts, i.e. visits to the gap in a trial, to locate the target
(Figure 5e; All trials combined, WT 2.5 ± 0.004, KO 1.8 ± 0.03, p > 0.05). Number of
attempts required to locate the target in successful trials did not differ across the
genotypes (p > 0.05), although during failures KO animals visited the gap significantly

33

less often than WT mice (Figure 5e, p < 0.05). Although the sensory deficit was not a
reflection of a general lack of motor activity on the task (see above), mobility of the KO
mice was significantly less than WT mice (Figure 5f; all trials combined, WT 7.7 ± 0.2,
KO 6.6 ± 0.2, p < 0.05).

Gap cross training (GCt). The sensory deficit in KO mice persisted even when the
animals were rewarded for gap-crossing and trained on the task for 3 continuous weeks
(Figure 5c). GCt increased the likelihood, for both WT and KO animals, to locate the
target object when it was placed at whisker distances (WT: sGC 0.15 ± 0.09, GCt 0.37 ±
0.19, p < 0.05; KO: sGC 0 ± 0, GCt 0.02 ± 0.01) or closer (WT: sGC 0.68 ± 0.12, GCt
0.96 ± 0.02, p < 0.05; KO: sGC 0.19 ± 0.10, GCt 0.62 ± 0.11, p < 0.01). Although KO
animals crossed significantly larger distances during GCt compared to sGC (Figure 5a,
b), their performance in the whisker distances was still impaired (p < 0.01). As in the
case of sGC, KO mice explored the gap for shorter periods than the WT mice (Figure 5c;
p < 0.01) and had reduced number of visits to the gap, attempting to find the target
(Figure 5e; p < 0.05). Furthermore KO mice were less mobile on the task (Figure 5f; p <
0.01). Increased duration of mobility and sensory exploration during failures, compared
to successful trials, argue that the sensory deficit observed in KO mice is not due to lack
of sensory exploration, motivation or a generalized motor deficit. Accordingly time it
took for the KO mice to complete successful trials were largely comparable to the WT
mice (Figure 5d; Successful trials: WT 41.8 ± 1.4, KO 33.3 ± 1.3, p > 0.05; Failures:
WT 84.6 ± 4.6, KO 118.4 ± 5.1, p > 0.05).

34

Figure 5. Impaired whisker function in male 5-HTT KO mice a) Top: Schematic view of the
experimental set-up. Animals tried to locate a target object (i.e. platform) placed after a gap
(Voigts et al., 2008). Their mobility is tracked using motion sensors. Bottom: Probability of
successful object localization and subsequent gap-cross. 5-HTT deletion impairs tactile sensation
(P<0.01,Kolmogorov-Smirnov test). b) Rewarded training for 3 weeks improves performance
but does not rescue the whisker deficit. Top: Experimental set-up is similar to Figure 6a with the
addition of computer controlled reward delivery ports. Bottom: Probability of gap-crossing (WT
vs KO, P<0.01, Kolmogorov-Smirnov test). c) Duration of exploration, i.e. time spent at the gap,
d) time animals required to travel between the two ends of the platform during successful trials or
the delay to return to the starting position after sensory exploration at the gap during failures, e)
number of attempts (i.e. visits to the gap) f) duration of mobility across genotypes and training
conditions. Please refer to the text for statistical comparisons. Error terms are standard errors of
the means.

DISCUSSION
Conditioned fear:
During training, mice receiving tone-footshock pairings showed progressively
increased freezing behavior. Genotype and sex did not significantly affect freezing
behavior during the training phase. During recall, mice conditioned to the tone showed

35

significantly increased freezing behavior to the tone. During recall testing, KO compared
to WT mice displayed increased freezing to the tone. Relevant to this study, evaluation
of footshock sensitivity has revealed no genotypic differences (Lira et al., 2003). This is
consistent with our observation and that of others (Wellman et al., 2007) of no genotypic
differences in freezing behavior during the training phase in mice that received
conditioned footshocks. This suggests that the incoming footshock related
somatosensory information should not be greater in the KO animals; thus the increased
freezing responses noted in KO compared to WT mice during recall are not mediated by
altered perception in the mouse footpad during training. In any case, prior reports of
hypoalgesic responses to noxious stimulation in other sensory modalities (visceral,
temperature, mechanical, inflammatory) would predict an impairment in fear responses
during training and subsequent recall (Hansen et al., 2011; Holschneider et al., 2010;
Vogel et al., 2003).
While genotypic effect of tone exposure on freezing behavior was greater in the
conditioned group, small genotypic differences in freezing were also apparent in the
control groups. This could mean that the handling stress may have sufficiently
'sensitized' KO animals to elicit a partial fear response. This is plausible considering that
stress has been proposed to interact with this gene in humans (Caspi et al., 2003), and 5-
HTT KO mice have been shown to respond to stressors that are insufficient to affect WT
mice (Tjurmina et al., 2002).
While sex seems to play a role in freezing to contextual fear (Maren et al., 1994),
there is still debate as to the extent sex plays during freezing to a discrete cue (Maren et

36

al., 1994; Pryce et al., 1999). In this preparation, there was no sex effect on freezing
behavior in either the WT or KO groups. This may reflect a genotype specific ceiling
effect on freezing behavior in this preparation. In support of this ceiling effect, another
experiment using the same parameters looking at genotypic effects of chronic stress in
males found the same genotypic effect during recall, but with no additional stress effect
(data not shown). It is possible that a more mild protocol would unmask effects of sex on
freezing behavior.

Exploratory-based measures of anxiety:
5-HTT KO mice have been shown to exhibit reduced exploratory behavior
(Holmes et al., 2003a) and greater anxiety (Adamec et al., 2006; Carroll et al., 2007;
Holmes et al., 2003a; Holmes et al., 2003b; Wellman et al., 2007) and depression-like
behaviors (Lira et al., 2003), as well as alterations in the responses of the
sympathoadrenal systems and hypothalamic-pituitary-adrenal axis to minor stressful
stimuli (Tjurmina et al., 2002). While modest phenotypic abnormalities on exploratory
and anxiety measures of anxiety were found in the open field, the significant results in the
light-dark exploration showed a mixed effect. Because these tests rely heavily on
somatosensory exploration, the measurement of anxiety may be influenced by known
genotypic deficits in the somatosensory cortex (Esaki et al., 2005; Persico et al., 2001) for
review (Gaspar et al., 2003).
Females showed increased exploration of novel environments. This effect was
mainly a result of increased exploration in WT females. It is unclear as to whether this

37

increase in exploratory behavior relates to decreased anxiety or if it has more to do with
general increase of activity in females (Archer, 1975; File, 2001). The interpretation of
these results are further complicated by the general lack of consistent sex effects on
anxiety levels across anxiety tests (Johnston and File, 1991; Palanza, 2001). These
discrepancies suggest that anxiety measures validated in male animals may measure
different variables in females (Johnston and File, 1991; Palanza, 2001). Overall female
KO and male KO mice showed very similar patterns of exploration and anxiety-like
behavior. The general lack of sex differences in exploration and anxiety like behavior in
KO animals may reflect the relatively greater impact of genotype than sex, resulting in a
ceiling effect (Olivier et al., 2008).

Home cage locomotor activity:
In contrast to a previous report showing 5-HTT KO mice to be hypolocomotory
(Holmes et al., 2002), this study found no significant difference in spontaneous home
cage locomotor activity. The differences in these two reports could relate to the length of
sampling. The current study looked at home cage locomotor activity over several weeks
following a week habituation period, whereas the prior report only measured home cage
activity over a 24 hour period following one day of acclimation (Holmes et al., 2002).
Shorter sampling of behavioral activity may have been influenced by anxiety-like
behavior, which is a characteristic of the 5-HTT KO animals (Holmes et al., 2003a; Pang
et al., 2011). No difference in home cage locomotion has also been shown in a study in
5-HTT KO rats (Linder et al., 2008). These results suggest that genotypic differences in

38

the open field and light-dark exploration are not due to changes in overall motor activity
levels.

Testing of whisker deficits:
To further explore the behavioral effects of documented abnormalities in the
somatosensory cortex of 5-HTT KO mice, an additional group of experimentally naïve
male mice were tested on a learning task dependent on intact whisker function. In this
task, the mouse was placed on one of two platforms with a variable gap-distance between
the platforms. In the presence of white noise and darkness, at distances where the mouse
could not easily touch with the paw or nose, the mouse had to rely on its whiskers to
successfully localize and cross to the opposing platform. Thus, this task allowed for
quantification of unrestrained whisker-based tactile exploration. This study confirmed
impaired whisker sensation in 5-HTT KO mice, a result which extends earlier anatomic
and electrophysiology reports of abnormalities in the somatosensory system (Esaki et al.,
2005; Persico et al., 2001) for review (Gaspar et al., 2003).

39

CONCLUSIONS
Genotype showed robust effects in the KO mice on fear behavior during fear
conditioning recall. The effect of genotype on exploratory-based measures of anxiety
was less apparent. Deficits in whisker function in 5-HTT KO mice may confound
measures of anxiety in exploratory-based anxiety tests (see also appendix A for results of
marble burying). Sex did not affect freezing behavior. Females did show increased
exploration of a novel environment, but this may have to do more with increased activity
in females.

40

CHAPTER TWO: CONDITIONING, GENOTYPE AND SEX
EFFECTS ON FUNCTIONAL CHANGES IN
CORTICOLIMBIC CIRCUITRY

INTRODUCTION
The 5-HTTLPR polymorphism has been associated with increased susceptibility
to anxiety (Canli et al., 2005; Furmark et al., 2004; Katsuragi et al., 1999; Lesch et al.,
1996) and mood symptoms in the face of environmental adversity (Caspi et al., 2003;
Kendler et al., 2005), but not all studies find an effect (Gillespie et al., 2005; Lang et al.,
2004; Munafo et al., 2009). One possible explanation for inconsistencies in linking the 5-
HTTLPR genetic variants with psychiatric conditions is the fact that genes alter
neurocircuitry, which then may only subtly affect overt behaviors (Hariri and
Weinberger, 2003). Because the relationship between genes and behavior is complex,
studies addressing brain function may provide greater insight into the effect of genes.
5-HTT related availability of 5-HT in the developing brain is related to the development
of key regions of the limbic system (Holmes, 2008; Homberg et al., 2010). Consistent
with the central role of 5-HT in neurodevelopment, evidence of robust 5-HTTLPR
influences on brain anatomy and function has emerged. In healthy individuals, the 5-
HTT ‘s’ allele has been associated with a 12-30% reduction in gray matter volume in
limbic regions critical for processing of negative emotion, including the anterior cingulate
cortex and amygdala (Pezawas et al., 2005; Scherk et al., 2009), as well as the

41

dorsolateral prefrontal cortex and hippocampus (Frodl et al., 2008; O'Hara et al., 2007).
On a functional level, several neuroimaging studies have found that healthy individuals
carrying a ‘s’ allele display amygdala hyper activation (Drabant et al., 2012; Hariri et al.,
2005; Hariri et al., 2006; Hariri et al., 2002; Heinz et al., 2005; Lau et al., 2009; Rao et
al., 2007) and altered activity in prefrontal regions (Drabant et al., 2012; Heinz et al.,
2005; Pezawas et al., 2005).
Morphological studies in 5-HTT KO mice also suggest a role for 5-HTT in the
development of corticolimbic structures. 5-HTT KO mice show increased spine density
in the amygdala (Nietzer et al., 2011; Wellman et al., 2007). While changes in neuronal
length in the infralimbic have been found, the direction of these changes (increase or
decrease) seem to be stress dependent (Nietzer et al., 2011; Wellman et al., 2007). While
these molecular studies in 5-HTT KO mice support human findings of abnormalities in
the amygdala and prefrontal cortex, a functional neuroimaging study during an emotional
task in 5-HTT KO mice could provide a direct translational bridge between the human
polymorphism associated brain functional abnormalities and the morphological studies
undertaken in 5-HTT KO mice.
The current study provides a detailed three-dimensional (3-D) map of functional
brain activation during fear-conditioned recall in the 5-HTT KO mouse, thereby
exploring the possibility of reverse translation of brain functional responses in rodents.
Specifically, we test the hypothesis that 5-HTT KO mice show an exaggerated limbic
activation during recall of a fear-conditioned tone. Because females may be more
sensitive to alterations in serotonergic tone (Booij et al., 2002; Khan et al., 2005;

42

Kornstein et al., 2000; Martenyi et al., 2001; Nishizawa et al., 1997; Young et al., 2009),
we hypothesize that genotypic differences in functional activation in corticolimbic
circuitry will be accentuated in females.
Brain mapping is performed using an autoradiographic method (Patlak et al.,
1984; Sakurada et al., 1978). Perfusion autoradiography fills a gap in the current
armamentarium of imaging tools in that it can deliver a 3-D assessment of functional
activation of the awake, nonrestrained animal, with a temporal resolution of ~5–10
seconds and a spatial resolution of 100 µm (Holschneider and Maarek, 2004;
Holschneider and Maarek, 2008). This distinguishes it from other histological methods
such as c-fos or cytochrome oxidase, which integrate brain responses over a duration of
hours to days, or electrophysiological recordings, which typically only target very limited
regions of the brain, or functional magnetic resonance imaging (fMRI) or positron
emission tomography (microPET), which provide whole brain analysis, but require
sedation of the animal.

METHODS
Animals:
Mice were bred at the university vivarium from pairs obtained from Taconic
(Taconic, Hudson, NY). Mice had been backcrossed onto a C57BL/6 background for
greater than 15 generations from an original mixed background [129/P1ReJ (ES cells),
C57BL/6J and CD-1] (Bengel et al., 1998; Salichon et al., 2001). Mice were weaned at 3
weeks, housed in groups of 3-4 on a 12-hour light/dark cycle (lights on at 0600) with

43

direct contact bedding and free access to rodent chow (NIH #31M diet) and water. At the
start of behavioral testing, animals were individually housed. Genotyping was performed
by Transnetyx, Inc. (Cordova, TN) from tail snips obtained post mortem with primer
sequences obtained from Taconic (m5-HTT-C: 5’ TGA ATT CTC AGA AAG TGC
TGT C 3’, m5-HTT-D: 5’ CTT TTT GCT GAC TGG AGT ACA G 3’, neo3a: 5’ CAG
CGC ATC GCC TTC TAT C 3’). All behavioral testing was conducted during the light
phase of the light/dark cycle (0930 to 1430). Animals had gone through the open field
and light dark exploration the week before surgery.

Functional brain mapping during tone exposure:
Surgery. Animals were anesthetized with isoflurane (2.0%). The ventral skin of the
neck was aseptically prepared and the right external jugular vein was catheterized with a
1-French silastic catheter (SAI infusion, Chicago, IL), which was advanced 1 cm into the
superior vena cava. The catheter was externalized through subcutaneous space to a
dorsal percutaneous port. The catheter was filled with 0.01 mL Taurolidine-Citrate lock
solution (SAI infusion, Chicago, IL) and was closed with a stainless steel plug (SAI
infusion, Chicago, IL).

Functional neuroimaging during conditioned fear recall. One minute after the start of
the tone exposure (see chapter 2), the radiotracer was injected intravenously at 1.0
mL/min using a mechanical infusion pump (Harvard Apparatus, Holliston, MA),
followed immediately by injection of the euthanasia solution. This resulted in cardiac

44

arrest within 5-10 seconds, a precipitous fall of arterial blood pressure, termination of
brain perfusion, and death. Brains were rapidly removed and flash frozen in
methylbutane/dry ice.

Autoradiography. Brains were sliced in a cryostat at -20
o
C in 20 µm sections, with an
interslice spacing of 140 µm. Slices were heat dried on glass slides and exposed to
Kodak Ektascan diagnostic film (Eastman Kodak, Rochester, NY USA) for 14 days at
room temperature along with twelve [
14
C] standards (Amersham Biosciences,
Piscataway, NJ). Autoradiographs were then digitized on an 8-bit gray scale using a
voltage stabilized light box (Northern Lights Illuminator, InterFocus Ltd., England) and a
Retiga 4000R charge-coupled device monochrome camera (Qimaging, Canada).
Cerebral blood flow (CBF) related tissue radioactivity was measured by the classic [
14
C]-
iodoantipyrine method (Patlak et al., 1984; Sakurada et al., 1978). In this method, there
is a strict linear proportionality between tissue radioactivity and CBF when the data is
captured within a brief interval (~10 seconds) after the tracer injection (Jones et al., 1991;
Van Uitert and Levy, 1978).

3-D reconstruction of the digitized autoradiographs. 3-D reconstruction has been
described in prior work (Nguyen et al., 2004). In short, regional CBF (rCBF) was
analyzed on a whole-brain basis using statistical parametric mapping (SPM, version
SPM5, Wellcome Centre for Neuroimaging, University College London, London, UK).
SPM, a software package was developed for analysis of imaging data in humans (Friston

45

et al., 1991), has recently been adapted by us and others for use in brain autoradiographs
(Dubois et al., 2008; Lee et al., 2005; Nguyen et al., 2004). A 3-D reconstruction of each
animal’s brain was conducted using 69 serial coronal sections (starting at slice bregma
2.98 mm) and a voxel size of 40 µm x 140 µm x 40 µm. Adjacent sections were aligned
both manually and using TurboReg, an automated pixel-based registration algorithm
(Thevenaz et al., 1998). After 3-D reconstruction, all brains were smoothed with a
Gaussian kernel (FWHM = 120 µm x 420 µm x 120 µm). The smoothed brains from all
groups were then spatially normalized to the smoothed reference brain (one “artifact free”
brain). Following spatial normalization, normalized images were averaged to create a
mean image, which was then smoothed to create the smoothed template. Each smoothed
original 3-D reconstructed brain was then spatially normalized into the standard space
defined by the smoothed template (Nguyen et al., 2004).

SPM. An unbiased, voxel-by-voxel analysis of whole-brain activation using SPM was
used for detection of significant changes in functional brain activation. Voxels for each
brain failing to reach a specified threshold (80% of the mean voxel value) were masked
out to eliminate the background and ventricular spaces without masking gray or white
matter. Global differences in the absolute amount of radiotracer delivered to the brain
were adjusted in SPM for each animal by scaling the voxel intensities so that the mean
intensity for each brain was the same (proportional scaling). Using SPM, a factorial
ANOVA was implemented at each voxel testing the null hypothesis that there was no
conditioning, genotype or sex effect, as well as the interaction between genotype and

46

conditioning, genotype and sex, and sex and conditioning (F
1, 91
, p < 0.05). Brain regions
were identified using coronal, sagittal and transverse views from the mouse brain atlas
(Franklin and Paxinos, 2008).

RESULTS
Fidelity of the alignment and 3D reconstruction process:
This was assessed by visual inspection of the internal structures viewed in different
orthogonal sections, as well as by visual inspection of the smoothness of the cortex after
rendering the surface using a tool in the SPM software. A sample brain reconstruction is
shown in figure 6.

Figure 6. Sample 3D reconstruction of a mouse brain from serial coronal autoradiographic slices
depicting cerebral perfusion patterns.

47

Main effects of conditioning, genotype, sex and conditioning on functional changes
of corticolimbic circuits (Figure 7, Table 1):
Conditioning. Conditioning altered rCBF in sensory, but not motor cortex.
Conditioning changed rCBF patterns in the prefrontal cortex (cingulate, retrosplenial and
prelimbic). Conditioning changed rCBF in the basolateral complex of the amygdala and
in the dorsal hippocampus.

Genotype. Genotype altered regional cerebral blood flow (rCBF) in motor and sensory
cortex including the somatosensory barrel field cortex. Genotype also altered rCBF in
the insular and prefrontal cortex (cingulate, retrosplenial). In limbic structures, genotype
altered rCBF patterns broadly in the amygdala and in the hippocampus (dorsal and
ventral).

Sex. Sex altered rCBF in motor and sensory cortex including the somatosensory barrel
field cortex. Sex also altered rCBF in the insular cortex. In the prefrontal cortex, sex
effected rCBF in the prelimbic and cingulate cortex and did not change rCBF in the
retrosplenial or infralimbic. Sex altered rCBF to the hippocampus (dorsal and ventral).
In the amygdala, sex only altered rCBF to the cortical amygdala.

48

Figure 7. Factorial analysis examining a main effect of genotype, sex or conditioning (p < 0.05).
Depicted are select coronal slices (anterior-posterior coordinates relative to bregma) of the
template brain. Colored overlays show statistically significant effects of genotype or
conditioning, but do not reflect the direction of the effect. Abbreviations are from Franklin and
Paxinos (2008) mouse atlas: BLA (basolateral amygdaloid complex), CoA (cortical amygdala),
Cg (cingulate cortex), dorsal hippocampus (dHPC), I (insular cortex), M1 (primary motor cortex),
M2 (secondary motor cortex), PrL (prelimbic cortex), RS (retrosplenial cortex), S1 (primary
somatosensory cortex), S1BF (primary somatosensory, barrel field), S2 (secondary
somatosensory cortex), ventral hippocampus (vHPC).

49

50

Conditioning, genotype, and sex interactions on functional changes of corticolimbic
circuits (Figure 8, Table 1):
Genotype x Conditioning. Genotype and conditioning interacted to effect rCBF in
sensory (including barrel field cortex), but not motor areas. Genotype also showed
conditioning dependent effects in the cingulate, infralimbic, prelimbic and insula. In
limbic areas, genotype and conditioning interacted to affect the amygdala and dorsal
hippocampus.

Sex x Conditioning. Sex by conditioning interactions were found in motor, but only
minimally in sensory regions. Sex by conditioning interactions were also found in the
cingulate, prelimbic and retrosplenial. Sex by conditioning interactions were also noted
in the amygdala and ventral hippocampus.

Genotype x Sex. There was genotype by sex interactions in the motor, sensory cortex
including the barrel field cortex, and insular cortex. In the prefrontal cortex, genotype
and sex showed an interaction in the cingulate cortex only.

51

Figure 8. Factorial analysis examining interactions between genotype, sex or conditioning (p <
0.05). Depicted are select coronal slices (anterior-posterior coordinates relative to bregma) of the
template brain. Colored overlays show statistically significant effects of genotype or
conditioning, but do not reflect the direction of the effect. Abbreviations are from Franklin and
Paxinos (2008) mouse atlas: BLA (basolateral amygdaloid complex), CoA (cortical amygdala),
Cg (cingulate cortex), dorsal hippocampus (dHPC), I (insular cortex), Il (infralimbic cortex), M1
(primary motor cortex), M2 (secondary motor cortex), PrL (prelimbic cortex), RS (retrosplenial
cortex), S1 (primary somatosensory cortex), S1BF (primary somatosensory, barrel field), S2
(secondary somatosensory cortex), ventral hippocampus (vHPC).

52

53

Figure 9. Effects of genotype on functional activity in response to a tone. Depicted are select
coronal slices (anterior-posterior coordinates relative to bregma) of the template brain. Colored
overlays show statistically significant increased (red) and decreased (blue) differences for each
comparison. Intragroup comparison were examined for genotype effects with respect to
conditioning and sex (MCON: KO vs. WT; FCON: KO vs WT; MCF: KO vs. WT; FCF: KO vs.
WT). Abbreviations are from Franklin and Paxinos mouse atlas (Franklin and Paxinos, 2008):
BLA (basolateral amygdaloid complex), CoA (cortical amygdala), Cg (cingulate cortex), dorsal
hippocampus (dHPC), I (insular cortex), Il (infralimbic cortex), M1 (primary motor cortex), M2
(secondary motor cortex), PrL (prelimbic cortex), RS (retrosplenial cortex), S1 (primary
somatosensory cortex), S1BF (primary somatosensory, barrel field), S2 (secondary
somatosensory cortex), ventral hippocampus (vHPC).

54

55

Figure 10. Effects of sex on functional activity in response to a tone. Depicted are select coronal
slices (anterior-posterior coordinates relative to bregma) of the template brain. Colored overlays
show statistically significant increased (red) and decreased (blue) differences for each
comparison. Intragroup comparison were examined for sex effects with respect to conditioning
and genotype (WTCON: F vs. M; WTCF: F vs. M; KOCON: F vs. M; KOCF: F vs. WT).
Abbreviations are from Franklin and Paxinos mouse atlas (Franklin and Paxinos, 2008): BLA
(basolateral amygdaloid complex), CoA (cortical amygdala), Cg (cingulate cortex), dorsal
hippocampus (dHPC), I (insular cortex), Il (infralimbic cortex), M1 (primary motor cortex), M2
(secondary motor cortex), PrL (prelimbic cortex), RS (retrosplenial cortex), S1 (primary
somatosensory cortex), S1BF (primary somatosensory, barrel field), S2 (secondary
somatosensory cortex), ventral hippocampus (vHPC).

56

57

Main
Effect
Main
Effect
Main
Effect

Gene
(G)
Sex (S) Cond
(C)
G x S G x C S x C
Cortex
Cingulate (Cg) #/# #/# #/# #/# #/- #/#
Infralimbic (IL) -/- -/- -/- -/- #/# -/-
Insula (I) #/# #/# -/- #/# #/# -/-
Motor: primary (M1) #/# #/# -/- #/# -/- #/#
secondary (M2) #/# #/# -/- #/# -/- #/#
Prelimbic (PrL) -/- #/# #/# -/- #/# #/#
Retrosplenial (RS) #/# -/- #/# -/- -/- #/#
Somatosensory: barrel field
(S1BF)
#/# #/# -/- #/# #/# -/-
primary (S1, non barrel
field)
#/# #/# #/# #/# #/# -/-
secondary (S2, non barrel
field)
#/# #/# #/# #/# #/# #/#
Subcortex
Amygdala: basal amygdala
complex (BLA)
#/# -/- #/# #/# #/- #/#
cortical amygdala (ACo) #/# #/# -/- -/- -/- -/-
Hippocampus: dorsal
(dHPC)
#/# #/# #/# #/# #/- -/-
ventral
(vHPC)
#/# #/# -/- #/# -/- #/#
Table 1. Significant changes in rCBF in the cortex and subcortex in the left and right
hemispheres (L/R). # p < 0.05, no significant change.

58

DISCUSSION
Effects of conditioning:
Activation of the amygdala occurs in stressful situations (LeDoux, 2000). The
amygdala receives signals of danger and sets in motion emotional, autonomic and
neuroendocrine coping mechanisms to respond the stress. In cued fear conditioning it is
believed that the amygdala incorporates the incoming sensory information and through its
connections elicits the behavioral, neurohumeral, and sympathetic responses
characteristic of fear states (Goosens and Maren, 2001). We found functional changes in
amygdala activation to be conditioning dependent, which is consistent with its proposed
role in fear conditioning.
While auditory fear conditioning can occur in the absence of the medial prefrontal
cortex (mPFC, infralimbic and prelimbic), evidence supports and important role of the
mPFC in modulating the expression and extinction of the fear response via the amygdala
(Sotres-Bayon and Quirk, 2010). The prelimbic is believed to be important for the
expression of conditioned auditory stimuli (Corcoran and Quirk, 2007). The infralimbic
is thought to play more of a role in creating the plasticity necessary for fear extinction
learning (Laurent and Westbrook, 2009). During fear conditioning recall, an effect of
conditioning was found in the prelimbic but not infralimbic cortex. This pattern of
functional changes is consistent with the purported dual roles of the infralimbic and
prelimbic in fear conditioning (Sotres-Bayon and Quirk, 2010).
The anterior cingulate has been shown to modulate the efficiency of fear related
learning (Bissiere et al., 2008), while the role of the retrosplenial (posterior cingulate) in

59

auditory fear conditioning remains unresolved (Keene and Bucci, 2008; Lukoyanov and
Lukoyanova, 2006). Consistent with the role of the cingulate in fear conditioning,
conditioning was shown to effect functional activation in the cingulate and retrosplenial
cortex.
There is a general consensus that the ventral hippocampus (vHPC) and dorsal
hippocampus (dHPC) are functionally distinct structures (Fanselow and Dong, 2010).
The vHPC is known to connect to the prelimbic and to the amygdala and believed to play
a role in the emotional response and acquisition of tone conditioning (Maren and Holt,
2004; Zhang et al., 2001). The dHPC is believed to play a role in spatial navigation and
contextual conditioning (Phillips and LeDoux, 1992). Somewhat contrary to the reported
role of vHPC and dHPC in tone conditioning, conditioning effects were found in dHPC
but not vHPC. In the vHPC, a conditioning by sex interaction was found. This means
that in the vHPC the effects of conditioning may be sex specific. Differences in the
dHPC may reflect differences in spatial navigation between conditioned and control
animals, which is reflected in differences in freezing behavior.
The insular cortex, which strongly innervates the amygdala, is thought to play an
integral role in sensorimotor integration (Hoistad and Barbas, 2008; Yasui et al., 1991).
Conditioning alone did not show functional changes in the insula, but did interact with
genotype to alter functional changes in the insula. This suggests that insular activation to
conditioning could be genotype specific. Differences in functional changes as a result of
conditioning were found in sensory regions. Conditioning specific effects in motor
regions was sex dependent. It is surprising that conditioning alone did not alter

60

functional changes in the motor cortex because conditioning significantly altered freezing
behavior.

Effects of genotype:
Evidence suggests that 5-HTT related 5-HT availability affects the development
and function in corticolimbic circuits (Holmes, 2008; Homberg et al., 2010). Functional
changes in the amygdala were strongly influenced by genotype. Genotypic effects in the
mPFC, however, were dependent on conditioning. This suggests that genotype robustly
influences functional changes in the amygdala regardless of task, which is consistent with
reports in human showing genotypic differences in the amygdala across a variety of task
conditions (Drabant et al., 2012; Hariri et al., 2005; Hariri et al., 2006; Hariri et al., 2002;
Heinz et al., 2005; Lau et al., 2009; Rao et al., 2007). Genotypic effects in the mPFC,
however, may be task dependent. This genotypic pattern of strong effects in the
amygdala and task dependent effects in the prefrontal cortex is consistent with
morphological abnormalities in the amygdala and infralimbic seen in 5-HTT KO mice
(Nietzer et al., 2011; Wellman et al., 2007). These results provide further evidence that
5-HTT genotype alters the efficiency of the mPFC stress coping circuitry (Homberg,
2012), which could underlie genotypic differences in stress reactivity.
In the anterior cingulate and retrosplenial cortex, functional changes showed a
main effect of genotype. Because the anterior cingulate modulates amygdala activation
(Petrovic et al., 2004), genotypic differences in the amygdala could result in abnormal
cortical modulation of amygdala activation.

61

Genotype affected functional changes in the vHPC and dHPC. As the vHPC is
believed to play an important role in anxiety behaviors, genotypic effects in the vHPC is
consistent with the notion the 5-HTT genotype alters circuitry important in fear and
emotion. Genotypic effects in dHPC were not seen in male only dataset (Pang et al.,
2011). While the effect of genotype showing up in the combined male/female sample
could reflect increased power, a sex by genotype interaction in dHPC activation suggests
that genotypic differences in the dHPC are accentuated in females and could reflect a
stronger genotypic effect in exploration of the female group. This is plausible given the
effect of sex on exploratory patterns, specifically in WT females (chapter one). The
effects of genotype on functional changes in the hippocampus is consistent with
genotypic differences in hippocampal anatomical studies in human 5-HTTLPR (Frodl et
al., 2008) and proteomic studies in 5-HTT KO mice (van den Hove et al., 2010).
Genotypic effects in the hippocampus could reflect serotonergic effects on the
morphofunctional development of the hippocampus and synaptic plasticity in the
hippocampus (Chubakov et al., 1993; Dai et al., 2008; Matsumoto et al., 2004).
Genotype strongly affected functional changes in the insula cortex. This
genotypic effect in functional changes in the insula could imply genotypic differences in
the processing of aversive sensory signals. Genotypic differences were found in motor
and sensory regions including the barrel field cortex (S1BF). These differences are
consistent with genotypic differences in freezing behavior and exploratory patterns.
Differences in S1BF could reflect differences in KO's anatomy and electrophysiological

62

function in the barrel field cortex (Esaki et al., 2005; Persico et al., 2001), which has been
shown to result in deficits in barrel dependent behavioral tasks in KO mice (Chapter one).
Brief mention should be made of the possibility that a neurotransmitter such as 5-
HT could modulate the sensitivity of vascular responses, which could be important in
imaging of genetic alterations in the serotonergic system. There is evidence that 5-HTT
is important in blood vessels and plays a role in regulating blood pressure (Ni and Watts,
2006), with 5-HT being known to have both vasoconstrictive and vasodilatory properties
(Ni and Watts, 2006). Because of the importance of 5-HTT in these processes, there is
the possibility that some of the results of the CBF are more general effects of 5-HT on
vasculature, rather than specific effects of the imaging paradigm itself. This is unlikely,
however, as small changes in blood pressure if they occur would likely have little effect
on changing CBF because of autoregulation. Furthermore, there is evidence, that lifelong
abnormalities in 5-HTT results in compensatory mechanisms in the vascular system; thus
acute versus chronic alterations in 5-HTT result in different effects. Indeed, in awake,
nonanesthetized 5-HTT KO rats there are no alterations in diurnal mean arterial pressure
and heart rates (Linder et al., 2008), but 5-HT has an increased potency during acute
administration in these animals (Linder et al., 2008).

Effects of sex:
Sex showed a main effect in the prelimbic cortex. Since the prelimbic cortex is
believed to play a role in biasing attention to emotional stimuli, this functional change
suggests that females may differently attend to emotional stimuli. Functional changes in

63

the prelimbic cortex also showed a sex by conditioning interaction, which further
suggests that sex effects how incoming sensory signals are attended to depending on the
emotional salience. Sex dependent effects of the prefrontal cortex have also been found
during fear extinction (Baran et al., 2010) and lesions to prefrontal cortex appear to have
sex specific effects on stress response (Buchanan et al., 2010). Sex altered functional
changes in the cingulate but not the retrosplenial cortex. Sex interacted with conditioning
and genotype to alter functional changes in the cingulate. Sex interacted with
conditioning to alter functional changes in the retrosplenial. Changes in functional
activation of the basolateral amygdala complex did not show a main sex effect.
However, it did show a sex by conditioning and sex by genotype interaction. This
suggests that sex alone does not play a strong role in functional changes in the amygdala,
but interacts with other factors to alter functional activation. This is consistent with
studies in humans, which show a sex by emotional valence effect in the amygdala
(Domes et al., 2010).
The pattern of functional changes in prelimbic and amygdala suggest that sex
differences in the processing of emotional stimuli may relate more to the differences in
emotional regulation rather than stress responsivity. This is consistent with studies in
humans suggesting that females show altered attentional bias to emotional stimuli (Sass
et al., 2010; Tan et al., 2011). Interestingly, in female mice freezing behavior correlates
positively with functional activation in the prelimbic, but only weakly with the amygdala
(data not shown). In males, freezing does not correlate with prelimbic activation, but
correlates positively with functional activation in the amygdala (data not shown).

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Functional changes in both the dHPC and vHPC showed an effect of sex. Since
the vHPC has been implicated in emotional processing, sex differences could underlie
differences in emotional behavior. Functional changes in the vHPC also showed an
interaction with both genotype and conditioning, which means that the addition of other
factors also influence vHPC activity. Functional changes in the dHPC also showed a sex
by genotype effect. Differences seen in the dHPC could relate to sex differences in
spatial navigation and exploration of novel environments (Bettis and Jacobs, 2009; Frick
and Gresack, 2003), which was found to be sex dependent in exploratory-based anxiety
tests (Chapter 2). The functional changes in the dHPC also showed a sex by genotype
effect. This sex by genotype effect was also seen in exploratory-based measures with
WT females showing increased exploration of the environment. Sex differences were
also found in the insula, motor and sensory regions, which could reflect differences in the
processing of emotional stimuli and exploration patterns.
Estrogen is known to alter cerebrovascular reactivity (Diomedi et al., 2001).
While estrogen effects hemodynamics, Dietrich and colleagues found that overall pattern
of activation did not change throughout the menstrual cycle (Dietrich et al., 2001). This
suggests that taking into account estrous phase would mainly affect the size of the effect
rather than the overall pattern of sex differences.

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CHAPTER THREE: GENOTYPE AND CONDITIONING
EFFECTS ON FUNCTIONAL CONNECTIVITY IN
CORTICOLIMBIC CIRCUITRY

INTRODUCTION
Studies of functional connectivity in human subjects have implicated the
corticolimbic system in normal emotional processing to a wide range of affective stimuli
(Kilpatrick et al., 2006; Labus et al., 2008; Wrase et al., 2003). Functional disconnection
between the medial prefrontal cortex and the amygdala is a widely invoked paradigm to
explain emotional dysregulation in a number of psychiatric disorders, including Major
Depression (Carballedo et al., 2011), Bipolar Disorder (Shah et al., 2009; Strakowski et
al., 2004), Panic Disorder (Ohrmann et al., 2010), and Posttraumatic Stress Disorder
(Gilboa et al., 2004; Rauch et al., 2000; Shin et al., 2005). Deep brain stimulation of
subgenual, anterior cingulate cortex results in dramatic remission of some previously
treatment-resistant patients suffering from major depression (Holtzheimer et al., 2012;
Johansen-Berg et al., 2008; Lozano et al., 2012; Mayberg et al., 2005). Animal studies
provide further evidence for the role of corticolimbic circuitry in the stress coping
response. Inactivation of the ventral medial prefrontal cortex increases learned
helplessness, which is a behavioral pattern reminiscent of animals exposed to stress
(Amat et al., 2005).

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In the corticoamygdala circuit, the amygdala plays a role in responding to
threatening stimuli and the prefrontal cortex plays a role in attentional bias towards or
away from threatening stimuli (LaBar et al., 1998; Phelps et al., 2004). Research shows
that 5-HTT related 5-HT availability affects development of corticolimbic circuitry
(Holmes, 2008; Homberg et al., 2010). In humans, individuals with the 's' version of 5-
HTTLPR show reductions in the uncinate fasciculus, the white matter tract connecting
the amygdala to the orbital and medial prefrontal cortex with the amygdala (Pacheco et
al., 2009). Alterations in functional connectivity between the prefrontal cortex and
amygdala has been shown in healthy ‘s’ carriers with increased coupling between
prefrontal region BA10 and the amygdala (Friedel et al., 2009; Heinz et al., 2005;
Pezawas et al., 2005), but decreased connectivity in the amygdala-perigenual anterior
cingulate circuit. Furthermore, the effects of 5-HTTLPR gene on functional connectivity
may differ in healthy controls and those with major depression (Friedel et al., 2009).
Friedel and collaborators found that in contrast to findings in healthy controls, in patients
with major depression the ‘s’ allele associated with decreased connectivity between the
amygdala and BA10 (Friedel et al., 2009). Connectivity in the default network also
appears decreased in ‘s’ children and adolescents (Wiggins et al., 2012). These 5-
HTTLPR associated results resemble the mechanism of behavioral control seen in
rodents (Homberg, 2012). Functional connectivity studies in 5-HTT KO mice could
provide a translational bridge between human findings and animal studies showing the
role 5-HT plays in the corticoamygdala system and in normal emotional processing.

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To understand organization of the underlying brain network, we performed an
interregional correlation based functional connectivity analysis. This is a well established
method, which has been applied to analyze rodent brain mapping data of multiple
modalities, including autoradiographic deoxyglucose uptake (Barrett et al., 2003;
Soncrant et al., 1986), autoradiographic CBF (Wang et al., 2011a; Wang et al., 2011b),
cytochrome oxydase histochemistry (Fidalgo et al., 2011), and fMRI (Schwarz et al.,
2007). We applied these methods to understand how genotype alters the interaction of
brain regions at the network level during retrieval of an auditory tone. In these studies,
correlations are calculated in an inter-subject manner, i.e. across subjects within a group.
This differs from the intra-subject cross correlation analysis often used on fMRI time
series data (Liang et al., 2011; Magnuson et al., 2011; Pawela et al., 2008). Therefore
cautions need to be taken when comparing results from different modalities and analytic
methods.

METHODS
Anatomical region of interest (ROI) were drawn manually in MRIcro (version 1.40,
http://cnl.web.arizona.edu/mricro.htm) on the left and right hemispheres over a template
brain made up of the normalized brains of 99 mice (48 males and 51 females) according
to the mouse brain atlas (Franklin and Paxinos, 2008). Through logical conjunction the
anatomical ROIs were combined with SPM clusters showing a main effect of
conditioning, genotype or genotype x conditioning created a functional ROI. Mean
optical density of each ROI was calculated for each animal using the Marsbar toolbox for

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SPM (version 0.42, http://marsbar.sourceforge.net/). Pearson's correlation coefficients
between each pair of ROIs were calculated across subjects within a group in Matlab
(version 6.5.1, Mathworks Inc., Natick, MA, USA) to construct a correlation matrix for
each group. Correlation matrices were plotted as heatmaps in Matlab. Pearson’s
coefficients (r) were then transformed into z-scores using the Fisher transformation. To
control Type I error caused by the large number of correlations computed, a jackknife
procedure following (Barrett et al., 2003) was implemented. For a group of n subjects, n
iterations were performed in which one subject was dropped sequentially and the
correlation matrix recalculated with the remaining n − 1 subjects. A correlation was
considered ‘reliably’ significant only if it was significantly different from zero (P < 0.05)
in all iterations.

RESULTS
Interregional correlation matrixes of rCBF was constructed for control and
conditioned fear in WT and KO mice and visualized as heat maps (Figures 8-11).
Significant correlations (p < 0.05) were interpreted as functional connections and marked
with white dots. The matrix is symmetrical across the diagonal line from the upper left to
lower right, which itself reflects the correlation of the ROIs to themselves.

Functional correlations in WT mice:
Control. In WT control animals, cortical modulatory structures including infralimbic,
prelimbic, cingulate and retrosplenial cortex showed strong positive correlations with

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each other with the exception of the infralimbic and retrosplenial regions. With the
exception of the retrosplenial cortex, this cluster also positively correlated with bilateral
motor cortex and left primary somatosensory cortex, and the striatum (dorsal CPu and
nucleus accumbens). Prefrontal regions negatively correlated with the right posterior
insula. The retrosplenial cortex and cingulate also positively correlated with vision and
auditory cortex. The infralimbic and prelimbic cortex showed negative correlations with
the right secondary somatosensory cortex.
The amygdala showed positive correlations within its structures, with the dorsal
hippocampus, with the left insula and dorsal raphe. Visual and auditory cortexes strongly
positively correlated with each other. This cluster also positively correlated with the right
ventral hippocampus and negatively correlated with the anterior insula, ventral CPu, the
dorsal hippocampus and the dorsal raphe.

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Figure 11. Interregional correlation matrix showing functional connectivity patterns in WT-CON
mice. Z-scores of Pearson's correlation coefficients are color-coded. Significant correlations (p <
0.05) determined by the jackknife procedure are marked with white dots. Abbreviations: L (left),
R (right), IL (infralimbic), PrL (prelimbic), Cg (cingulate), RS (retrosplenial cortex), S1 (primary
somatosensory cortex), S2 (secondary somatosensory cortex), au (auditory cortex), aI (anterior
insula), pI (posterior insula), NaC (nucleus accumbens), dCPu (dorsal caudate putamen), vCPu
(ventral caudate putamen), BLA (basolateral amygdala complex), BMA (basomedial amygdala),
dHPC (dorsal hippocampus), vHPC (ventral hippocampus), MnR (median raphe), DR (dorsal
raphe).

Conditioned Fear. In comparison to control WT mice, the strong interconnectivity in
cortical modulatory structures was refined such that infralimbic and prelimbic clustered
and the cingulate and retrosplenial clustered. The positive correlations with these regions
and striatal regions, motor cortex and primary somatosensory cortex and negative

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correlations with right posterior insula were largely lost. Positive correlations between
the cingulate and retrosplenial and visual and auditory cortex were maintained.
Conditioned animals did show additional negative correlations between the retrosplenial
and the insula and the amygdala. The positive correlations within the amygdala were
attenuated in conditioned animals compared to unconditioned WT controls. New
negative correlations between the amygdala and the visual cortex emerged. In
conditioned animal, strong positive correlations between motor and sensory regions
emerged. New positive correlations between this cluster with auditory and insular
regions and negative correlations with the dorsal hippocampus emerged. The insula
cortex also became more positively correlated within itself.

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Figure 12. Interregional correlation matrix showing functional connectivity patterns in WT-CF
mice. Z-scores of Pearson's correlation coefficients are color-coded. Significant correlations (p <
0.05) determined by the jackknife procedure are marked with white dots. Abbreviations: L (left),
R (right), IL (infralimbic), PrL (prelimbic), Cg (cingulate), RS (retrosplenial cortex), S1 (primary
somatosensory cortex), S2 (secondary somatosensory cortex), au (auditory cortex), aI (anterior
insula), pI (posterior insula), NaC (nucleus accumbens), dCPu (dorsal caudate putamen), vCPu
(ventral caudate putamen), BLA (basolateral amygdala complex), BMA (basomedial amygdala),
dHPC (dorsal hippocampus), vHPC (ventral hippocampus), MnR (median raphe), DR (dorsal
raphe).

Functional correlations in KO mice:
Control. In control 5-HTT KO animals, cortical modulatory structures were positively
correlated with each other including the infralimbic with the prelimbic, the prelimbic
with the cingulate and the cingulate with the retrosplenial. This cluster also positively
correlated with the nucleus accumbens, left dorsal hippocampus, the median and dorsal

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raphe and negatively correlated with motor, sensory, the insula, ventral caudate putamen
and parts of the amygdala. The infralimbic and prelimbic also negatively correlated with
visual and auditory cortex. Motor, somatosensory cortex, auditory cortex and the insula
showed strong positive correlations between these structures. This cluster also formed
strong positive correlations with the ventral caudate putamen and the right basal lateral
amygdala complex and negative correlations with the dorsal hippocampus and the
median and dorsal raphe. The amygdala showed very little positive intercorrelation
between its substructures.

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Figure 13. Interregional correlation matrix showing functional connectivity patterns in KO-CON
mice. Z-scores of Pearson's correlation coefficients are color-coded. Significant correlations (p <
0.05) determined by the jackknife procedure are marked with white dots. Abbreviations: L (left),
R (right), IL (infralimbic), PrL (prelimbic), Cg (cingulate), RS (retrosplenial cortex), S1 (primary
somatosensory cortex), S2 (secondary somatosensory cortex), au (auditory cortex), aI (anterior
insula), pI (posterior insula), NaC (nucleus accumbens), dCPu (dorsal caudate putamen), vCPu
(ventral caudate putamen), BLA (basolateral amygdala complex), BMA (basomedial amygdala),
dHPC (dorsal hippocampus), vHPC (ventral hippocampus), MnR (median raphe), DR (dorsal
raphe).

Conditioned Fear. The pattern of correlations in 5-HTT KO mice that were conditioned
to the tone largely reflected the patterns seen in the 5-HTT KO control mice, but the
correlations were generally attenuated to those seen in control animals. Positive
correlations within cortical modulatory structures were reflective of that seen in KO
animals, except that that retrosplenial separated out more in the conditioned KO animals

75

then it did in control KO animals. The negative correlations between this cluster and
motor, sensory, visual cortex, auditory cortex were greatly attenuated. This cluster made
wider but less robust connections to the striatum. Functional correlations with the
amygdala were greatly altered, with slightly positive correlations between the infralimbic
and prelimbic and the amygdala and negative correlation between the cingulate and the
amygdala. The positive correlations between the prefrontal cluster and the raphe were
lost.
The strong positive correlations between motor, sensory cortex and auditory
cortex remained in the conditioned animals and extended to include the retrosplenial
cortex and visual cortex. The positive correlations between this cluster the insula and
negative correlations with the dorsal hippocampus were attenuated. The positive
correlations between this cluster and the ventral caudate putamen and parts of the
amygdala were lost as was the negative correlations between this cluster and the dorsal
raphe.

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Figure 14. Interregional correlation matrix showing functional connectivity patterns in KO-CF
mice. Z-scores of Pearson's correlation coefficients are color-coded. Significant correlations (p <
0.05) determined by the jackknife procedure are marked with white dots. Abbreviations: L (left),
R (right), IL (infralimbic), PrL (prelimbic), Cg (cingulate), RS (retrosplenial cortex), S1 (primary
somatosensory cortex), S2 (secondary somatosensory cortex), au (auditory cortex), aI (anterior
insula), pI (posterior insula), NaC (nucleus accumbens), dCPu (dorsal caudate putamen), vCPu
(ventral caudate putamen), BLA (basolateral amygdala complex), BMA (basomedial amygdala),
dHPC (dorsal hippocampus), vHPC (ventral hippocampus), MnR (median raphe), DR (dorsal
raphe).

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DISCUSSION
Functional correlations in WT mice:
Control. In WT control mice, a positive corticostriatal core was revealed between
prefrontal cortical regions, including infralimbic, prelimbic, cingulate, retrosplenial and
the nucleus accumbens and dorsal CPu. Within this corticostriatal core, the prefrontal
regions also positively correlated with motor and primary somatosensory cortex. Wang
et al., 2011a reported similar findings of positive correlations in corticostriatal core in
freely moving control rats during a passive step down avoidance task. A correlation
between prefrontal cortex, frontal cortex, nucleus accumbens core and striatum were also
reported in rats in an awake resting state (Soncrant et al., 1986). These results are
reminiscent to findings in the human brain default network, which has been suggested to
exist in rats (Lu et al., 2012). Although the function of the default network is still
unclear, deficits in this circuit have been implicated in a variety of neuropsychiatric
disorders (Lu et al., 2012).

Fear conditioned. Following footshock training, WT mice showed reorganization of
brain functional connectivity. Intrastructural positive connections as well as cortico-
subcortical connections were greatly reduced in number and magnitude. Specifically,
conditioned WT mice showed attenuated interregional correlations within prefrontal
regions and almost complete loss of correlation between prefrontal regions and the
striatum, motor and primary somatosensory cortex. Similar task-evoked disconnection
has been previously reported in rats (Wang et al., 2011b), as well as in the human

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literature (Biswal et al., 1995; Deco et al., 2011; Greicius et al., 2003; Raichle et al.,
2001; Raichle and Snyder, 2007). At the same time, new connectivity patterns emerged
characterized by increased intra-cortical connectivity within motor and sensory regions.
This cluster also showed new positive correlations with auditory cortex and insula (which
showed increased intra-insular connectivity) and negative correlations with the dorsal
hippocampus. This connectivity pattern could suggest increased sensorimotor integration
during cues signaling potential threats.

Functional correlations in 5-HTT KO mice:
Control. Robust genotypic effects were apparent even in control animals. The following
differences between KO compared to WT mice were noted: 1) decreased prefrontal
connectivity to the amygdala, motor and sensory cortex, visual auditory cortical regions
and the insula 2) decreased connectivity within the amygdala 3) increase in functional
connectivity within motor and sensory regions and within the insula, 4) increased
connectivity of motor and sensory regions to the insula and the caudate putamen, as well
as decreased connectivity of the motor and sensory regions to the dorsal hippocampus
and 5) increase in functional connectivity within the raphe nuclei (MnR, DR) and
decreased connectivity between the raphe and motor and sensory cortex, the insula and
the amygdala.
These findings show a large effect of genotype on the organization of neural
circuits during an auditory cue not signaling any kind of danger. In fact, 5-HTT KO
control mice (no exposure to footshock) demonstrated correlation patterns within

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prefrontal cortical modulatory regions and functional correlations between motor,
sensory, auditory and insular cortex that were reminiscent of WT conditioned mice.
While the implications of this are unclear, it suggests that 5-HTT KO mice process and
attend to incoming sensory information differently than WT mice.
Brownings and colleagues describe two neural circuits involved in biasing
attention towards sensory stimuli: the amygdala and the prefrontal cortex. When
attention is given to a particular stimulus, these systems alter activation in the
corresponding sensory and association cortices (Browning et al., 2010). In humans, 5-
HTTLPR genotype has been implicated in attention processing of emotional stimuli
(Beevers et al., 2009; Fox et al., 2009; Kwang et al., 2010; Perez-Edgar et al., 2010;
Pergamin-Hight et al., 2012). A recent meta-analysis showed that low 5-HTT function
relates to increased attention towards negative stimuli (Pergamin-Hight et al., 2012). The
results of this study further suggest that "baseline" neural circuitry involving sensory
systems is greatly affected by genotype, which has also been shown in human studies
(Wiggins et al., 2012).
Additionally, the raphe made many more correlations with brain structures in the
5-HTT KO mice than in the WT mice. Since serotonergic neuron projections originate in
the raphe and autoregulatory 5-HT1A receptor density have been shown to be altered in
5-HTT KO mice (Li, 2006), these differences could reflect differences in the serotonergic
response.

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Fear conditioned. The pattern of correlations in 5-HTT KO mice that were conditioned
to the tone largely reflected the patterns seen in the 5-HTT KO control mice, but the
correlations were generally attenuated compared to those seen in control animals. This
task-evoked disconnection was also noted in WT mice. While intracorrelations between
the prefrontal regions, as well as the negative correlations between the medial prefrontal
regions and the basolateral amygdala were attenuated in conditioned KO compared to
control KO mice, this reorganization was less robust than that seen in WT mice. The
positive connectivity between the prefrontal cluster and the raphe was lost during
conditioning. The negative correlations with this prefrontal cluster and motor, sensory,
visual cortex, auditory cortex were greatly attenuated and a new increased positive
functional connectivity emerged between the posterior cingulate cortex (retrosplenial
cortex) with motor and sensory areas. This suggests that in the presence of a cue
signaling threat, robust changes occur in how the medial frontal cortex interacts with
other brain structures.

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CONCLUSIONS

The results of these studies contribute to the literature addressing the effects of
loss of 5-HTT function on anxiety, motor and sensory behavior and neural function in
corticolimbic and somatosensory systems in 5-HTT KO mice. Additionally, these studies
provide several novel findings including 1) evidence that genotypic effects on sensory
systems could contribute to different behavioral effects in conditioned versus exploratory
based anxiety tests, 2) 3-D maps of functional changes in 5-HTT KO mice and 3)
correlational based measures of genotype effects on functional connectivity patterns.
5-HTT KO mice displayed robust increases in freezing behavior during a cued
conditioned recall test, which is consistent with the theory that these mice display
increased anxiety like behaviors. In exploratory-based anxiety tests, however, genotypic
effects were minimal or even paradoxical. Paradoxical effects on anxiety like behavior in
5-HTT KO mice were also found in marble burying (appendix A). Further testing of
home cage locomotor activity did not reveal any deficits in male 5-HTT KO mice,
suggesting that reduced locomotor activity in these mice is specific to novel
environments. Tests of whisker function, however, demonstrated behavioral deficits
related to reported abnormalities in the somatosensory cortex of 5-HTT KO mice.
Differences in whisker function could alter exploratory behavior independent of
differences in anxiety levels and support the use of cation when interpreting behavior in
this mouse model.

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Genotype significantly effected functional activation in circuitry involved in
emotional processing including the amygdala, hippocampus, insula, cingulate, and
retrosplenial cortex. There was a genotype x conditioning interaction in the prelimbic
cortex. Additionally genotypic effects were found in motor and somatosensory regions
suggest genotypic effects in somatosensory function and processing of stimuli.
Genotypic effects found in the barrel field cortex support the findings in chapter one of
abnormalities in barrel dependent tasks and add to the literature showing morphological
and electrophysiological deficits in 5-HTT KO mice barrel function. Connectivity
studies provided further evidence of robust genotypic effects on the organization of
neural circuits involved in emotion and somatosensory function. 5-HTT KO mice
displayed greater connectivity patterns at baseline then WT mice including increased
interconnectivity within motor, sensory and insula cortex and between this cluster and the
caudate putamen. 5-HTT KO mice also showed decreased prefrontal connectivity to the
amygdala, motor and sensory cortex, visual and auditory cortical regions and the insula.
There was an increase in functional connectivity within the raphe nuclei and decreased
connectivity between the raphe and motor and sensory cortex, the insula and the
amygdala.
While genotypic effects on anxiety behavior were a mixed bag of results the
imaging studies provided robust evidence of genotypic effects on neural activation and
connectivity. The differences in the effect of the gene on anxiety behavior and neural
activation relates to how genes exert their effects. Genes alter molecular processes that
then result in changes in neural circuits. Changes in neural processes may or may not

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affect overt behaviors; thus the effects of a gene on behaviors are complex. This has led
to imaging genetics to bridge the gene to syndrome pathway. In many ways, 5-HTTLPR
has become the ‘poster child’ for imaging genetics because of the robustness of the effect
of the gene on amygdala activation and connectivity between emotional modulatory
regions and the amygdala. Similar patterns of morphological changes in corticolimbic
structures have also been reported in 5-HTT KO mice. My studies provide an
intermediate translational bridge further suggesting that genotype exerts robust effects on
corticolimbic and somatosensory circuits.
Changes in neural circuitry as a result of 5-HTT related 5-HT predisposes
individuals to neural activity patterns that could be hyperresponsive to stressful stimuli.
Furthermore, differences in somatosensory processing could alter the processing of
emotional stimuli. Taken together, these two effects could mean that lifelong reduction
in 5-HTT function results in abnormal processing of emotional stimuli and a heightened
response in emotional regions to stressful stimuli. This means that the presence of the
low expressing gene does not result in an individual having increased mood and anxiety
symptoms per se (i.e. the genotype does not equal an anxious or depressed phenotype),
but in the presence of a stressor individuals with lifelong reduction in the function of 5-
HTT would show increased susceptibility to mood and anxiety disorders. Indeed the
phenotypic effects of this gene has been shown to be stress dependent and more robust
genotypic effects on anxiety behavior were found in chronically stressed male 5-HTT KO
compared to WT mice (data not shown). This stress susceptibility rather than a direct
increase in mood and anxiety phenotypes could explain why imaging findings are much

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more robust than behavioral phenotypes and suggest that imaging is a more sensitive
measure than behavioral phenotyping.
These reverse translational findings provide further evidence for the utility of
imaging genomics and serve as a translational bridge between human neuroimaging
studies and morphological studies in 5-HTT KO mice. Furthermore, through
strengthening the construct validity of the model neuroimaging could provide a more
direct understanding of morphological changes underlying changes in neural function.
This is useful as morphological studies are hard to undertake in human subjects.
Additionally, future studies could also address a stress by genotype effect on structure,
function and plasticity in the brain. As stress is hard to model in humans, the 5-HTT KO
mouse model could provide a better understanding of how this gene and stress interact to
alter brain structure, function and plasticity.
The greatest effects of sex on anxiety behavior were subtle and found mainly
during exploration of novel environments. In contrast to the hypothesis, sex differences
in exploration were attributed to increased activation in WT females with little to no
differences in 5-HTT KO mice. Female compared to male WT mice showed increased
exploration of novel environments, but there was little to no difference in male and
female KO animals. In fear conditioning the robust genotypic effect was replicated in
female animals, but there was no added effect of sex. The relative lack of genotype x sex
interactions on anxiety like behavior is different than hypothesized, but may reflect a
ceiling effect of the gene on exploration of novelty, as has been previously noted by
others (Kalueff et al., 2007; Olivier et al., 2008). Similar to what was seen in the

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genotype to phenotype effect, sex also showed much more robust effects at the circuitry
level rather than at the behavioral level. While sex did not significantly alter freezing
behavior, sex showed robust changes in functional activation patterns in motor and
sensory cortex, prefrontal (prelimbic), hippocampus, insula and amygdala.
While sex differences in stress susceptibility and prevalence of stress reactivity
disorders have been known for a while, females were traditionally excluded from
research or lumped in with male participants. This has led to a lack of understanding of
emotional processing in females and how this might differ from males. The pattern of
activation within the amygdala and within prelimbic and cingulate (medial prefrontal)
cortex suggest that sex may affect emotional modulatory regions more than the amygdala
itself. This pattern supports the interesting notion that sex differences in prevalence of
stress reactivity of the amygdala may arise from differences in emotional regulation
rather than from emotional reactivity. This increased understanding could be explored in
humans and could effect treatment. If emotional regulation were aberrant in females,
perhaps females with anxiety and depression would benefit more from treatments that
incorporate emotional regulation strategies. Looking at sex differences in extinction
learning could be an interesting way to further tease out these processes. Research into
extinction learning is increasingly focused on prefrontal modulation of the amygdala and
suggests that extinction is new learning rather than forgetting. If regulatory processes in
emotional regulation were sex dependent than I would expect to see robust changes
during extinction learning.

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Conditioning significantly altered functional brain activation in regions implicated
in the fear response including the amygdala, prelimbic, cingulate cortex, retrosplenial
cortex, sensory regions and dorsal hippocampus. Activation in the ventral hippocampus
and motor regions showed a sex by conditioning interaction. Functional connectivity
patterns also showed reorganization as a result of conditioning. In WT mice,
conditioning attenuated interregional correlations within prefrontal regions and between
prefrontal regions and the striatum, motor and primary somatosensory cortex. At the
same time, new connectivity patterns emerged characterized by increased intra-cortical
connectivity within motor and sensory regions and between these regions and auditory
cortex and insula (which showed increased intra-insular connectivity) and negative
correlations with the dorsal hippocampus. This connectivity pattern suggests increased
sensorimotor integration during cues signaling potential threats. Overall, the patterns of
connectivity were very similar, but attenuated in KO conditioned compared to KO
control animals. Importantly, the connections between the prefrontal cortex and other
regions were greatly attenuated, as were the connections between the raphe and other
brain regions. This suggests that in KO mice the presence of an emotionally valenced
cue alter connectivity in regions implicated in the stress response.
Overall, the results of my studies provide strong evidence that 5-HTT KO and sex
greatly alter neural circuitry, but only show modest changes on anxiety behavior. These
results further suggest that changes occur at a neural circuitry level, which then may or
may not affect overt behavior; thus imaging may provide a more sensitive measure than
behavioral phenotyping alone.

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APPENDIX A: GENOTYPE EFFECTS ON MARBLE
BURYING

METHODS
A separate group of male mice (n=11/group) were tested in a marble burying
paradigm (Gorton et al.). Each mouse was placed in a novel cage filled with one inch of
cozy critter super shavin's bedding (International Absorbants Inc., Ferndale, WA).
Twenty-five small blue glass marbles (10-12 mm diameter) were clustered in the center
of the cage. Mice were placed in the front of the cage facing the marbles and allowed to
explore for thirty minutes. Thereafter mice were returned to their home cage and the
number of marbles buried (>2/3 of the marble buried with bedding) was counted. Group
averages of marbles buried were compared using a t-test (two tailed, p<0.05).

RESULTS AND DISCUSSION
Marble burying is a test of 'defensive behavior' (Njung'e and Handley, 1991) that
is reliant on burrowing (Gyertyan, 1995). Male 5-HTT KO mice buried less marbles then
WT mice (Figure 15). This 'paradoxical' (nonanxious) response in the KOs has been
previously reported a result which has been previously reported (Kalueff et al., 2006;
Line et al.).

112

Figure 15. Marble Burying 5-HTT KO mice compared to WT mice buried significantly less
marbles placed in a novel cage; *p < 0.001. Error bars represent standard error of the mean.

113

APPENDIX B. GENOTYPE EFFECTS ON CIRCADIAN
RYTHMICITY IN MALE 5-HTT KO MICE

INTRODUCTION
The mammalian circadian clock located in the suprachiasmatic nucleus (SCN) is
thought to be modulated by serotonin (5-HT) (Prosser et al., 1993). Reduction in
response to photic phase shifts by 5-HT is thought to occur through inhibition of the
release of glutamate from retinal terminals, as well as by decreasing the responsiveness of
retinorecipient cells in the SCN (Rea and Pickard, 2000; Ying and Rusak, 1994). It is
well-known that serotonergic agonists can phase shift the circadian clock in the SCN in
vitro (Prosser et al., 1993) and in vivo (Challet et al., 1998; Edgar et al., 1993).
Furthermore, acute pharmacological increases in 5-HT have been shown to attenuate the
responsivity of animals to photic phase shifts (Challet et al., 2001; Gannon and Millan,
2007) and destruction of serotonergic fibers increases the magnitude of photic phase
shifts (Bradbury et al., 1997). What is less clear are the effects of lifelong increases in 5-
HT levels on circadian rhythms and phase shifts.
In this study, we examine the effect of lifelong elevated 5-HT levels on circadian
rhythms in locomotion in the serotonin transporter knockout (5-HTT KO) mouse model.
5-HTT KO mice have provided insight into the effects 5-HTT has on physiological
function and behavior (for review see Murphy and Lesch, 2008). These mice have
greatly reduced, but not wholly absent 5-HT clearance (Baganz et al., 2008; Zhou et al.,
2002), an observation similar, though not identical, to findings in the human subjects

114

carrying the low expressing forms ('s' or 'L
G
') of the serotonin transporter length
polymorphic region (5-HTTLPR) (Greenberg et al., 1999). On a behavioral level, 5-HTT
KO mice and humans with the low-expressing form of 5-HTTLPR display increased
susceptibility to depressive behavior (Alexandre et al., 2006; Caspi et al., 2010), and an
increased stress hormone response (Gotlib et al., 2008; Tjurmina et al., 2002).
Abnormalities of circadian rhythms, sleep architecture, and phase advances or delays are
commonly associated with mood disorders (Manber and Chambers, 2009; Mistlberger et
al., 2000). Changes in sleep quality and insomnia are noted in association with the 5-
HTTLPR polymorphism (Brummett et al., 2007; Deuschle et al., 2010). Alterations in
sleep architecture, specifically increased frequency and duration of rapid eye movement
sleep (REM) (Alexandre et al., 2006; Wisor et al., 2003), have also been reported in 5-
HTT KO mice. To our knowledge, this is the first report examining basal circadian
rhythms and photic phase shifting in the 5-HTT KO mouse. Since the phase response
curve for serotonergic agonists appears to have a larger phase advance region than delay
region (Edgar et al., 1993), we hypothesized that 5-HTT KO mice would be relatively
phase-advanced in constant conditions relative to WT mice. Furthermore, we
hypothesized that the photic phase shift would be attenuated in KO animals.

METHODS
Animals:
5-HTT KO mice were backcrossed onto a C57BL/6 background for greater than
15 generations from an original mixed background [129/P1ReJ (ES cells), C57BL/6J and

115

CD-1] (Bengel et al., 1998; Salichon et al., 2001). Mice were bred at USC from pairs
obtained from Taconic (Taconic, Hudson, NY, USA). Prior to the start of experiments,
mice were housed in groups of three to four animals under standard conditions. At the
start of circadian recordings adult male mice were individually housed. Genotyping was
confirmed by Transnetyx, Inc. (Cordova, TN, USA) from tail snips obtained post mortem
with primer sequences obtained from Taconic (m5-HTT-C: 5’ TGA ATT CTC AGA
AAG TGC TGT C 3’, m5-HTT-D: 5’ CTT TTT GCT GAC TGG AGT ACA G 3’,
neo3a: 5’ CAG CGC ATC GCC TTC TAT C 3’).

Circadian Rhythms:
A modified Aschoff type II design was employed to measure basal circadian
rhythms and response to a circadian time (CT) 14 light pulse. Transparent cages (31 cm x
18 cm x 13 cm) containing the individually housed mice (5-HTT KO: n = 11, average age
= 14.5 ± 0.5 weeks; WT: n = 14, average age = 15.7 ± 0.6 weeks) were placed within
frames containing three infra-red photo beams spaced 8.8 cm apart (San Diego
Instruments, San Diego, CA, USA). Locomotion indicated by photo beam breaks was
recorded electronically using PAS software (San Diego Instruments, San Diego, CA,
USA). After one week of habituation to the room, baseline recording (12 hour light, 12
hour dark; LD 12:12) was started. After four weeks on the LD 12:12 schedule, animals
were given a light pulse 14 hours after light onset (one hour duration, CT 14, 150 lux).
Mice were maintained in constant darkness for three weeks while activity continued to be
recorded.

116

Behavioral Analysis:
Periods, amplitudes, activity counts, and acrophases were analyzed separately for
the light/dark schedule and constant darkness conditions using ClockLab (Actimetrics,
Wilmette, IL, USA). Means for the KO and WT mice were compared using a t-test for
independent groups (significance defined as p < 0.05). To minimize carry over effects
from the light pulse, the first five days post light pulse were considered transient days and
excluded from the free running analysis. Period was determined by measuring the slope
of a regression line on the actogram in ClockLab and by the F-periodogram (Actimetrics,
Wilmette, IL, USA). An amplitude measure was calculated using the F-periodogram in
ClockLab (Actimetrics, Wilmette, IL, USA). Briefly, a signal to noise ratio was
generated by dividing the modified F-value at the dominant period by the noise value at
that period. Activity counts were automatically generated based on the number of beam
breaks. The acrophase was determined from the time at which the maximum in the
locomotor rhythm occurred in either the light/dark cycle or constant darkness. Phase
shifts were determined by comparing the difference in the fitted regression lines before
and after a light pulse was given.

RESULTS
No significant differences in amplitude (KO = 1.46 ± .08; WT = 1.5 ± .08), period
(KO = 23.99 hrs ± .01 hrs; WT = 23.99 hrs ± .00 hrs), acrophase (KO = 19.98 hrs ± 0.15
hrs; WT = 20.00 hrs ± 0.14 hrs) or total activity counts (KO = 1010 ± 42; WT = 1141 ±
79) were seen between WT and KO mice during the standard 12:12 light-dark cycle (p >

117

0.05). There was also no significant difference in amplitude (KO = 1.36 ± .04; WT =
1.29 ± .06), period (KO = 23.84 hrs ± .03 hrs; WT = 23.77 hrs ± .02 hrs ), acrophase (KO
= 21.20 hrs ± 0.22 hrs; WT = 20.38 hrs ± 0.34 hrs) or total activity counts (KO = 962 ±
77; WT = 867 ± 89) between WT and KO mice during constant darkness (p > 0.05). The
mean phase delay to a light pulse was significantly attenuated in KO compared to WT
mice (Figure 1, KO = -1.00 hrs ± .25 hrs; WT = -1.78 hrs ± .21 hrs, t = -2.4, p < 0.05).

118

Figure 16. Double plotted actograms Sample of double plotted actograms of homecage
locomotor activity in two WT and two KO mice. After four weeks on standard 12:12 light-dark
schedule, animals were given a light pulse (indicated by the arrow) at CT 14. Thereafter, mice
were maintained in constant darkness for three weeks while activity continued to be recorded.

DISCUSSION
Since adult 5-HTT KO mice exhibit elevated basal extracellular 5-HT over that of
WT controls (Mathews et al., 2004), we expected to see differences in the organization of
circadian rhythms in this preparation. In general, we found the 5-HTT KO mice to have
surprisingly normal homecage locomotor activity and circadian rhythms (both in LD

119

12:12 and constant darkness). KO mice compared to WT mice did not show significant
differences in acrophase in constant darkness. This is different from what we
hypothesized based on knowledge of acute administration of serotonergics (Edgar et al.,
1993). This provides further evidence that lifelong alterations in serotonergic function do
not have the same effect as acute alterations. This might represent compensatory changes
in the 5-HTT KO mice, which result in a normalization of the locomotor and circadian
patterns, even in a state of elevated 5-HT.
A previous report found 5-HTT KO mice to be hypolocomotory (Holmes et al.,
2002). In this study, however, we found no significant difference in spontaneous
homecage locomotor activity, which is consistent with studies of 5-HTT KO rats (Linder
et al., 2008). The current study looked at homecage locomotor activity over several
weeks, whereas a prior report looked at a 24 hour period following one day of
acclimation (Holmes et al., 2002). Shorter sampling of behavioral activity may have
been influenced by anxiety-like behavior, which is a characteristic of the 5-HTT KO
animals (Holmes et al., 2003; Pang et al., 2011).
We did see a significant attenuation in the photic phase shift in the 5-HTT KO
mice. This is consistent with data from acute increases in 5-HT neuronal activity, which
have been shown to attenuate photic phase shifts (Challet et al., 2001; Gannon and
Millan, 2007; Mistlberger et al., 2002). Diminished phase shifts to nocturnal light pulses
have also been reported in mice lacking the 5-HT 1B receptor (Sollars et al., 2006).
Since the 5-HT-1B receptor is desensitized or downregulated in the 5-HTT KO mouse
(Shanahan et al., 2009), it is possible that the attenuation of the photic phase shift in this

120

study reflects a similar functional change in that receptor at some point in the afferent
input to the SCN. The chronic effects of heightened 5-HT tonus would then be quite
similar to the acute effect of such elevation, i.e. a reduced photic phase shift (Rea and
Pickard, 2000; Ying and Rusak, 1994).

CONCLUSIONS
Basal circadian parameters and homecage locomotor activity remained unchanged
in 5-HTT KO mice. This may reflect compensatory changes that normalize these
patterns. Challenge with a light pulse, however, unmasked a diminished ability to
respond with a phase shift.

121

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Abstract (if available)

Abstract In humans, the serotonin transporter linked polymorphic region (5-HTTLPR) results in altered transcriptional efficiency of the transporter gene, as well as uptake of serotonin (5-HT) by neurons. The low expressing `s' allele of this polymorphism has been linked to increased susceptibility to stress and altered connectivity of corticolimbic neural circuits, the disruption of which is widely invoked to explain emotional dysregulation in anxiety and mood disorders. ❧ Serotonin transporter (5-HTT) knockout (KO) mice offer a promising model for psychiatric research as close parallels exist between the human polymorphism and the mouse model at the level of serotonergic profile, behavior, physiological function, and stress hormone response. Although much work has gone into characterizing 5-HTT KO mice, little work to date has examined functional activation patterns and connectivity of circuits involved in emotional regulation in these mice. The experiments outlined in this dissertation address this gap in knowledge by examining sex and genotypic differences in the underlying functional activation of corticolimbic circuits. The inclusion of females in this study provides important information about how sex differences in serotonergic function alter brain function. Functional connectivity analysis is also undertaken in a combined male/female groups to determine genotypic effects on a network level. ❧ Overall, the experiments contained in this dissertation contribute novel information about the role of sex and 5-HTT genotype in anxiety like behavior and function of corticolimbic circuits. Specifically, this is the first study using whole brain functional perfusion mapping in the 5-HTT KO mouse model. Furthermore, these studies support sex differences in functional regulation of the prefrontal-amygdala network in a fear-conditioned paradigm. These studies establish the translational value of functional brain mapping endpoints and suggest greater specificity than the behavioral endpoints. Future studies could address the underlying molecular and structural changes accompanying functional changes seen in 5-HTT KO mice.

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Asset Metadata

Creator Pang, Raina D. (author)

Core Title Genotype and sex effects on anxiety behavior and functional activation in corticolimbic circuits in serotonin transporter knockout mice (5-HITT KO)

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 05/30/2012

Defense Date 05/03/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag amygdala,functional neuroimaging,OAI-PMH Harvest,serotonin transporter knockout mice

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Holschneider, Daniel P. (committee chair), Celikel, Tansu (committee member), Jakowec, Michael (committee member), Watts, Alan G. (committee member)

Creator Email rpang@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-43601

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