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Pharmacokinetics and developmental effects of exposures to selective serotonin reuptake inhibitor (SSRI) antidepressants

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Pharmacokinetics and developmental effects of exposures to selective serotonin reuptake inhibitor (SSRI) antidepressants

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Content PHARMACOKINETICS AND
DEVELOPMENTAL EFFECTS OF EXPOSURES TO
SELECTIVE SEROTONIN REUPTAKE INHIBITOR
(SSRI) ANTIDEPRESSANTS
JUAN C . HUEZO VELASQUEZ

A DISSERTATION PRESENTED TO
THE FACULTY OF THE GRADUATE SCHOOL OF
THE UNIVERSITY OF SOUTHERN CALIFORNIA
IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE

DOCTOR OF PHILOSOPHY

IN

NEUROSCIENCE

AUGUST 2017

COPYRIGHT 2017 JUAN C VELASQUE Z
II
EPIGRAPH

Stuart rose from the ditch, climbed into his car, and started up the road that led
toward the North...As he peeked ahead into the great land that stretched before
him, the way seemed long. But the sky was bright, and he somehow felt he was
headed in the right direction.

— E.B. White, Stuart Little (1945)

III
DEDICATION

For my supportive family
and friends that have become family

To my grandparents for their unparalleled vivacity
and my mother Ana for inspiring me to always strive for
excellence, for believing and reminding me that there
is nothing I could not do

IV
ACKNOWLEDGEMENTS

My deepest gratitude goes to my brilliant advisor, Dr. Alexandre Bonnin, who
expertly guided me through my doctoral training and shared with me the
excitement (and sometimes frustration) of over 5 years of research. As scientists,
we take a journey into the unknown where we are trained to be critical thinkers,
consider all possibilities, push the boundaries of our limitations to overcome
diverse challenges, and emerge as problem solvers. I will be forever grateful for
Alex’s mentorship in this process. His insight and creativity were a source of
inspiration and drive over the course of my work, and are qualities I hope to
emanate forward. From sharing my excitement for results of important
experiments, exchanging ideas to take my research to greater heights, and even
long-winded discussions about the world, Alex always kept an open door and
fostered an environment that was challenging but also fun.
I have been fortunate to learn from the exceptional scientists on my guidance
and dissertation committees—Dr. Karen Chang, Dr. Daniel Campbell, Dr. Ruth
Wood, Dr. Jean Shih, and Dr. Andrew MacKay—whose thoughts and comments
have enabled my own growth as a scientist. I must also thank my undergraduate
mentors Dr. Curtis Loer at the University of San Diego and Dr. Dan Gibbs at the
Salk Institute for initially introducing me to scientific research.
I am also indebted to my fellow lab members for providing a stimulating
environment in which I have learned and grown. I am grateful to have worked
alongside Nick Goeden, Jenny King, and Jonathan and Matthew Wang. I am
particularly grateful for the insight, encouragement, and friendship of Yen Chan,
Ligia Galindo, and Clarissa James. Day after day and into the evening hours, I
could not have asked for a better team of colleagues to work benchside, to
troubleshoot experiments, and celebrate successes with. I am also thankful for

V
the visits and insights provided by Dr. Juli Wu and the refreshing enthusiasm
brought by the collaboration with Dr. Skyla Herod and her team. I feel very
fortunate to have also collaborated with Dr. Thierry Fournier and Dr. Sophie Gil at
their labs in Paris.
I am grateful to the funding agencies that have supported my research:
National Institutes of Health, USC, the Neuroscience Graduate Program, and the
Chateaubriand Fellowship. I am grateful to the Zilkha Neurogenetic Institute for
providing me with funding through the Zach Hall Travel Award in order to travel to
the Society for Neuroscience meeting.
On a personal note, I have endless gratitude for all of my family and friends.
In particular, I thank my grandparents Emerito and Maria Elena, my mother Ana,
my brothers Salvador and Rene, and the friends that offered me unwavering
commitment and encouragement: Terese, Anna, Roy, and Jacob Leyrer; Ruth,
Matt, and Jordan Toopes, thank you for welcoming and accepting me into your
families as one of your own; and my friends and classmates at USC and my
1647 family. I am thankful for all of their patience listening to my scientific
theories, encouragement when I worked through seemingly insurmountable
challenges, support when I had to live abroad, for taking and celebrating my
successes as their own, for their flexibility when lab took over my weekends:
words fall short of how grateful I am for all your support, love, and ability to keep
me laughing and persevering forward. A special thank you to Jordan and Ellie for
joining me in this adventure and making my days brighter with your optimism and
endless tail wags, respectively. Thank you for helping me remember that our
work as scientists truly matters; although science is often difficult and sometimes
frustrating, it is also absolutely necessary. I am grateful for the encouragement to
not lose sight of the positive impact I can make through science and how special
and privileged I am to work on understanding the brain.

VI
TABLE OF CONTENTS
EPIGRAPH II
DEDICATION III
ACKNOWLEDGEMENTS IV
LIST OF FIGURES IX
LIST OF TABLES XI
ABSTRACT XII
CHAPTER 1: INTRODUCTION TO DEPRESSION AND ANTIDEPRESSANT
USE DURING PREGNANCY 1
CHAPTER 1 POINTS OF INTEREST 2
1.1 ABSTRACT 3
1.2 INTRODUCTION 4
1.3 SEROTONIN AND FETAL BRAIN DEVELOPMENT 6
1.4 PRENATAL EXPOSURES AND LONG-TERM CONSEQUENCES 8
1.5 IMPACT OF SSRIS ON FETAL DEVELOPMENT MAY DEPEND ON
ROUTES OF EXPOSURE DURING PREGNANCY 13
1.6 SEROTONIN AND THE NEURODEVELOPMENTAL PROGRAMMING OF
MENTAL DISEASES 17
CHAPTER 2: FROM MOTHER TO FETUS: A METHODOLOGICAL
APPROACH TO STUDY PLACENTAL SSRI TRANSFER EFFECTS 19
CHAPTER 2 POINTS OF INTEREST 20
2.1 ABSTRACT 21
2.2 INTRODUCTION 22
2.2.1 SEROTONIN AND FETAL BRAIN DEVELOPMENT 24

VII
2.3 MATERIALS, EQUIPMENT, AND SETUP 31
2.3.1 EX VIVO PERFUSION OF THE MOUSE PLACENTA FOR
BIDIRECTIONAL DRUG TRANSPORT STUDIES 31
2.3.2 EX VIVO PERFUSION OF THE MOUSE PLACENTA FOR
BIDIRECTIONAL DRUG TRANSPORT STUDIES 35
2.4 METHODS 36
2.4.1 EX VIVO PERFUSION OF THE MOUSE PLACENTA FOR
BIDIRECTIONAL DRUG TRANSPORT STUDIES 36
2.4.2 IN VIVO STUDIES OF SSRI TRANSFER TO THE FETAL COMPARTMENT 41
2.5 NOTES AND EXPECTED RESULTS 43
2.5.1 EX VIVO PERFUSION OF THE MOUSE PLACENTA FOR
BIDIRECTIONAL DRUG TRANSPORT STUDIES 43
2.6 CONCLUSION 47
CHAPTER 3: MATERNAL PHARMACOKINETICS AND FETAL DISPOSITION
OF (±)-CITALOPRAM DURING MOUSE PREGNANCY 48
CHAPTER 3 POINTS OF INTEREST 49
3.1 ABSTRACT 50
3.2 INTRODUCTION 51
3.3 RESULTS 53
3.3.1 PREGNANCY AFFECTS THE DISPOSITION AND PHARMACOKINETICS
OF CIT 53
3.3.2 DOSE AFFECTS CIT DISTRIBUTION AND CLEARANCE DURING
PREGNANCY 56
3.3.3 PREGNANCY STAGE AFFECTS DISPOSITION AND
PHARMACOKINETICS OF CIT 60
3.3.4 CIT RAPIDLY REACHES THE FETAL CIRCULATION AND BRAIN AFTER
MATERNAL ADMINISTRATION 63
3.3.5 FETAL CIT DISPOSITION IS INDEPENDENT OF PLACENTAL/FETAL
SERT EXPRESSION 65
3.3.6 FETAL EXPOSURE TO DCIT IS DEPENDENT ON PREGNANCY STAGE 67
3.3.7 GESTATIONAL AGE DEPENDENT CHANGES IN CIT METABOLISM 69
3.4 DISCUSSION 72
3.5 MATERIALS AND METHODS 79
3.6 SUPPORTING INFORMATION 89

VIII
CHAPTER 4: COMBINATORIAL EFFECTS OF PRENATAL EXPOSURES TO
MATERNAL STRESS AND ANTIDEPRESSANT (CITALOPRAM) ON FETAL
BRAIN SEROTONERGIC NEURODEVELOPMENT IN MICE 92
CHAPTER 4 POINTS OF INTEREST 93
4.1 INTRODUCTION 94
4.1.1 THE CASE OF ASD AND PRENATAL SSRI EXPOSURE 95
4.1.2 UNTANGLING THE EFFECTS OF DEPRESSION FROM SSRI EFFECTS 96
4.2 RESULTS 97
4.2.1 DEPRESSION- AND ANXIETY-LIKE BEHAVIORS FOLLOWING
GESTATIONAL CUS ARE REVERSED BY CIT ADMINISTRATION
DURING PREGNANCY 97
4.2.2 EXPOSURE TO MATERNAL CUS AND CIT DIFFERENTIALLY AFFECT
NEUROCHEMISTRY AND NEURAL ARCHITECTURE OF FETAL
FOREBRAIN 5-HT 101
4.2.3 DEVELOPMENTAL EFFECTS OF CUS EXPOSURE ON HINDBRAIN 5-HT
ARE REVERSED BY CIT ADMINISTRATION 104
4.2.4 PRELIMINARY DIFFUSION MAGNETIC RESONANCE IMAGING OF THE
EMBRYONIC MOUSE BRAIN FOLLOWING GESTATIONAL EXPOSURE
TO CUS AND CIT 107
4.3 DISCUSSION 109
4.4 MATERIALS AND METHODS 115
CHAPTER 5: CONCLUSION AND FUTURE DIRECTIONS 124
5.1 OVERVIEW 124
5.2 SUMMARY OF FINDINGS 126
5.3 FUTURE DIRECTIONS 131
REFERENCES 133

IX
LIST OF FIGURES

FIGURE 1.1 TREATMENT OF DEPRESSION DURING PREGNANCY WITH SSRIS
IS ASSOCIATED WITH VARYING PREGNANCY OUTCOMES 10
FIGURE 1.2 THE EFFECTS OF SSRIS ON FETAL BRAIN DEVELOPMENT MAY
BE INDUCED THROUGH DIFFERENT PATHWAYS 16
FIGURE 2.1 EFFECTS OF PRENATAL EXPOSURES TO STRESS AND SSRIS ON
THE FETAL BRAIN 29
FIGURE 2.2 SCHEMATICS OF THE EX VIVO DUAL PERFUSION SYSTEM 33
FIGURE 2.3 CHROMATOGRAPHIC CONDITIONS FOR THE DETECTION OF
SSRIS. 40
FIGURE 2.4 TRANSPLACENTAL TRANSFER OF CIT AND DCIT 44
FIGURE 2.5 IN VIVO MATERNAL-FETAL TRANSFER OF CIT AND DCIT 45
FIGURE 2.6 IMMUNOHISTOCHEMITRY OF THE FETAL BRAIN 46
FIGURE 3.1 CIT SERUM CONCENTRATION-TIME PROFILES IN NON-
PREGNANT AND PREGNANT MICE 55
FIGURE 3.2 MATERNAL CIT SERUM CONCENTRATION-TIME PROFILES IN
GD18 MICE 58
FIGURE 3.3 MATERNAL SERUM CIT CONCENTRATION-TIME PROFILES IN
PREGNANT MICE OF DIFFERENT GESTATIONAL STAGES 61
FIGURE 3.4 FETAL SERUM AND BRAIN CIT CONCENTRATION-TIME
PROFILES DURING GESTATION 64
FIGURE 3.5 FETAL AND PLACENTAL SERT EXPRESSION DOES NOT AFFECT
CIT CONCENTRATIONS 66
FIGURE 3.6 FETAL SERUM AND BRAIN DCIT CONCENTRATION-TIME
PROFILES DURING GESTATION 68
FIGURE 3.7 DETERMINATION OF DCIT SOURCES TO THE FETAL
COMPARTMENT 71
FIGURE S3.1 MATERNAL SERUM DCIT CONCENTRATION-TIME PROFILES
DURING PREGNANCY 89
FIGURE 4.1 GESTATIONAL CHRONIC UNPREDICTABLE STRESS
EXPERIMENTAL SCHEDULE 97
FIGURE 4.2 GESTATIONAL CUS INDUCES DEPRESSION- AND ANXIETY-LIKE
BEHAVIORS ON PREGNANT MICE 99

X
FIGURE 4.3 FETAL FOREBRAIN 5-HT CONCENTRATION AND
SEROTONERGIC OUTGROWTH ARE DIFFERENTIALLY
AFFECTED BY GESTATIONAL EXPOSURE TO CUS AND CIT 102
FIGURE 4.4 EFFECTS ON FETAL HINDBRAIN 5-HT FOLLOWING
GESTATIONAL EXPOSURE TO CUS AND CIT 106
FIGURE 4.5 DIFFUSION MAGNETIC RESONANCE IMAGING (DMRI) IN
THE E17 EMBRYONIC BRAIN FOLLOWING MATERNAL CUS
AND/OR CIT 108

XI
LIST OF TABLES

TABLE 3.1 COMPARISON OF CIT PHARMACOKINETICS IN NON-PREGNANT
AND PREGNANT GD18
FD
MICE 56
TABLE 3.2 EFFECT OF WEIGHT-ADJUSTED DOSE ON PHARMACOKINETIC
PARAMETERS OF CIT IN PREGNANT MICE 59
TABLE 3.3 GESTATIONAL AGE-DEPENDENT PHARMACOKINETICS OF CIT
AT GD14 AND GD18 IN MICE 62
TABLE S3.1 BIEXPONENTIAL EQUATION TERMS FOR MATERNAL CIT
CONCENTRATION-TIME PROFILE MODELS 90
TABLE S3.2 BODY WEIGHTS FOR ADULT CD-1 MICE (NON-PREGNANT) AND
PREGNANT DAMS AT GD14 AND GD18 91

XII
ABSTRACT

Despite extensive research efforts, the neurobiology and pathophysiology of
Major Depression Disorder (MDD) remains poorly understood. On a global scale,
mental disorders are among the leading causes of illness-induced disability
throughout adulthood, of which depression is the most prevalent, especially in
women of reproductive age (Global Burden of Disease Report & World Health
Organization 2013). Considering the chronic nature of depression and its
substantial impact, there is great need for improved understanding of the etiology
and pathophysiology of MDD.
Drugs like selective serotonin reuptake inhibitors (SSRIs) that are indicated
for the treatment of neuropsychiatric conditions such as depression and anxiety
target serotonergic mechanisms. During pregnancy, a time when the prevalence
rates of depression are ~15%, treatment with SSRIs is of major concern due to
the potential effects on the development of the serotonergic system. Since 5-HT
is implicated in multiple processes that orchestrate critical functions during brain
development, altering serotonin levels or serotonergic system development could
impact this delicate process. Consistent with a potential role in the fetal
programming of adult mental disorders, basic and epidemiological findings have
linked developmental disruption of 5-HT signaling to diverse functional disorders
in adulthood.

XIII
The work described in this dissertation aims to investigate how prenatal
exposures affect serotonin and fetal brain development. Of special interest is the
role of the placenta, which synthesizes 5-HT reaching the developing fetal
forebrain early in gestation. Additionally, it takes a focused look into the
pharmacokinetics of SSRIs in pregnancy, uncovering a pathway by which fetal
drug exposures are regulated during development. Finally, this thesis also begins
to distinguish between the independent effects induced by maternal stress from
those that are pharmacologically induced, a much needed approach to start to
address the clinical dilemma of SSRI treatment during pregnancy.

1
1

INTRODUCTION TO DEPRESSION AND
ANTIDEPRESSANT USE DURING PREGNANCY

Juan C Velasquez, Nick Goeden, and Alexandre Bonnin

Neuroscience Graduate Program and Zilkha Neurogenetic Institute, Department
of Cell and Neurobiology, Keck School of Medicine, University of Southern
California,
Los Angeles, CA 90089

This chapter has been expanded and modified from the 2013 publication:
Placental serotonin: implications for the developmental effects of SSRIs and
maternal depression. Frontiers in Cellular Neuroscience. 7:47.

INTRODUCTION TO DEPRESSION AND
ANTIDEPRESSANT USE DURING PREGNANCY

2
CHAPTER 1 POINTS OF INTEREST

Serotonin (5-HT) is implicated in multiple psychiatric disorders and is arguably
the most studied brain neurotransmitter. In spite of this notion, serotonergic
system development and the role of 5-HT in brain development and disease
remains a challenging question without definitive answers. While serotonin is a
focus of investigation in the study of depression, anxiety, and Autism Spectrum
Disorders (ASDs), it is often investigated in advanced disease stages and in the
context of the effects of selective serotonin reuptake inhibitor (SSRI)
antidepressants. Nevertheless, accumulating evidence demonstrates that 5-HT
plays multiple crucial roles during critical periods of brain development, which are
very distinct from 5-HT functions in the adult brain. This chapter lays the
contextual groundwork for this dissertation, reviewing the multiple, diverse roles
of 5-HT in fine-tuning fetal brain development and presenting the idea that
affecting this delicate balance has long-term consequences throughout the
lifespan. Some highlights presented include:
§ The placenta is the primary source of 5-HT for the forebrain in early
gestation
§ The placenta as a link between early genetic and environmental
perturbations that affect brain development via 5-HT output
§ SSRI treatment of maternal depression during pregnancy may target the
placenta, affect its physiology, and ultimately impact fetal health and
development
INTRODUCTION TO DEPRESSION AND
ANTIDEPRESSANT USE DURING PREGNANCY

3
1.1 ABSTRACT

In addition to its role in the pathophysiology of numerous psychiatric disorders,
increasing evidence points to serotonin (5-HT) as a crucial molecule for the
modulation of neurodevelopmental processes. Recent evidence indicates that
the placenta is involved in the synthesis of 5-HT from maternally derived
tryptophan (TRP). This gives rise to the possibility that genetic and environmental
perturbations directly affecting placental TRP metabolism may lead to abnormal
brain circuit wiring in the developing embryo, and therefore contribute to the
developmental origin of psychiatric disorders. In this review, we discuss how
perturbations of the placental TRP metabolic pathway may lead to abnormal
brain development and function throughout life. Of particular interest is prenatal
exposure to maternal depression and antidepressants, both known to alter fetal
development. We review existing evidence on how antidepressants can alter
placental physiology in its key function of maintaining fetal homeostasis and have
long-term effects on fetal forebrain development.
INTRODUCTION TO DEPRESSION AND
ANTIDEPRESSANT USE DURING PREGNANCY

4
1.2 INTRODUCTION

There is a wealth of evidence suggesting that serotonin (5-HT) plays a critical
role in many neurodevelopmental processes. Basic and epidemiological studies
link disruption of the 5-HT pathway to a host of developmental and functional
disorders, yet direct evidence of the molecular mechanisms underlying these
perturbations remains lacking, especially in humans. Studies in animal models
have indicated that 5-HT is a key modulator of neuronal cell proliferation,
migration, and brain wiring during fetal and early postnatal development (Brezun
and Daszuta, 1999, 2000; Azmitia, 2001; Kindt et al., 2002; Banasr et al., 2004;
Bonnin et al., 2007; Brezun and Daszuta, 2008). Furthermore, genetic and
environmental disruption of 5-HT receptor function during critical periods of fetal
brain development in mice lead to behavioral abnormalities throughout life, such
as adult anxiety disorders (Gaspar et al., 2003; Holmes et al., 2003a, 2003b;
Ansorge et al., 2004; Nordquist and Oreland, 2010; Morelli et al., 2011; Garbett
et al., 2012; Malkova et al., 2012). Interestingly however, there is sparse
evidence of specific associations between 5-HT receptor gene mutation or
dysfunction and mental illness in humans (Gingrich and Hen, 2001; Gaspar et al.,
2003; Segman et al., 2003).
Generally weak phenotypes in single receptor knockout mice and the
existence of 15 different receptor subtypes for 5-HT suggest that genetic
alteration of one specific subtype may be compensated for by the presence of
other pharmacologically and functionally similar receptors (e.g. 5-HT1B and
1.2 INTRODUCTION

5
5-HT1D receptors; see Van Kleef et al., 2012). Basic studies were able to alter
function of several receptors simultaneously during restricted, critical time
periods, thus potentially preventing compensatory signaling through other
receptors and leading to clear phenotypes (Ansorge et al., 2004; Bonnin et al.,
2007).
What is common to all receptor subtypes is their endogenous ligand, 5-HT.
Therefore, altered 5-HT tissue concentration may lead to generalized disruption
of signaling through more than one receptor type simultaneously. This possibility
is supported by dramatic effects from the pharmacological disruption of 5-HT
synthesis in early experiments, contrasting with mild effects of single receptor
knockout models (van Kleef et al., 2012).
Recent results show that 5-HT signaling, and thus extracellular levels of 5-HT,
play a crucial role in the thalamocortical wiring of the fetal forebrain by affecting
netrin-1 mediated axonal guidance (Bonnin et al., 2007, 2011). Thus, altered 5-
HT concentration in the fetal brain tissue, in addition to signal/receptor
interaction, may have far-reaching developmental and functional consequences
(Bonnin and Levitt, 2012). A recent study showed that the fetal forebrain
accumulates placentally derived serotonin during early pregnancy (Bonnin et al.,
2011), a period during which axons are experiencing active outgrowth and
guidance. The role of placental metabolism of 5-HT from maternally derived
TRP, its potential genetic and environmental perturbations, and their downstream
consequences are currently under intense investigation.
INTRODUCTION TO DEPRESSION AND
ANTIDEPRESSANT USE DURING PREGNANCY

6
1.3 SEROTONIN AND FETAL BRAIN DEVELOPMENT

Serotonergic neurons are one of the most ubiquitous circuits in the mammalian
brain, forming early during fetal development, and innervating essentially the
entire central nervous system. The early presence of 5-HT, as well as the
proposed maternal origin of 5-HT, has led to the hypothesis that 5-HT may be an
essential growth and regulatory factor for the fetal brain during critical periods of
development (Lauder and Krebs, 1976; Lidov and Molliver, 1982; Gaspar et al.,
2003; Bayard et al., 2007; Bonnin et al., 2011; Migliarini et al., 2012). This is
supported by the idea that disruption of the 5-HT signaling system is a key
developmental component for a number of neuropsychiatric disorders, such as
schizophrenia, affective disorders, anxiety, and autism (Chugani et al., 1999;
Whitaker-Azmitia, 2001; Sodhi and Sanders-Bush, 2004; Bonnin and Levitt,
2012). Genetic mouse models have shown that excess levels of 5-HT in the
brain, obtained by knocking out the transporter (SERT; Slc6a4) or monoamine
oxidase-A (MAO-A) genes, which are involved in the re-uptake and degradation
of 5-HT respectively, lead to abnormal development of topographically organized
whisker-barrel fields in the somatosensory cortex (Cases et al., 1996; Persico et
al., 2001). Furthermore, recent studies have shown that increased activity of the
serotonergic pathway may lead to abnormal cortical development and neuronal
migration (Janusonis et al., 2004; Riccio et al., 2009). On the other hand, 5-HT
depletion through the use of Pet1 knockout mice, in which there is a dramatic
reduction of serotonergic neuron number and differentiation, shows no
1.3 SEROTONIN AND FETAL BRAIN DEVELOPMENT

7
identifiable gross brain malformations, despite evidence of later behavioral and
functional deficits (Hendricks et al., 2003; Liu et al., 2010). Similarly, targeted
inactivation of tryptophan hydroxylase 2 (Tph2), the rate-limiting enzyme for the
synthesis of 5-HT specifically in the brain, in the mouse model has been
demonstrated to produce behavioral and functional deficits. However, lack of 5-
HT did not lead to obvious cellular or histological abnormalities in the brain
(Savelieva et al., 2008; Alenina et al., 2009; Yadav et al., 2009). Nevertheless,
the more recent analysis of a knock-in mouse line, in which the brain-specific
Tph2 gene was replaced by an eGFP reporter, showed significant abnormalities
in serotonergic innervation in several regions of the rostral brain (Migliarini et al.,
2012). Combined, these data suggest that specific circuits are finely tuned to 5-
HT during their initial formation, including the serotonergic system itself. The next
logical question is to determine if, and how, 5-HT signaling during development is
impacted by genetic and environmental perturbations shown to be associated
with increased risk of neuropsychiatric disorders.
Recent work suggests that the maternal and placental source of 5-HT may be
a critical link between early genetic and environmental perturbations and their
impact on fetal brain development. Consequently, exposure to pharmacological
or environmental insults, combined with genetic factors that disrupt maternal or
placentally derived 5-HT may have profound and long-lasting consequences on
the developing brain, leading to a host of neuropsychiatric disorders thought to
have developmental origins.
INTRODUCTION TO DEPRESSION AND
ANTIDEPRESSANT USE DURING PREGNANCY

8
In the next section, we discuss how particular environmental and
pharmacological insults such as exposure to maternal depression and
antidepressants during pregnancy may impact fetal brain development, taking
into account the potential effects on the maternal-fetal interface function.

1.4 PRENATAL EXPOSURE S AND LONG -TERM CONSEQUENCES

Major Depression Disorder (MDD) is a devastating mood disorder that
indiscriminately affects individuals of all backgrounds and ages, and is common
even in women during gestation. In fact, the prevalence of MDD is about 15%
during pregnancy, and Selective Serotonin Reuptake Inhibitors (SSRIs) are the
primary pharmacologic intervention (Oberlander et al., 2006). Despite an unclear
safety profile and a lack of well-controlled safety studies, an estimated 13% of
pregnant women are prescribed an SSRI antidepressant during all or part of their
pregnancy (Cooper et al., 2007). This common off-label use is warranted for its
beneficial effects of improving maternal mood and relieving symptoms of
depression, which presumably lead to better pregnancy outcomes. Due to their
high use and unknown safety, there is high surveillance of SSRIs by the U.S.
Food and Drug Administration, which has placed some SSRIs in Pregnancy
Category D, indicating demonstrated risks to the fetus (Greene, 2007).
Recent epidemiological studies suggest that fetal exposure to maternal SSRI
therapy is implicated in disturbing several physiological and cognitive domains
during fetal development. Their prescribed use is associated with increased
1. 4 PR ENATAL EXPOSURES AND LONG TERM CONSEQUENCES

9
prevalence of preterm delivery, intrauterine growth restriction, and
neurobehavioral disturbances in infants (Oberlander et al., 2009).

Additionally,
fetal SSRI exposure has been shown to increase risks of Postnatal Adaptation
Syndrome, low Apgar scores, Persistent Pulmonary Hypertension of the
Newborn, long term changes in cardiac morphology and physiology,
gastrointestinal abnormalities, Autism Spectrum Disorders, and postnatal
language learning deficits in humans (Fig. 1.1)

(Cohen et al., 2000; Simon, 2002;
Laine et al., 2003; Källén, 2004; Chambers et al., 2006; Levinson-Castiel et al.,
2006; Oberlander et al., 2006; Louik et al., 2007; Talge et al., 2007; Cooper et
al., 2007; Calderon-Margalit et al., 2009; Lund et al., 2009; Merlob et al., 2009;
Pedersen et al., 2009, 2010; Reis and Källén, 2010; Hadjikhani, 2010a; Kornum
et al., 2010; Croen et al., 2011b; Nijenhuis et al., 2012a, 2012b; Nordeng et al.,
2012; Weikum et al., 2012; Yonkers et al., 2012a; Haskell et al., 2012; Jimenez-
Solem et al., 2012).
Leaving maternal MDD untreated to avoid the potential teratogenicity of
SSRIs also poses significant risks. The anguish and psychological distress
accompanied by untreated MDD induces considerable maternal stress, one of
the earliest adverse experiences with long-term effects on the offspring. Several
animal and human studies show that maternal stress or depression disrupt fetal
neurobehavioral development and affect cognitive, emotional and behavioral
outcomes throughout childhood (Peters, 1990; Hayashi et al., 1998; Talge et al.,
2007; Homberg et al., 2010).
INTRODUCTION TO DEPRESSION AND
ANTIDEPRESSANT USE DURING PREGNANCY

10
Figure 1.1 Treatment of depression during pregnancy with SSRIs is associated
with varying pregnancy outcomes
(A) While every gestational stage of SSRI exposure has been implicated in increased
risks for cognitive, physiological, or developmental teratogenicity, the period of
exposure is an important factor that appears to influence clinical outcomes in the
offspring. We limited this list to outcomes that have been the focus of several
epidemiological studies in recent years and for which differential exposure data during
pregnancy was available. (B) Untreated maternal depression and stress have been
associated with several risks that affect cognitive and developmental outcomes. While
associations are not generally correlated to specific trimesters, exposure to untreated
maternal depression or stress during pregnancy pose adverse risks to fetal health and
development.Study Selection and Data Extraction Studies were selected if they had
clearly identified maternal SSRI exposure for specific trimesters of pregnancy and
assessed neonatal outcomes. Epidemiological studies that included medium-to-large
number samples exposed to different SSRI drugs were selected. Direct comparison of
absolute odds ratio values across these studies is not possible due to varying specific
study designs, adjustments for level of maternal depression and various
sociodemographic and lifestyle factors, drug dosages, length of exposure, and SSRI
treatment options. *PPHN, Persistent pulmonary hypertension of the newborn
1. 4 PR ENATAL EXPOSURES AND LONG TERM CONSEQUENCES

11
Children exposed to the stress induced by depressed mothers are also at
increased risk of developmental delay, impaired language development, and
even low IQ scores

(Fig.1.1) (Deave et al., 2008; Paulson et al., 2009). The
impact of maternal depression on newborns has effects that last beyond infancy,
as one-third of school-aged children of depressed mothers suffer from
depression and anxiety disorders (Pilowsky et al., 2006). Beyond childhood,
animal studies have shown that neonatal SSRI exposure suppresses adult
serotonergic signaling and elicits depressive- and anxiety-like behaviors in
adulthood (Ansorge et al., 2008a; Shanahan et al., 2009).
Maternal depressive states and prenatal exposure to SSRIs both alter fetal
health. For the developing fetus, associated risks stem from both the untreated
illness and the treatment itself, underscoring a therapeutic risk-benefit dilemma:
SSRI treatments that safeguard maternal health have adverse effects on the
developing fetus, but leaving maternal depression untreated also poses various
significant, adverse risks.
Several perspectives have been offered to account for how some psychiatric
disorders may arise from the disruption of particular neurotransmitter systems
during development. Disruption during sensitive developmental periods may
have lasting effects expressed during adulthood, and since 5-HT signaling
participates in several developmental programs (see above), dysfunction of the
5-HT system may be implicated in the etiology of several mental disorders in
humans, particularly in MDD. Genetic studies in mice show that transient
INTRODUCTION TO DEPRESSION AND
ANTIDEPRESSANT USE DURING PREGNANCY

12
developmental disruption the 5-HT system by exposure to SSRIs results in long-
term behavioral abnormalities and increased anxiety in adult offspring (Ansorge
et al., 2004; Maciag et al., 2006; Ansorge et al., 2008; Oberlander et al., 2008).
Not only does neonatal SSRI exposure reduce serotonergic signaling, but also
elicits a down regulation in midbrain expression of Tph2, an essential enzyme in
the serotonin synthesis pathway (Maciag et al., 2006).
As mentioned above, studies in animal models point to evidence that 5-HT
influences mammalian nervous system development. Disruption of 5-HT
signaling has several important implications, namely in the modulation of axonal
guidance mechanisms that establish precise fetal brain circuits (Gross et al.,
2002a; Bonnin et al., 2006a, 2007b). Because embryonic thalamocortical axons
(TCAs) express SERT and accumulate 5-HT, serotonin is able to shape the
outgrowth and synaptic connectivity of their projections (Bonnin et al., 2012).
SSRIs target and block SERT with high affinity, and have been shown to directly
affect serotonergic modulation of TCA responses to the guidance cue netrin-1 in
vitro. Presence of the SSRI Citalopram (racemic mixture of (R)- and (S)-
enantiomers) switched TCA response to attractive or repulsive guidance cues by
impacting the direction of their projections (Bonnin et al., 2012). Moreover, mice
with genetically disrupted SERT function, which may serve as a model for
chronic SSRI exposure, display changes in neuronal cytoarchitecture, 5-HT
function and neurobehaviors, all components that have developmental origins
(Oberlander et al., 2009). Genetic studies in mice show that disruption of 5-HT
1. 5 IMPACT OF SSRIS ON FETAL DEVELOPMENT

13
receptors during a restricted period of pre- and postnatal development results in
long-term behavioral abnormalities (Gross et al., 2002a). Taken together, these
results suggest that SSRIs could induce topographical shifts in important circuits
of the fetal brain, thus constituting a possible mechanism that gives rise to certain
mental illnesses by altering circuit-formation and ultimately, proper brain function
later in life.

1.5 IMPACT OF SSRIS ON FETAL DEVELOPMENT MAY
DEPEND ON ROUTES OF EXPOSURE DURING PREGNANCY

The placenta is essential for ensuring the growth and survival of the fetus during
development. Not only does it support fetal homeostatic functions, but also
serves as the essential source of 5-HT for the fetal forebrain during a transient,
critical period of development (Bonnin et al., 2011; Bonnin and Levitt, 2012). The
placenta is able to synthesize 5-HT from a maternal TRP precursor in both mice
and humans (Bonnin et al., 2011; Bonnin and Levitt, 2012; Goeden and Bonnin,
2013). This exogenous source of 5-HT is available to the fetal brain during
developmental milestones including cortical neurogenesis, cell migration, and
circuit formation (Bonnin et al., 2011). Therefore, proper placental function during
gestation may be essential for the 5-HT modulation of neurodevelopment.
The placenta may play a major role between SSRIs exposure and their
associated teratogenicity during gestation. Since the fetal brain acquires
placenta-derived 5-HT during a critical period of widespread axonal outgrowth,
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14
the effects of SSRIs on fetal brain development may be through an indirect
pathway that affects proper placental physiology, resulting in indirect,
downstream effects on the fetus. Although it is not clear whether SSRI exposure
induces physiological changes in the placenta, its high expression of SERT
support the notion that SSRIs would retain their high binding affinity in this organ
(Ganapathy et al., 1993; Yavarone et al., 1993; Shearman et al., 1998; Verhaagh
et al., 2001). If blocking SERT function alters placental 5-HT synthesis and/or
transport to the fetus, or maternal 5-HT degradation, SSRI treatments could be
teratogenic primarily by altering placental physiology. The placenta’s key function
of maintaining fetal homeostasis may thus be compromised and have long-term
effects on fetal forebrain development.
Alternatively, SSRIs may be able to readily cross the placenta and enter the
fetal circulation, where they could directly target the developing brain’s
serotonergic system. While there is some evidence of SSRIs crossing the
placenta, studies have focused on umbilical cord concentrations at birth in
humans (Hostetter et al., 2000a; Hendrick, 2003; Sit et al., 2011). Several
commonly used SSRIs such as Citalopram, Fluoxetine, and Paroxetine were
shown to cross the placental barrier at term, with various efficiencies (e.g. mean
ratios of umbilical cord to maternal serum concentrations ranged from 0.29 to
0.89)

(Hendrick, 2003). These studies give a snapshot of maternal-fetal SSRI
transplacental transport at birth; however, there is no data earlier in gestation,
particularly when the fetal brain may be most susceptible to disruptions of 5-HT
1. 5 IMPACT OF SSRIS ON FETAL DEVELOPMENT

15
signaling. Such data is difficult to obtain in humans, rendering studies in animal
models as crucial and necessary to providing key insights.
The impact of SSRIs on fetal brain development may therefore result from
direct actions on the fetal brain, indirect actions on placental or maternal
physiology or, more likely, a combination of all these routes (Fig. 1.2). Ongoing
efforts to measure transplacental transfer and effects on placental physiology of
SSRIs throughout the course of pregnancy in mice, and to determine the drugs
biodistribution in the fetus, will help determining precisely how they affect fetal
brain development.
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16

Figure 1.2 The effects of SSRIs on fetal brain development may be
induced through different pathways.
Direct (A) or indirect pathways may affect placental (B), maternal (C), or
both maternal and placental physiology (D), ultimately resulting in
downstream effects on the fetus. Direct effects (A) suggest that SSRIs
readily cross the placenta and enter the fetal circulation, where they
would directly target the developing brain’s serotonergic system.
Alternatively, physiological changes in the placenta (B), or delivery of
maternal factors essential for the developing fetal brain (C) may be
affected through indirect pathways. The combination of both direct and
indirect pathways inducing adverse effects on the fetal brain may also be
possible (D). Under the influence of varying maternal, fetal and placental
(maternal-fetal combination) genetic susceptibilities (DNA double helix
symbol), the effects of SSRI exposure at different pregnancy stages may
lead to diverse developmental outcomes.
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17
1.6 SEROTONIN AND THE NEURODEVELOPMENTAL
PROGRAMMING OF MENTAL DISEASES

Transient disruption of essential signaling events during critical developmental
periods may have lasting effects that are expressed throughout life. The
serotonergic system steers neurodevelopment through the key modulation of
neurogenesis, cell migration, and brain wiring that give rise to proper brain
function. With a diversity of molecular targets on which to focus, it makes sense
that perturbations of 5-HT signaling have been implicated in the pathogenesis of
diverse neurodevelopmental disorders. The perturbations of the 5-HT
neurotransmitter system during development, whether directly on the fetal brain
or on its placental modulation during early gestation, may have long lasting
developmental and physiological consequences. Risk factors, both genetic and
environmental, that alter 5-HT concentration in the fetal brain tissue may thus
ultimately pose far-reaching functional consequences throughout life.
Fetal exposures to SSRIs and maternal stress induced by MDD are early
exposures that have been associated with various diseases affecting
physiological and cognitive domains. The heterogeneity and diversity of different
disease outcomes is informed by the length and developmental period of adverse
exposures, in addition to fetal genetic susceptibilities. Together with the altered
fetal brain 5-HT signaling caused by SSRI exposure in different stages, the
influence of maternal, fetal and placental (maternal-fetal; see Fig.1.2) genetics
could possibly lead to different disease states. The manifestation of several
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18
mental disorders associated with serotonin dysfunction, namely MDD, ASD, and
other psychiatric illnesses may thus require multiple events of environmental,
genetic, and their interactions, to occur.
While the associated risks from fetal SSRI exposure continue to be
elucidated, the mechanisms of 5-HT neurodevelopmental disruptions, and how
they ultimately lead to adult-onset disorders need further study. There is also a
clinical demand for effective and safe treatment of maternal MDD, taking into
consideration the effects of drug therapy on the safety of the developing fetus.

19
2

FROM MOTHER TO FETUS: A
METHODOLOGICAL APPROACH TO STUDY
PLACENTAL SSRI TRANSFER EFFECTS

Juan C Velasquez and Alexandre Bonnin

Neuroscience Graduate Program and Zilkha Neurogenetic Institute, Department
of Cell and Neurobiology, Keck School of Medicine, University of Southern
California,
Los Angeles, CA 90089

Published 2015 in Walker ,DW (Ed.), Prenatal and Postnatal Determinants of
Development. New York: Humana Press, pp. 245-262.

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CHAPTER 2 POINTS OF INTEREST

As discussed in Chapter 1, several neurodevelopmental processes are
modulated by 5-HT, which has a placental origin in early pregnancy. Investigating
placental perturbations with a focus on how its physiology is affected is an
approach that can be used to study the downstream structural and functional
effects on the development of the fetal brain. The idea that disorders manifested
in postnatal and adult life may have origins during prenatal development
implicates placental function as a key player. This chapter describes a careful
technical approach that details innovative methods to study placental physiology
and its role in development. Applying the study of transplacental transfer kinetics
of SSRI antidepressants and their potential impact on fetal brain 5-HT as an
example, we provide theoretical background and detailed experimental
approaches that can be customized by other investigators for different studies.
Highlighted are the following techniques:
§ Ex vivo dual perfusion of the mouse placenta
§ Designing in vivo studies of maternal to fetal SSRI transfer
§ High Pressure Liquid Chromatography for the determination of SSRI drug
concentrations and biogenic monoamine analyses in biological samples
§ Immunohistochemistry of fetal brains to study serotonergic system
development

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21
2 . 1 ABSTRAC T

A host of neurodevelopmental processes are modulated by serotonin (5-HT), a
molecule also implicated in the etiology of diverse psychiatric disorders. Prenatal
exposures that affect serotonergic signaling and the developing 5-HT system are
increasingly associated with multiple long-term repercussions for the offspring.
Both maternal depression and antidepressant treatments have been shown to
affect fetal neurodevelopment during pregnancy, possibly through alterations of
5-HT levels that are otherwise precisely set by placental and endogenous
sources. The result of such dysregulation impacts a variety of critical signaling
pathways, and eventually leads to long-term effects on postnatal function. This
chapter provides investigators with details of recently developed methods that
can be applied to the study of how maternal-fetal transfer of therapeutic drugs,
such as selective serotonin reuptake inhibitors (SSRIs), cross the placenta and
impact fetal brain circuit.
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2.2 INTRODUCTION

Serotonergic signal transduction throughout the nervous system involves 17
different serotonin (5-hydroxytryptamine; 5-HT) receptors subtypes, each unique
in cellular and molecular aspects, tissue distribution, and gene expression (Anon,
2002; Hoyer et al., 2002). Despite innate differences within the serotonergic
receptor family, they all have 5-HT as their ligand in common. The structural and
functional features of the serotonergic system, namely its abundant and
widespread anatomical projections, along with diverse receptor subtypes, provide
great insight into how 5-HT is involved in almost every imaginable
developmental, physiological, and behavioral process throughout life. It follows
that altered 5-HT tissue concentration may potentially lead to generalized
aberrant signaling through more than one receptor subtype simultaneously,
having widespread effects across several domains.
Many drugs indicated for the treatment of symptoms observed in
neuropsychiatric conditions such as major depression, anxiety, schizophrenia
and obsessive-compulsive disorder target serotonergic mechanisms (Gorman
and Kent, 1999; Hu et al., 2006; Stein et al., 2006; Blasi et al., 2013). This befits
the notion that 5-HT is implicated in the regulation of multiple processes
orchestrating cognitive, behavioral, and physiological functions. Most
interestingly, before its role in adult neurotransmission, 5-HT signaling plays
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23
critical functions during brain development; an idea that stems from the early
appearance of serotonergic neurons, axons and receptors in the brain (Lidov and
Molliver, 1982; Lebrand et al., 1996a, 1998; Brüning and Liangos, 1997; Brüning
et al., 1997; Gaspar et al., 2003). Consistent with a potential role in the fetal
programming of adult mental disorders, basic and epidemiological findings have
linked developmental disruption of 5-HT signaling to diverse functional disorders
in adulthood (Chugani et al., 1999b; Whitaker-Azmitia, 2001b; Sodhi and
Sanders-Bush, 2004b; Bonnin and Levitt, 2012a; Harrington et al., 2013, 2014;
Velasquez et al., 2013). In particular, animal studies have shown that pre- and
early postnatal exposure to selective serotonin reuptake inhibitors (SSRIs), which
inhibit the 5-HT transporter (SERT; Slc6a4) activity and reuptake of 5-HT in
neurons and other cell types, induces increased anxious behaviors much later in
the adult offspring (Gaspar et al., 2003; Holmes et al., 2003a, 2003b; Ansorge et
al., 2004a; Nordquist and Oreland, 2010; Malkova et al., 2012). As discussed in
the next sections, the molecular mechanisms by which this early developmental
exposure has long-term consequences on adult brain function remain largely
unknown. As more mechanistic influences of 5-HT signaling on various aspects
of fetal brain development and their long-term consequences are being
uncovered, this signaling pathway will undoubtedly become a central tenet of the
developmental programming of adult mental disorders (Bonnin and Levitt, 2011;
Ganu et al., 2012; Oberlander, 2012).
2.2 INTRODUCTION

24
This chapter also provides brief discussions on the development of the
serotonergic system and its relevance to the fetal programming of adult mental
disorders. Of particular interest is prenatal exposure to maternal depression and
antidepressants, both suggested to alter fetal brain development. Additionally, it
outlines innovative techniques that can be applied to the study of gestational
exposure to therapeutic drugs such as SSRIs on placental physiology and fetal
brain development.

2 .2.1 SEROTONIN AND FETAL BRAIN DEVELOP M ENT

Serotonergic development is a complex, multi-organ process with specific spatial
and temporal patterns of receptors expression, which has emerged as a powerful
influence on the causality of several neuropsychiatric disorders (Bonnin et al.,
2006b; Bonnin and Levitt, 2012a; Velasquez et al., 2013). This focus has
increased further in light of the early presence of 5-HT and the recent discovery
of the placental origin of 5-HT in the fetal brain (Bonnin et al., 2011; Bonnin and
Levitt, 2012a). Serotonergic neurons generate one of the most ubiquitous circuits
in the mammalian brain. 5-HT cell bodies emerge early in fetal development
(~embryonic day (E)10 in mice) and cluster in the raphe nuclei; over the following
days of murine fetal development, 5-HT neurons rapidly form vast axonal
projections that essentially innervate all regions of the neural axis in a caudal-to-
rostral gradient (Pattyn et al., 2004; Hawthorne et al., 2010).

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Intriguingly, 5-HT itself and several of its receptors are already present in the
rostral part of the fetal forebrain before a single 5-HT axon has reached the area
(prior to E14.5) (Bonnin et al., 2006b, 2007a; van Kleef et al., 2012). This led to
the discovery that the fetal forebrain accumulates placentally synthesized 5-HT
during early pregnancy (Bonnin et al., 2011; Bonnin and Levitt, 2012a). Thus, the
placenta acts as an exogenous source of 5-HT for the forebrain at a time when
the endogenous 5-HT system has yet to fully mature.

2.2.2 THE POTENTIAL IMPACT OF PRENATAL EXPOSURES

5-HT is a key trophic factor during fetal and early postnatal development,
modulating critical histogenic processes such as neuronal cell proliferation,
migration, and brain circuit wiring (Brezun and Daszuta, 1999, 2000, 2008;
Whitaker-Azmitia, 2001b; Kindt et al., 2002; Banasr et al., 2004; Bonnin et al.,
2007a). Transient genetic (e.g. 5-HT1A receptor knockout) and pharmacological
(e.g. exposure to SSRIs) disruption of 5-HT signaling during critical periods of
fetal and early postnatal brain development lead to long-term behavioral
abnormalities, such as increased anxiety in adulthood (Gross et al., 2002b;
Ansorge et al., 2008a; Oberlander et al., 2009). These far-reaching functional
consequences may arise due to abnormal serotonergic system development
leading to, or originating from, altered 5-HT concentration and aberrant
ligand/receptor interactions in fetal brain tissue. Thus, genetic and environmental
factors that alter levels of 5-HT during pregnancy may lead to abnormal signaling
2.2 INTRODUCTION

26
in the developing fetal brain, a potential mechanism for the developmental origins
of adult mental disorders. Such is a possibility for about 15% of pregnant women
that are diagnosed with Major Depression Disorder (MDD) during gestation, a
leading cause of disability worldwide (Oberlander et al., 2006; Suri et al., 2007;
Olivier et al., 2013). Despite an unclear safety profile, a lack of well-controlled
safety studies, and assignments to Pregnancy Categories C and D by the US
Food and Drug Administration, about 9% of pregnant women diagnosed with
MDD in the United States are prescribed a SSRI antidepressant during all or part
of their pregnancy, a figure that has been steadily on the rise (Cooper et al.,
2007; Bourke et al., 2014b).
In the adult brain, SSRIs inhibit SERT-mediated 5-HT reuptake activity,
resulting in increased 5-HT concentration at the synapse, which contributes to
relieving symptoms of maternal depression (Hiemke and Härtter, 2000). For the
fetus however, since SERT is expressed early in the brain (e.g. E12 in raphe
serotonergic neurons and E14 in thalamic neurons) (Lebrand et al., 1996a;
Narboux-Nême et al., 2008), early exposure to SSRIs is likely to alter overall fetal
tissue concentrations of 5-HT, resulting in broad effects on 5-HT signaling
(Brüning and Liangos, 1997; Brüning et al., 1997; Lebrand et al., 1998).
Developmental alterations of 5-HT signaling in vivo have been shown to affect
the formation of major axonal circuits in the fetal brain. For instance, the genetic
manipulation of two G
i
-protein coupled 5-HT receptors (htr1b and htr1d)
expression by in utero electroporation at E12.5, specifically in developing
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27
thalamic neurons, led to abnormal thalamocortical axon (TCA) pathway formation
four to six days later (Bonnin et al., 2007a). This was shown to arise from 5-HT
receptor-mediated changes in intracellular cAMP in thalamic neurons, which
modulates the response of growing TCAs to guidance cues during initial pathway
formation. Together with the finding that the commonly-prescribed SSRI
Citalopram directly affects the response of TCAs to guidance cues in vitro
independently of 5-HT signaling (Bonnin et al., 2012), these results suggest that
fetal brain exposure to SSRIs may directly or indirectly alter the formation of TCA
pathways. Thus, fetal brain exposure to maternally-ingested SSRIs may
potentially lead to functional alterations of the circuits that underlie social,
emotional, and cognitive higher functions in the offspring (Bonnin et al., 2012).
For the fetus, the maternal depressive state is one of the earliest adverse
experiences resulting in poor pregnancy outcomes and an induction of long-term
adverse effects (Bonari et al., 2004; Moses-Kolko et al., 2005; Oberlander et al.,
2006, 2009; Cooper et al., 2007; Deave et al., 2008; Davis and Sandman, 2012)
(Figure 2.1A). The dilemma arises when the both the pharmacological
intervention to alleviate the maternal condition and the illness itself pose serious
risks to fetal health, an unfavorable scenario in which a risk-benefit decision must
be made. Accumulating epidemiological studies show increased risks for a
variety of physiological, cognitive, and developmental disorders (extensively
reviewed here (Olivier et al., 2013; Velasquez et al., 2013)), including the recent
increased associated risk for Autism Spectrum Disorders (Croen et al., 2011b;
2.2 INTRODUCTION

28
Harrington et al., 2014). Although important, these studies cannot delineate
between the risks brought about by SSRIs from those attributed to the underlying
maternal depressive disorder (Figure 2.1A, B).
As with maternal depression, SSRI use during pregnancy has often been
associated with poor pregnancy outcomes (Simon, 2002; Källén, 2004; Lund et
al., 2009; Sit et al., 2011; Yonkers et al., 2012b). To explain how maternal SSRI
therapy may affect brain development in the offspring of mothers coping with
depression, some studies suggest that they cross the placenta into the fetal
circulation, although this evidence is based on drug measures taken from
umbilical cord blood at birth and transfer studies at term in the human perfused
placental cotyledon model (Hostetter et al., 2000a; Heikkine et al., 2002;
Hendrick, 2003; Sit et al., 2011). Like the fetus, the placenta in is a state of
developmental flux in both structure and function, and its progressive remodeling
has yet to be considered in the context of transplacental transport earlier in
gestation when highly sensitive developmental milestones are taking place
(Mikheev et al., 2008; Sitras et al., 2012; Uusküla et al., 2012). In any event,
once in the fetal compartment, SSRIs may directly target brain SERT and affect
baseline 5-HT levels and signaling, thereby impacting early (neuronal
differentiation) or late (axon growth and guidance) phases of brain development
(Figure 2.1C, D).
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29

Alternatively, SSRIs may impact placental physiology in such a way that the
effects indirectly impact the fetus. Since the fetal brain acquires placenta-derived
5-HT during a critical period of widespread axonal outgrowth, the effects of
SSRIs on fetal brain development may be through an indirect pathway that
Figure 2.1 Effects of Prenatal Exposures to Stress and SSRIs on the Fetal
Brain
Effects of SSRIs on fetal brain development may be through direct or indirect
pathways. (A) in untreated maternal depression, the fetal brain may be
continuously exposed to maternal stress effects. (B) SSRIs may relieve maternal
stress and indirectly prevent its effects on fetal brain development. At the same
time, SSRIs may transfer through the placenta, reach the fetal brain, and alter (C)
early (e.g. neuronal differentiation; or (D) late (e.g. axon growth and guidance)
phases of fetal brain development. SSRIs = Selective Serotonin Reuptake
Inhibitors, ctx = cortex, DR = Dorsal Raphe, DT = Dorsal Thalamus
2.2 INTRODUCTION

30
affects placental tryptophan metabolism to 5-HT, resulting in downstream effects
on the fetus. Although it is not clear whether SSRI exposure induces
physiological changes in the placenta, its high expression of SERT support the
notion that SSRIs would have high binding affinity in this organ (Ganapathy et al.,
1993; Yavarone et al., 1993; Shearman, 1998; Verhaagh et al., 2001). If blocking
SERT function alters placental 5-HT synthesis and/or transport to the fetus, the
placenta's key function of maintaining fetal homeostasis may be compromised by
SSRIs.
In summary, the impact of SSRIs on fetal brain development may result from
direct actions on the fetal brain, indirect actions on placental or maternal
physiology or, more likely, a combination of all these routes. Exposure to SSRIs
at a time surrounding critical developmental milestones may very well have
profound, long-lasting implications on offspring brain function; most importantly,
these effects have yet to be considered in the context of the underlying maternal
depression, which may significantly impact brain development and function if left
untreated. Thus, the question arises as to whether SSRIs cross the placenta,
and if so, to what extent do they access the fetal blood compartment and reach
the fetal brain at different stages of pregnancy? The next sections describe
innovative ex vivo and in vivo methods to explore these questions and to begin to
assess the mechanisms by which in utero exposure to antidepressants may alter
neurodevelopmental processes.

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31
2.3 MATERIALS, EQUIPMENT, AND SETUP

One of the major challenges of studying of how extrinsic factors impact
development during pregnancy is the physiologically dynamic state of pregnancy.
Developmental programs are precisely modulated both in time and space; a
particular developmental process may thus be affected at certain time points but
not others. A rigorous, multi-faceted set of techniques, all done at various
developmental time-points of interest, are necessary to gain meaningful insight
into gestational conditions. The ex vivo perfusion of the mouse placenta
procedure provides the framework for studying placental drug transport,
independently of maternal and fetal metabolism. In contrast, in vivo studies of
SSRI exposures are designed to quantify the pharmacokinetics of drug transfer
to the fetal compartment, and the influences of maternal and fetal metabolism on
these parameters. These observations can be correlated with
immunohistochemical analyses of fetal brain architecture. In following the
experimental protocols described below, any animal tissue should be collected in
compliance with legislative and institutional requirements.

2.3.1 EX VIVO PERFUSION OF THE MOUSE PLACENTA FOR
BIDIRECTIONAL DRUG TRANSPORT STUDIES
This procedure provides the framework for directly studying transplacental
passive/active molecular transport and the downstream consequences this might
have for the developing fetus. Ex vivo perfusion systems offer a reliable,
2.3 MATERIALS , EQUIPMENT , AND SETUP

32
reproducible method for studying acute physiological responses of an organ to
various environmental manipulations while maintaining the cellular organization,
compartmentalization and three-dimensional structure intact. The perfusion
procedure, which can be completed in 4–5-hours, allows for integrated,
physiological studies of de novo synthesis and metabolism and transport of
materials across the live mouse placenta.
The system can be used to measure the short-term maternal-fetal transfer
pharmacokinetics of individual SSRIs at different stages of gestation,
independent of maternal and fetal metabolism. Following microcannulations of
placental vasculature, perfusion solutions of known composition are infused into
the maternal and fetal sides of an isolated mouse placenta that is maintained in a
thermostated, oxygenated chamber (Figure 2.2). Peristaltic pumps keep
perfusion media flowing at steady, physiological rates uniquely adapted to the
maternal and fetal circulations. For maternal-fetal transfer, the fetal perfusion
output can be collected through the umbilical vein for analysis (Figure 2.2C).
The perfusion system theory and setup, along with a list of equipment,
materials, procedural notes, and troubleshooting guidelines, have been
extensively detailed in both a protocols publication (Goeden and Bonnin, 2013)
and a textbook chapter (Goeden and Bonnin, 2014). This chapter will thus focus
on customizing this technique for the study of transplacental transfer of SSRIs for
pharmacokinetic studies.
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Figure 2.2 Schematics of the ex vivo dual perfusion system
The artificial maternal circulation consists of a multi-channel, fast-switching peristaltic
perfusion system (pump #1) (A) connected to the uterine artery on the maternal side of
the placenta. The artificial fetal circulation consists of a single-channel peristaltic
perfusion system (pump #2) connected to the umbilical artery (B) and a multi-channel
negative-pressure peristaltic collection system (pump #3) connected to the umbilical
vein (C) on the fetal side of the placenta. SSRI = Selective Serotonin Reuptake
Inhibitor, HPLC-FLD = High Pressure Liquid Chromatography coupled with
Fluorescence Detection.
2.3 MATERIALS , EQUIPMENT , AND SETUP

34
2.3.1.1 REAGENTS
§ M199 medium without phenol red (Corning Cellgro, Cat. No. 90-050-PB)
§ Bovine Serum Albumin (AMRESCO, Cat. No. 0332)
§ Heparin (Sigma-Aldrich, Cat. No. H3149)
§ Glucose (BDH, Cat. No. BDH8005)
§ Dextran40 (TCI, Cat. No. D1448)
§ Sodium bicarbonate (BDH, Cat. No. BDH0280)
§ L-glutamine (Alfa Aesar, Cat. No. A14201)
§ Fast green dye (Haleco, Cat. No. 19143)
§ Phosphoric acid (EMD Millipore, Cat. No. 4809391000)
§ Citalopram hydrobromide (Tocris, Cat. No. 1427)
§ Desmethylcitalopram hydrobromide (Sigma-Aldrich, Cat. No. D-047)
§ Fluoxetine hydrochloride (Sigma-Aldrich, Cat. No. F132)
§ Norfluoxetine hydrochloride (Sigma-Aldrich, Cat. No. F133)
§ Paroxetine hydrochloride (Sigma-Aldrich, Cat. No. 1500218)
§ Antipyrine (Sigma-Aldrich, Cat. No. 10790)
§ Potassium phosphate monobasic (Sigma-Aldrich, Cat. No. P0662)
§ Acetonitrile, HPLC Grade (EMD Millipore, Cat. No. AX0145)
§ Perchloric acid (EMD Millipore, Cat. No. 1005171000)

2.3.1.2 EQUIPMENT
§ SpeedVac Concentrator (Thermo-Savant SPD1010, ThermoFisher
Scientific, Massachusetts, USA)
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35
§ High Pressure Liquid Chromatography System (Eicom 700, Eicom
Corpotation, Kyoto, Japan)
§ Fluorescence Detector (Shimadzu RF-20A-XS, Shimadzu, Kyoto, Japan)

2.3.2 EX VIVO PERFUSION OF THE MOUSE PLACENTA FOR
BIDIRECTIONAL DRUG TRANSPORT STUDIES
SSRIs given during pregnancy may reach the fetal brain and alter various critical
phases of fetal brain development. Direct drug action on the fetal brain requires
that SSRIs cross the maternal-fetal placental barrier. In this context, the effect of
SSRIs on brain development largely depends on the transfer of SSRIs to the fetal
compartment. In vivo maternal-fetal transfer measures following timed drug
exposures are useful to measure detailed profiles of maternal metabolism and
the disposition of the drug of interest and its metabolites to the fetus.

2.3.2.1 REAGENTS
§ Physiological saline (BD, Cat. No. 221819)
§ Citalopram hydrobromide (Tocris, Cat. No. 1427)
§ Desmethylcitalopram hydrobromide (Sigma-Aldrich, Cat. No. D-047)
§ Fluoxetine hydrochloride (Sigma-Aldrich, Cat. No. F132)
§ Norfluoxetine hydrochloride (Sigma-Aldrich, Cat. No. F133)
§ Paroxetine hydrochloride (Sigma-Aldrich, Cat. No. 1500218)
§ Antipyrine (Sigma-Aldrich, Cat. No. 10790)
§ Perchloric acid (EMD Millipore, Cat. No. 1005171000)
2.3 MATERIALS , EQUIPMENT , AND SETUP

36
§ Potassium phosphate monobasic (Sigma-Aldrich, Cat. No. P0662)
§ Acetonitrile, HPLC Grade (EMD Millipore, Cat. No. AX0145

2.3.2.2 EQUIPMENT
§ High Pressure Liquid Chromatography System (Eicom 700, Eicom
Corpotation, Kyoto, Japan)
§ Fluorescence Detector (Shimadzu RF-20A-XS, Shimadzu, Kyoto, Japan).

2.4 METHODS

2.4.1 EX VIVO PERFUSION OF THE MOUSE PLACENTA FOR
BIDIRECTIONAL DRUG TRANSPORT STUDIES
The placenta perfusion setup, method, and procedure should be followed
according to previous publications (Goeden and Bonnin, 2013, 2014). This
section focuses on the media preparation customized for the simulated maternal
and fetal blood supplies for transfer studies, in addition to instruction for perfusion
sample preparation and analysis using High Pressure Liquid Chromatography
coupled with Fluorescence Detection (HPLC-FLD).

FROM MOTHER TO FETUS : A METHODOLOGICAL APPROACH
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37
Maternal circulation solution
§ M199 Medium without phenol red
§ Bovine Serum Albumin (2.9g/dL)
§ Heparin (20 IU USP/mL)
§ Dextran40 (7.5g/L)
§ Glucose (1g/L)
§ Sodium bicarbonate (2.2g/L)
§ L-glutamine (100mg/L)
§ Fast-green dye (0.001%)
§ pH 7.3 with 1M phosphoric acid
Fetal circulation solution
§ M199 Medium without phenol red
§ Bovine Serum Albumin (2.9g/dL)
§ Heparin (20 IU USP/mL)
§ Dextran40 (30g/L)
§ Glucose (0.5g/L)
§ Sodium bicarbonate (2.2g/L)
§ L-glutamine (100mg/L)
§ Fast-green dye (0.001%)
§ pH 7.3 with 1M phosphoric acid
Pharmacological substances of interest may be added to either maternal or
fetal solutions above according to the directional transfer study of interest. Unlike
in vivo studies which would have to rely on the use of radioactively-labeled
molecules, the technique and detection method allow the versatility for maternal-
fetal or fetal-maternal transplacental transfer of native (unlabeled) compounds.
Antipyrine (1µg/mL), a freely diffusible substance across the placental barrier,
should also be included as a measure of perfusion efficiency and as an internal
positive control (Heikkinen et al., 2003; Mathiesen et al., 2010).

2.4.1.1 PERFUSION SAMPLE PREPARATION AND ANALYSIS
Once perfusion samples have been collected at the desired intervals, these can
be stored at -80ºC until time of analysis. Investigators should be aware of drug
2.4 METHODS

38
degradation resulting from freeze-thaw cycles and length of storage (which
should be measured independently). This section outlines sample preparation
procedure and HPLC-FLD chromatographic conditions.

Sample preparation
1. Thaw perfusion sample on ice.
2. Measure sample volume and add acetonitrile (1:1 v/v) to deproteinate the
sample.
3. Vortex sample and incubate at room temperature for 10-minutes.
4. Separate the precipitated proteins by centrifugation at 5,000-rpm for 10-
minutes.
5. Collect and measure the supernatant volume.
6. Evaporate sample in SpeedVac concentrator at room-temperature.
Evaporation time will vary depending on sample starting volume.
7. Using a 0.2M perchloric acid with 100μM EDTA-2Na solution, resuspend
evaporated sample in half of the supernatant volume (Step 5) following
acetonitrile extraction (this corresponds to the original sample volume
without a need for dilution adjustments or dilutions affecting limit of
detection).
8. Analyze sample by HPLC-FLD.
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39
9. Standard calibration curves can be made using stock compounds
appropriately diluted in a solution composed of 0.2M perchloric acid with
100μM EDTA-2Na.

HPLC-FLD chromatographic conditions
HPLC-FLD analysis can be performed using an Eicom 700 system coupled to a
Shimatzu RF-20AX fluorescence detector. An Eicompak SC-30DS C
18
reversed-
phase column packed with 3-μm silica particles (3.0 x 100 mm I.D.) is used as
the analytical column. A 10 μL aliquot of extracted sample is injected onto the
column and eluted with a mobile phase of 10 mM KH
2
PO
4
/Acetonitrile (3:1 v/v)
(pH= 4.0 with 1 M phosphoric acid), at a flow rate of 500 μL/min. Using the same
system, other analytic conditions can be customized for each SSRI, each
molecule having a specific chromatographic profile (Figure 2.3).

Determination of Antipyrine
The perfusion samples can be assessed for antipyrine concentration with the use
of a spectrophotometer. Since the perfusion samples consist of a modified cell
culture medium, several endogenous molecular species may generate
interfering, background measures. Therefore, to quantify maternal-fetal transfer
of antipyrine, fetal solution without antipyrine must be extracted and prepared as
above in the Sample preparation subsection. This solution should be used as a
blank and to prepare the calibration standard dilutions from stock antipyrine. The
2.4 METHODS

40
spectrophotometer detection wavelength should be set at 240 nm. This method
provides a quick, reliable assay of antipyrine quantification with a limit of
detection of ~10 ng/mL. If needed, higher sensitivity can be achieved with an
HPLC system coupled to ultraviolet (UV) detection (Menjoge et al., 2011).

Figure 2.3 Chromatographic Conditions for the Detection of SSRIs
Different drugs in the SSRI class can be detected by making minor modifications
to the detection wavelengths of the HPLC-FLD system and the acetonitrile
composition of the mobile phase. Each compound will have a specific
chromatographic signature varying in retention time and sensitivity. The
technique allows for highly sensitive assays that grant investigators great
versatility for quantifying different species of interest. SSRI = Selective Serotonin
Reuptake Inhibitor, NFLX = Norfluoxetine, FLX = Fluoxetine, PRX = Paroxetine,
CIT = Citalopram, DCIT = Desmethylcitalopram

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41
2.4.2 IN VIVO STUDIES OF SSRI TRANSFER TO THE FETAL
COMPARTMENT
Animal treatments
Working with a timed-pregnant dam at a developmental time of interest,
administer drug of interest at a dosage shown to provide efficacious therapeutic
effects in mice. For the SSRI Citalopram, our lab has performed intraperotineal
(i.p.) injections with 20 mg/kg of body weight resuspended and filter-sterilized in a
physiological saline volume of 0.01 mL/g of drug (Cryan et al., 2004; Crowley et
al., 2006). Additionally, we have also administered SSRIs orally via drinking
water. due to different pharmacokinetics and dynamics.

Dissections
Following a timed exposure to the drug, pregnant dams are anesthetized with a
lethal dose of isofluorane. Before performing a cervical dislocation and
caesarean section, the dam is assessed for loss of corneal reflex and lack of
responsiveness to toe pinch. A midline incision then is made through the skin
over the abdomen using sterile scissors. Using a second pair of sterile scissors
and forceps, the underlying muscle layer is then cut, exposing the uterine horns,
which are then carefully removed and placed in a sterile petri dish with cold
Phosphate Buffer Saline (PBS). As a secondary measure to ensure euthanasia,
the diaphragm of the dam is severed following removal of the uterus. A cardiac
puncture is performed with a 25 gage syringe needle to collect a maternal blood
sample for drug metabolism analysis (usually ~ 1 mL per dam). Using new sterile
2.5 NOTES AND EXPECTED RESULTS

42
instruments and under a dissection microscope, the fetuses are removed from
the uterine horns, placentas isolated and flash frozen in liquid nitrogen prior to
storage at -80ºC. The embryos are then decapitated and the fetal blood collected
through the exposed carotid and jugular vasculature using a pipette; the blood
from at least 10 embryos per dam is collected and pooled into a tube on ice. The
brains can then be dissected and flash-frozen before storing at -80ºC. Both the
maternal and the fetal blood should be left on ice for 30-minutes prior to
centrifuging at 2,000 x g for 10-minutes at 4ºC. The sera (supernatant) is then
collected and stored at -80ºC.

Sample preparation
1. Prepare extraction buffer, composed of 0.2M perchloric acid, 100μM
EDTA-2Na and 100ng/mL isoproterenol, to use as an internal standard.
2. Add 0.5 mL of ice-cold extraction buffer per 100 mg of tissue. For fetal
blood serum, add equal amounts of sample and extraction buffer (1:1 v/v).
Maternal blood serum requires a higher dilution with extraction buffer (1:3
v/v).
3. Tissue should be homogenized or sonicated until it is fully suspended in a
homogeneous solution. Time and sonication power will vary depending on
the age and tissue type, but consistency across similar samples must be
maintained.
4. Denature the protein by keeping the homogenate in an ice bath for 30 min
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43
5. Spin at 20,000xg for 15-min at 4°C
6. Remove and measure the supernatant volume, analyze by HPLC-FLD
using the chromatographic conditions outlined in section 3.1.2 above
7. Standard curves can be made using stock compounds appropriately
diluted in the extraction buffer solution (Step 1)
8. Quantify drug measures, adjust for sample dilutions and internal standard.
9. Measure protein concentration in each sample using a Bovine Serum
Albumin (BSA) assay.

2 . 5 NOTES AND EXPECTED RESULTS

2.5.1 EX VIVO PERFUSION OF THE MOUSE PLACENTA FOR
BIDIRECTIONAL DRUG TRANSPORT STUDIES
Analysis of perfusion samples can be used to study the pharmacokinetic profile
of bidirectional transfer of drugs across the placenta. Following HPLC-FLD
analysis, the transplacental transfer percentage (TPT) of each drug and
associated metabolite is calculated using the following equation: TPT = (C
f
x S
f
x
100) / (C
m
x S
m
); where C
f
is the concentration in fetal venous outflow, S
f
is the
fetal flow rate (3-5 µL/min – depending on age (Goeden and Bonnin, 2013)), C
m

is the SSRI concentration in maternal arterial inflow and S
m
is the maternal flow
rate (16-20 µL/min). The transplacental transfer index (TI) (i.e. the ratio of
transfer between SSRI and antipyrine - used as internal standard) is calculated
by dividing the TPT
(SSRI)
by the TPT
(antipyrine).
Based on preliminary data obtained
2.5 NOTES AND EXPECTED RESULTS

44
with the SSRI CIT ex vivo (Figure 2.4), we observe differential transfer between
the parent drug and its metabolite throughout the perfusion at E18. Ex vivo
maternal-fetal TPT measures can then be correlated to short-term in vivo
measures of fetal blood and brain tissue drug concentrations (see below) for the
estimation of drug metabolism and disposition to the fetal compartment.

Figure 2.4 Transplacental Transfer of CIT and DCIT.
An E18 CD-1 mouse placenta was perfused with 1500ng/mL of CIT and 500ng/mL
of DCIT through the maternal input side (uterine artery) for 120-min. The fetal
perfusion outputs were collected through the umbilical vein and analyzed for
quantification of CIT and DCIT. Transplacental transfer (TPT) ratios were measured
every 10 min for 120 min after a 30 min stabilization period (lower traces). Upper
traces: steady input of CIT and DCIT measured simultaneously through the output
side of the uterine artery; n=2 independent perfusions. CIT = Citalopram, DCIT =
Desmethylcitalopram.
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45
2 . 5 . 2 IN VIVO STUDIES OF SSRI TRANSFER TO THE FETAL COMPARTMENT
Measures of in vivo maternal-fetal transfer from our lab show that maternal CIT
and its metabolite DCIT reach the fetal blood stream and fetal brain within 15-min
following i.p. injection of 20 mg/kg CIT at E18 (Figure 2.5). This technique can
be applied to obtain a comprehensive pharmacokinetic profile of maternal
metabolism and transfer to the fetus across several time points. Furthermore, it
can be used to assess gestational differences in in terms of drug disposition to
the fetal compartment.

Figure 2.5 In Vivo Maternal-Fetal Transfer of CIT and DCIT
A CD-1 timed-pregnant mouse was injected i.p. with 20mg/kg of
Citalopram (CIT). After 30-minutes, maternal and fetal blood
serum along with placental and fetal brain tissue were harvested.
These samples were prepared for HPLC-FLD analysis for
quantification of CIT and its metabolite desmethylcitalopram
(DCIT). i.p. = intraperotenial, HPLC-FLD = High Pressure Liquid
Chromatography coupled with Fluorescence Detection.
2.5 NOTES AND EXPECTED RESULTS

46
While the determination of SSRI concentrations in the fetal blood and brain
are indications of in vivo exposures, the question of the biological impact that
different exposure paradigms and maternal treatment regimens have on fetal
development still remains. Examination of the fetal brain architecture is one way
to determine how SSRIs and maternal stress affect the development of the
serotonergic system and TCA formation. Immunohistochemical (IHC) imaging
studies (Figure 2.6) and quantification of immunofluorescence distribution allow
high-resolution visualization and accurate identification of 5-HT neurons (5-HT+)
and axons and TCAs (netrin-G1a+) throughout the rostro-caudal extent of the
Figure 2.6 Immunohistochemitry of the Fetal Brain
Antibody staining for 5-HT (red) and Netrin-G1a expressing thalamocortical
axons (TCA; green) of CD-1 fetal mouse brain at E18. After dissection, brains
were fixed overnight and dehydrated through sucrose gradients prior to
embedding. The fetal brain was then sliced in 20-μm sections and stained.
Netrin-G1a signal has been amplified with a Tyramide Signal Amplification (TSA)
kit. In the rostral forebrain, 5-HT-positive axons can be visualized in the medial
forebrain bundle (MFB). The path of thalamocortical axons from the thalamus
(Th), through the internal capsule (IC) to the cortex (Ctx) is clearly visible.
Relative immunofluorescence distribution analysis can be used to quantify the
effects of drug treatment on 5-HT and TCA axonal pathway formation.
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47
fetal brain (Bonnin et al., 2007a). These measures can be done in 20 µm-thick
coronal and sagittal sections encompassing the whole fetal brain, as previously
described (Eagleson et al., 2005; Bonnin et al., 2007a, 2011). Specific regions of
interest can be delimited for quantification: relevant structures include 1) from the
medial cortex to the claustrum (for netrin-G1a+ TCAs), and 2) from the
hypothalamus midline to the internal capsule, including the medial forebrain
bundle (for 5-HT+ axons) (Figure 2.6).

2.6 CONCLUSION

Transient disruptions of essential signaling events during critical developmental
periods may have lasting effects that are expressed throughout life. The
ubiquitous role of 5-HT signaling in brain development and adult brain function
befits that early perturbations of the serotonergic system are increasingly
implicated in the etiology of several neuropsychiatric conditions. In particular,
developmental perturbations of serotonergic signaling by in utero exposure to
maternal depression and SSRIs may affect the formation of functional
somatosensory circuits such as thalamocortical axon pathways. The techniques
described in this chapter were designed to provide direct functional correlation
between in utero exposure to therapeutic drugs such as SSRIs and
developmental perturbations resulting in abnormal formation of
serotonergic/thalamocortical axon pathways that may ultimately have far-
reaching functional consequences throughout life.

48
3

MATERNAL PHARMACOKINETICS AND FETAL
DISPOSITION OF (±) -CITALOPRAM DURING
MOUSE PREGNANCY

Juan C Velasquez, Nick Goeden, Skyla M. Herod*, and Alexandre Bonnin

Neuroscience Graduate Program and Zilkha Neurogenetic Institute, Department
of Cell and Neurobiology, Keck School of Medicine, University of Southern
California, Los Angeles, CA 90089;
*
Department of Biology and Chemistry,
Azusa Pacific University, Azusa, CA USA 91702

Published 2016 in ACS Chemical Neuroscience pp. 245-262.

MATERNAL PHARMACOKINETICS AND FETAL
DISPOSITION OF CIT DURING MOUSE PREGNANC Y

49
CHAPTER 3 POINTS OF INTEREST

So far, this dissertation has explored 5-HT as a critical modulator of fetal
development, the crucial role of the placenta in maintaining fetal homeostasis,
and presented a detailed innovative approach to studying placental physiology
and measuring fetal outcomes. The chapter ahead applies some of these
techniques among others, to explore the detailed pharmacokinetics of the SSRI
Citalopram during pregnancy. This subject matter is particularly relevant, as it
provides insight into answering questions posed earlier in chapter 1: how do fetal
SSRI exposures might affect 5-HT development through the highly dynamic time
of pregnancy? Key points highlighted in this chapter are:
§ CIT and its metabolite desmethylcitalopram (DCIT) are rapidly transferred
from the maternal to the fetal circulation and reach the fetal brain
§ The pharmacokinetics of CIT and the extent of fetal exposure are
dependent on pregnancy stage
§ The exploration of the serotonin transporter (SERT) on fetal CIT
disposition
§ Time-dependent fetal metabolic capacity of CIT and its significant
repercussions

MATERNAL PHARMACOKINETICS AND FETAL
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50
3.1 ABSTRACT

While selective-serotonin reuptake inhibitor (SSRI) antidepressants are
commonly prescribed in the treatment of depression, their use during pregnancy
leads to fetal drug exposures. According to recent reports, such exposures could
affect fetal development and long-term offspring health. A central question is how
pregnancy-induced physical and physiological changes in mothers, fetuses, and
the placenta influence fetal SSRI exposures during gestation. In this study, we
examined the effects of gestational stage on the maternal pharmacokinetics and
fetal disposition of the SSRI (±)-citalopram (CIT) in a mouse model. We
determined the maternal and fetal CIT serum concentration-time profiles
following acute maternal administration on gestational days (GD)14 and GD18,
as well as the fetal brain drug disposition. The results show that pregnancy
affects the pharmacokinetics of CIT and that maternal drug clearance increases
as gestation progresses. The data further show that CIT and its primary
metabolite desmethylcitalopram (DCIT) readily cross the placenta into the fetal
compartment, and fetal exposure to CIT exceeds that of the mother during
gestation 2 hours after maternal administration. Enzymatic activity assays
revealed that fetal drug metabolic capacity develops in late gestation, resulting in
elevated circulating and brain concentrations of DCIT at embryonic day (E)18.
Fetal exposure to the SSRI CIT in murine pregnancy is therefore influenced by
both maternal gestational stage and embryonic development, suggesting
potential time-dependent effects on fetal brain development.
MATERNAL PHARMACOKINETICS AND FETAL
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51
3.2 INTRODUCTION

An increasing number of women are prescribed selective serotonin (5-HT)
reuptake inhibitor (SSRI) antidepressants to treat depression during pregnancy
(Evans et al., 2001; Government of Canada, 2005; Cooper et al., 2007; Belmaker
and Agam, 2008; Deave et al., 2008; Davalos et al., 2012; Velasquez et al.,
2013). This pharmacological intervention presents a well-recognized clinical
conundrum, namely the accumulating concerns that developmental abnormalities
in the offspring may arise from fetal exposure to maternal depression and to
SSRIs (Hanley and Oberlander, 2012; Shea et al., 2012; Olivier et al., 2013,
2015; Velasquez et al., 2013). Few studies have focused on the consequences
of prenatal SSRI exposure on fetal neurodevelopment, but recent evidence
points to increased risks of autism spectrum disorders and postnatal language
learning deficits (Croen et al., 2011a; Weikum et al., 2012; Velasquez et al.,
2013; Harrington et al., 2014). Importantly, developmental outcomes appear to
depend on the type of SSRI used and the pregnancy stage of SSRI exposure,
suggesting the existence of sensitive time periods for the fetal programming of
specific disorders (Velasquez et al., 2013; Altieri et al., 2015). These
observations raise questions about the safety of SSRIs during pregnancy and
how the factors that affect fetal drug exposures influence short- and long-term
offspring health outcomes.
There are highly complex and dynamic maternal physical and physiological
changes taking place during pregnancy, a unique condition that also involves the
3.2 INTRODUCTION

52
progressive development of the placenta and fetus (Loebstein et al., 1997;
Dawes and Chowienczyk, 2001; Anderson, 2005; Feghali and Mattison, 2011;
Isoherranen and Thummel, 2013). Pregnancy (and its developmental stage) can
thus affect the maternal pharmacokinetics of drug absorption, distribution,
metabolism, and elimination, in addition to transplacental transfer and the extent
of fetal exposure (Evseenko et al., 2006; Myllynen et al., 2009; Pollex et al.,
2009; Ververs et al., 2009; Ploeger et al., 2010; Prouillac and Lecoeur, 2010;
Nicoletto and Rinaldi, 2011; Dilworth and Sibley, 2013). Therefore, evaluating the
impact of pregnancy on these pharmacological factors will provide insight into
how they relate to fetal drug exposures potentially leading to different neonatal
outcomes.
§ The maternal, placental, fetal, and genetic factors that determine fetal SSRI
exposures in the context of pregnancy remain largely unknown. Studies in
humans have largely focused on the maternal pharmacokinetics of SSRIs,
showing that maintaining a fixed dose results in low plasma concentrations
(Heikkinen et al., 2002, 2003; Freeman et al., 2008) in addition to kinetic
changes in drug absorption, distribution, metabolism, and clearance(Wadelius
et al., 1997; Hostetter et al., 2000b; Anderson, 2005; Lattimore et al., 2005;
Deligiannidis et al., 2014). The present study characterizes pregnancy-
induced changes in the pharmacokinetics of the widely prescribed SSRI (±)-
citalopram (CIT) during gestation in mice, and investigates how these
influence fetal drug exposure. We first compared the pharmacokinetics of CIT
MATERNAL PHARMACOKINETICS AND FETAL
DISPOSITION OF CIT DURING MOUSE PREGNANC Y

53
§ in non-pregnant and pregnant mice at gestational day (GD) 14 and 18. We
then investigated the disposition of CIT to the fetus by quantifying placental
drug transfer and the extent of fetal exposure following maternal
administration. Lastly, we investigated whether the maternal and fetal
metabolism of CIT is dependent on gestational age. The data reveal
important changes in the maternal and fetal pharmacokinetics of CIT and its
major metabolite during development in mice

3.3 RESULTS

3.3.1 PREGNANCY AFFECTS THE DISPOSITION AND
PHARMACOKINETICS OF CIT
To test whether pregnancy affects CIT pharmacokinetics, we administered a
single, fixed dose (FD) of CIT (0.6 mg) to non-pregnant mice and pregnant mice
at GD18 (termed GD18
FD
). Blood serum concentrations in both groups showed
biexponential decreases overtime from 3.5 min to 3.5 h post administration
(Figure 3.1). Pharmacokinetic analysis revealed that blood serum CIT reached
higher peak concentrations (C
0
) in non-pregnant female mice than in pregnant
GD18
FD
mice (P=0.0002; Table 3.1). Consistent with lower serum CIT
concentrations measured throughout the 3.5 h time course (Figure 3.1), a
significant 25% reduction in the AUC of serum CIT was observed in GD18
FD

dams compared to non-pregnant mice [t(4) = 10.97; P=0.0004; Table 3.1].
Although time course analyses from both groups showed similar CIT absorption
3.3 RESULTS

54
phases (1.67 vs 1.76; t(4) = 2.16; P=0.0968; non-pregnant and GD18
FD
,
respectively), the distribution rate constant (α) during pregnancy was
considerably lower than in non-pregnant mice (t(4) = 95.4; P<0.0001; Supp.
Table 3.1), as reflected by an increase in the volume of distribution (V
D
; t(4) =
3.40 ;P=0.0273; Table 3.1). Additionally, the elimination rate constant (β) was
higher in pregnant than non-pregnant mice (t(4) = 5.54; P=0.0052; Supp. Table
3.1), consistent with the higher clearance (C
L
) measured at GD18
FD
(t(4) = 12.9;
P=0.0002; Table 3.1). The half-life of CIT measured in pregnant dams was
slightly lower than in non-pregnant female mice (t(4) = 3.22; P=0.0323; Table
3.1).

MATERNAL PHARMACOKINETICS AND FETAL
DISPOSITION OF CIT DURING MOUSE PREGNANC Y

55

Figure 3.1 CIT serum concentration-time profiles in non-
pregnant and pregnant mice
A fixed dose (FD) of CIT (0.6 mg) was administered ip to non-
pregnant mice (n) and pregnant mice at GD18 (GD18
FD
; ).
Mice were sacrificed 3.5 min to 3.5 h following drug
administration and CIT serum concentrations were determined
by HPLC. Data represent means ± SD (N=3 mice per time
point). The insert (upper right) shows a semi-logarithmic plot of
serum CIT concentrations over time. Both pregnant and non-
pregnant female mice show bi-exponential decreases in serum
CIT concentrations over time.
3.3 RESULTS

56
Table 3.1 Comparison of CIT pharmacokinetics in non-pregnant and
pregnant GD18
FD
mice
The CIT serum concentration time-courses were fitted to a two-compartment
model to estimate the following pharmacokinetic parameters: C
0
: peak
concentration, t½: half-life; AUC: area under the concentration-time curve; V
D
:
volume of distribution; C
L
: clearance. Data are reported as the means ± SDs (N =
3 mice per time point). Statistical differences were determined by unpaired two-
tailed Student’s t-tests.
a,
Statistically significant differences (P<0.05) between
groups. FD = fixed dose.

3.3.2 DOSE AFFECTS CIT DISTRIBUTION AND CLEARANCE
DURING PREGNANCY
The results above indicate that pregnancy affects the pharmacokinetics of CIT in
mice. We next investigated whether CIT pharmacokinetics were different in
pregnant dams when given a weight-adjusted dose. Pregnant mice receiving a
weight adjusted (20 mg/kg of body weight, corresponding on average to 1.17 ±
0.05 mg CIT) dose (GD18) had significantly higher serum C
0
than those
administered a fixed, non- weight-adjusted dose (GD18
FD
) (0.6 mg – equivalent
to 20 mg/kg dose given to non-pregnant mice) [t(4) = 23.6; P<0.0001; Table 3.2].
Serum CIT concentrations were higher at every time point in GD18 compared to

MATERNAL PHARMACOKINETICS AND FETAL
DISPOSITION OF CIT DURING MOUSE PREGNANC Y

57
GD18
FD
mice (Figure 3.2A), consistent with a significant increase in AUC (t(4) =
27.1; P=0.0004; Table 3.2). Since the C
0
and AUC estimates are representative
of peak CIT concentrations and total drug exposures overtime, we normalized
these parameters to the amount of CIT injected. The measured serum CIT
concentrations were each divided by injected drug amounts followed by fitting to
a two-compartment model. We no longer found significant differences between
groups for the above parameters after dose-normalization (C
0
; t(4) = 2.56
P=0.0648; AUC; t(4) = 2.30; P=0.0937) (Figure 3.2B, Table 3.2). The CIT half-life
was unaffected by the dose administered (t(4) = 2.65; P=0.0599; Table 3.2).
When compared to the GD18
FD
group, an increase in V
D
(t(4) = 7.89; P=0.0014;
Table 3.2), as well as a reduction in CIT C
L
(t(4) = 12.9; P=0.0002; Table 3.2)
was observed in GD18 mice receiving a weight-adjusted dose. These
observations are consistent with significant differences observed in distribution
and elimination rate constants (α; t(4) = 5.38; P=0.0057; β; t(4) = 4.65; P=0.0097;
Supp. Table 3.1). The differences in t½, V
D
, and C
L
between the GD18 and
GD18
FD
groups remained unchanged after dose-normalization.

3.3 RESULTS

58

Figure 3.2 Maternal CIT serum concentration-time profiles in GD18 mice
(A) GD18 Pregnant mice received a weight-adjusted dose of CIT (n; GD18; 20 mg/kg
of body weight, ip) or a non-pregnant-equivalent fixed dose ( ; GD18
FD
; 0.6 mg, ip).
Maternal serum was collected from 3.5 min to 3.5 h after administration, and CIT
concentration was measured by HPLC (N = 3 dams per time point). (B) Serum
concentration-time profiles were normalized to the dose received by dividing the
measured CIT serum concentrations by the injected drug amount. Data are shown as
the means ± SD (N = 3 mice per time point). Inserts (upper right) show semi-
logarithmic plots of the data. FD = fixed dose
MATERNAL PHARMACOKINETICS AND FETAL
DISPOSITION OF CIT DURING MOUSE PREGNANC Y

59

Table 3.2 Effect of weight-adjusted dose on pharmacokinetic parameters of
CIT in pregnant mice
The CIT concentration-time profiles were fitted to a two-compartment model to
estimate all pharmacokinetic parameters. Dose-normalized (dn) C
0
and AUC
were also calculated (C
0dn
; AUC
dn
). Data are reported as the means ± SD (N = 3
mice per time point). Statistical differences were determined by an unpaired
Student’s t-test.
a
, indicates statistically significant differences (P<0.05) between
groups. FD = fixed dose.

3.3 RESULTS

60
3.3.3 PREGNANCY STAGE AFFECTS DISPOSITION AND
PHARMACOKINETICS OF CIT
The results thus far show that drug dosage and pregnancy affect CIT
pharmacokinetics in mice. Significant maternal physical and physiological
changes occur progressively throughout pregnancy, therefore we next
investigated if CIT pharmacokinetics are affected by pregnancy stage (Loebstein
et al., 1997; Dawes and Chowienczyk, 2001; Anderson, 2005; Isoherranen and
Thummel, 2013). Mice at GD14 and GD18 received a single, weight-adjusted
CIT dose (20 mg/kg of body weight). Within each group, maternal serum CIT
concentrations decreased over time in a biexponential fashion (Figure 3.3). The
GD18 mice had higher concentrations of CIT at every time point (Figure 3.3A),
consistent with a significantly higher AUC (t(4) = 15.9; P<0.0001; Table 3.3).
Similar differences were observed after dose normalization of these parameters
(Table 3.3). There was a significant increase (22%) in V
D
as gestation advanced
from GD14 to GD18 (t(4) = 4.97; P=0.0076; Table 3.3). Drug clearance was also
affected by gestational stage, as GD14 mice had significantly higher C
L
than
GD18 mice (t(4) = 8.58; P=0.0010; Table 3.3).

MATERNAL PHARMACOKINETICS AND FETAL
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61

Figure 3.3 Maternal serum CIT concentration-time profiles in pregnant mice of
different gestational stages
(A) Pregnant mice received a weight adjusted CIT dose (20 mg/kg) at GD14 ( ) or GD18
(n). Mice were sacrificed 3.5 minutes to 3.5 hours following drug administration and CIT
serum concentrations were measured by HPLC. Data represent the means ± SD serum
CIT concentration (N = 3 mice per time point). The insert (upper right) shows a semi-
logarithmic plot of the data. (B) Serum concentration-time profiles were normalized to the
dose received by dividing the measured CIT serum concentrations by the injected drug
amount. Data are shown as the means ± SD (N = 3 mice per time point). Inserts (upper
right) show semi-logarithmic plots of the data. FD = fixed dose

3.3 RESULTS

62

Table 3.3 Gestational age-dependent pharmacokinetics of CIT
at GD14 and GD18 in mice.
Following CIT administration (20 mg/kg ip), concentration-time
profiles were fitted to a two-compartment model to estimate all
pharmacokinetic parameters. Dose-normalized (dn) C
0
and AUC
were also calculated. Data are reported as the means ± SD (N = 3
mice per time point). Statistical differences were determined by an
unpaired Student’s t-test.
a
, indicates statistically significant
differences (P<0.05) between groups
MATERNAL PHARMACOKINETICS AND FETAL
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63
3.3.4 CIT RAPIDLY REACHES THE FETAL CIRCULATION AND
BRAIN AFTER MATERNAL ADMINISTRATION
The effect of pregnancy stage on maternal CIT pharmacokinetics may induce
differential exposure of the fetus to the drug over time. Therefore, we measured
the kinetics of CIT concentrations in fetal serum and brain at embryonic (E) day
14 and E18 after a single weight-adjusted maternal injection (20 mg/kg, ip). At
both embryonic stages, peak fetal serum CIT concentrations were detected
within similar time frames (15 min following maternal drug injection (Figure
3.4A)). In addition, fetal serum CIT concentrations decreased bi-exponentially
over time at both E14 and E18. The E18 fetal group had consistently higher
serum CIT concentrations than the E14 group (Figure 3.4A). In all experiments,
fetal sex was determined by SRY genotyping and embryo positions in the uterine
horns were recorded. The reported pharmacokinetic measures were not affected
by either parameter. The fetal/maternal CIT serum concentration ratios (F:M)
calculated throughout the 3.5 h period, showed that fetal exposure to CIT
exceeded that in mothers 2 h after injection at both gestational ages (F:M >1),
and that F:M were similar between the E14 and E18 groups (F
6,24
= 1.42;
P=0.2011; Figure 3.4B). To test if and how fast the fetal brain is exposed to
maternally administered CIT, fetal brain drug concentrations were measured over
time and normalized to total protein concentrations. Results show that fetal brain
CIT concentrations decreased mono-exponentially over time at both ages, with
the E18 group having consistently higher brain tissue CIT concentrations
compared to the E14 group (Figure 3.4C).
3.3 RESULTS

64

Figure 3.4 Fetal serum and brain CIT concentration-time profiles during gestation
Pregnant dams at GD14 and GD18 were administered a single ip injection of CIT (20 mg/kg)
and CIT concentrations in fetal serum and brain were measured by HPLC. (A) Data show
the means ± SD CIT concentration measured in the fetal serum over time from 3.5 min to 3.5
h (N = 3 dams per time point, 5-8 pooled fetal samples per dam). The insert (upper right)
shows a semi-logarithmic plot of the data. (B) Fetal/maternal CIT concentration ratios (F:M)
were not significantly different between the GD14 and GD18 groups (F
6,24
= 1.42; P=0.2011;
2-way ANOVA followed by Bonferroni adjustment for multiple comparisons). (C) Mean ± SD
CIT concentration measured in fetal brain tissue from 15 min to 3.5 h (N = 3 dams per time
point, 3 fetal brains per dam). The CIT concentrations were normalized to total fetal brain
protein concentrations.
MATERNAL PHARMACOKINETICS AND FETAL
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65
3.3.5 FETAL CIT DISPOSITION IS INDEPENDENT OF
PLACENTAL/FETAL SERT EXPRESSION
Maternally administered CIT rapidly reaches the fetal blood and brain. While
passing from the mother to the fetus, the primary binding target for CIT in the
placenta is the serotonin transporter (SERT; Slc6a4), which is expressed by
placental syncytiotrophoblastic cells of fetal origin (Ganapathy et al., 1993;
Yavarone et al., 1993; Shearman et al., 1998; Verhaagh et al., 2001; Bottalico et
al., 2004; Viau et al., 2009). Placental SERT could act as a local reservoir that
limits drug transfer to the fetal compartment. Yet, the influence of binding to
placental SERT on the transfer and fetal disposition of CIT is unknown. Here,
SERT heterozygous (HET) dams were crossed with SERT HET males to
generate SERT wildtype, HET, and knockout (KO) embryos and placentas. To
test whether CIT transfer and fetal disposition are influenced by placental/fetal
SERT expression, HET dams were injected with CIT (20 mg/kg; ip) at GD18 and
CIT concentrations were measured in the serum and brains of fetuses of each
genotype. Individual fetal blood and brain collections were performed 1.5 h post
maternal drug administration. The HPLC analyses showed no effect of fetal
genotype on CIT serum or fetal brain tissue concentrations (serum: F
2,12
= 0.34;
P=0.7100; brain: F
2,12
= 1.03; P=0.3851; Figure 3.5).

3.3 RESULTS

66

Figure 3.5 Fetal and placental SERT expression does not affect CIT
concentrations
Fetal serum (A) and brain (B) CIT concentrations in wildtype (WT), SERT
heterozygous (HET), and SERT knockout (KO) embryos. The SERT HET
dams were crossed with SERT HET males and the former received a single
CIT injection (20 mg/kg) at GD18. Individual E18 fetal samples were collected
1.5 h post-administration and CIT concentrations were measured by HPLC.
Data show the means ± SD CIT concentration (N = 2 dams, 8 total fetal
collections per dam). Brain CIT concentrations were normalized to total fetal
brain protein.

MATERNAL PHARMACOKINETICS AND FETAL
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67

3.3.6 FETAL EXPOSURE TO DCIT IS DEPENDENT ON
PREGNANCY STAGE
As uncovered above, fetuses are rapidly exposed to significant concentrations of
maternally administered CIT at E14 and E18. In adult mice and humans, CIT is
metabolized to the long half-life, SERT-inhibiting metabolite DCIT (Hiemke and
Härtter, 2000). During pregnancy, CIT metabolism could therefore result in
significant and long-term fetal exposure to this biologically active metabolite. We
quantified concentrations of DCIT in maternal and fetal serum samples and
brains collected after a single maternal administration of CIT (20 mg/kg; ip). In
fetal serum, peak DCIT concentrations were reached faster at E14 (1 h) than E18
(2.5 h) (Figure 3.6A). However, when taking into account DCIT concentrations
measured in the maternal serum (Suppl. Figure 3.1), the calculated
fetal/maternal DCIT serum concentration ratios were higher at E18 vs. E14
fetuses 2 h after injection (F
6,24
= 13.69; P=0.0166 - <0.0001) (Figure 3.6B). In
the fetal brain, overall DCIT concentrations were higher at E18 than E14,
consistent with significantly higher mean AUCs (35.5 ng´h/mL E14 vs. 62.8 E18;
(F
2,6
= 15.6; P=0.0159; Figure 3.6C). Fetal brain DCIT concentrations started to
decrease 2.5 h (at E14 and E18) after maternal CIT injection (Figure 3.6C).

3.3 RESULTS

68

Figure 3.6 Fetal serum and brain DCIT concentration-time profiles during gestation
Pregnant dams were administered 20 mg/kg CIT ip. The DCIT concentrations in maternal
serum, and fetal serum and brain were measured by HPLC. (A) Data show the means ± SD.
The DCIT concentrations were measured in fetal serum from 3.5 min to 3.5 h (N = 3 dams per
time point, 5-8 pooled fetal samples per dam) at E14 and E18. (B) Fetal/maternal (F:M) DCIT
serum concentration ratios. Statistical differences in F:M concentration ratios were analyzed by
2-way ANOVA followed by Bonferroni adjustment for multiple comparisons. *P<0.05; **P<0.01;
****P<0.0001. (C) Data show the mean ± SD protein-normalized DCIT concentrations in fetal
brain tissue from 15 min to 3.5 h following maternal CIT administration (N = 3 dams per time
point, 3 fetal brains per dam).
MATERNAL PHARMACOKINETICS AND FETAL
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69
3.3.7 GESTATIONAL AGE DEPENDENT CHANGES IN CIT
METABOLISM
Comparatively higher exposure of fetuses to DCIT at E18 than E14 occurred and
these differences could result from differential rates of maternal, fetal, and/or
placental metabolism between the two ages. To address these possibilities, we
first tested if the placenta itself metabolizes CIT. Live E18 mouse and term
human placentas were perfused ex vivo with 500 ng/mL CIT continuously infused
through the maternal uterine artery. The CIT and DCIT concentrations were
measured in the fetal eluates harvested in 10 min intervals through the umbilical
vein over a 120 min perfusion period. Here, CIT was detected at every time point
in fetal eluates (Fig. 3.7A), indicating that the parent drug readily crosses the live
placenta (TI = 0.93 ± 0.55), consistent with the in vivo results. Importantly, DCIT
was not detected in the fetal eluate at any time point (Fig. 3.7A). In addition, in
vitro assays showed no significant CIT to DCIT metabolic capacity of placental
microsomes at any age tested (Fig 3.7B). In a separate set of experiments, we
measured DCIT transplacental transfer by perfusing live E18 placentas with a
maternal solution containing DCIT only (500 ng/mL). The results showed that
maternal DCIT readily crossed the placenta (TI = 0.20 ± 0.96) (Figure 3.7C).
These findings indicate that the placenta does not metabolize CIT to DCIT, but
allows rapid maternal-fetal DCIT transfer. The maternal compartment is therefore
the direct source of DCIT to the fetus.
To test whether maternal CIT metabolism changes during the course of
pregnancy, we measured DCIT generation in GD14 and GD18 maternal liver
3.3 RESULTS

70
microsomal preparations. The data indicate that the rate of CIT to DCIT
metabolism in the maternal liver was similar at GD14 and GD18 (Fig. 3.7D),
consistent with in vivo measures showing similar maternal serum DCIT
concentrations at both pregnancy stages (AUC; GD18 = 611 ± 66.37 ng x h / mL
vs GD14 = 417.5 ± 52.47) [t(2) = 3.234; P=0.0838] (Suppl. Fig. 3.1).
These results indicate that variations in maternal CIT metabolism cannot
account for the higher DCIT fetal exposure measured in late pregnancy. The
remaining possibility was that the rate of CIT to DCIT metabolism in the fetus
might be higher at E18 than E14. We compared the rate of DCIT generation from
fetal liver microsomes at E14 and E18. There was no measurable DCIT
generation from E14 fetal liver microsomes throughout the 2 h incubation period,
whereas there was a significant rate of CIT to DCIT conversion from E18 fetal
liver microsomes (Fig. 3.7E).

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71

Figure 3.7 Determination of DCIT sources to the fetal compartment
(A) Human and mouse CIT (500 ng/mL) and Antipyrine (20 mg/mL) were perfused through the
human decidual surface of intact cotyledons of term placentas and through the uterine artery
on the maternal side of E18 mouse placentas. 10-minute collection fractions were harvested
over 120 min through the fetal output. After a 40 minute period of stabilization, there is steady
state transfer of CIT. Over a 60 min period of stable perfusion, the average transplacental
transfer (TPT) of CIT in term placentas is significantly higher than that of E18 placentas (p <
0.0001). However, the transfer index (TI; transfer ratio between CIT and antipyrine) is not
different (p = 0.6939). DCIT was not detected in both term and E18 fetal output samples at any
time point. ( N = 3 human placentas; N = 4 mouse placentas). Additionally, CIT and DCIT (n)
concentrations were measured by HPLC). (B) Placental microsomes were isolated at E14 and
E18 and incubated with CIT (500 ng/mL). Samples were collected at 20 min intervals over a 2
h period and CIT (left panel; expressed as percent of input) and DCIT (right panel; GD14 ( ),
GD18 (n) concentrations were measured by HPLC. (C) The GD18 placentas were perfused ex
vivo with 500 ng/mL DCIT in the maternal side. Perfusion samples were collected every 10 min
over a 2 h time period on the maternal (top) and fetal (bottom) sides and DCIT concentrations
were measured by HPLC (N = 3 perfusions). (D) Maternal and (E) fetal liver microsomes were
isolated at E14 ( ) and E18 (n) and incubated with CIT (500 ng/mL). Data indicate the mean
+/- SD DCIT concentrations measured by HPLC in samples at 20 min intervals over a 2 h
period (N = 2 independent experiments per gestational stage).
3.4 DISCUSSION

72
3.4 DISCUSSION

The purpose of the present study was to determine if pregnancy induces stage-
dependent changes in the pharmacokinetics and fetal disposition of CIT in mice.
We found that the maternal disposition and serum pharmacokinetics of CIT were
affected by pregnancy. The C
0
and AUC reductions observed in GD18
FD
dams
are related to the physical changes occurring during pregnancy, namely the
increases in maternal blood volume and total body weight as reflected by a
higher V
D
(Figure 3.1, Table 3.1). Additionally, our results show that drug C
L
is
increased during pregnancy, which is consistent with observations made for
other therapeutic drugs in clinical and pre-clinical studies (Wadelius et al., 1997;
Anderson, 2005; Zhou et al., 2010; Shuster et al., 2014). The increased C
L
may
also be explained by pregnancy-induced elevation in cardiac output, glomerular
filtration rate, and heightened activity of the cytochrome P450 enzymes involved
in CIT metabolism during pregnancy (CYP2D6, CYP3A4, CYP2C19) (Anderson,
2005; Feghali and Mattison, 2011; Mrazek et al., 2011; Shea et al., 2012;
Isoherranen and Thummel, 2013). Combined with the fetal metabolic capacity
emerging in late pregnancy (Figure 3.7E), these observations may account for
the reduction in CIT terminal half-life in GD18
FD
compared to non-pregnant
female mice. All together, these factors lead to the observed reduction of
circulating CIT in the maternal serum at GD18. In the clinical setting, similar
decreases in maternal plasma concentrations of CIT or other SSRIs have been
suggested to cause reduced therapeutic efficacy of these drugs during
MATERNAL PHARMACOKINETICS AND FETAL
DISPOSITION OF CIT DURING MOUSE PREGNANC Y

73
pregnancy (Milne and Goa, 1991; Hostetter et al., 2000b; Heikkinen et al., 2002,
2003; Lattimore et al., 2005; Freeman et al., 2008; Deligiannidis et al., 2014).
We next focused on determining whether CIT pharmacokinetics were affected
by pregnancy status by adjusting CIT doses to dam body weights. The weight-
adjusted dose administered to GD18 dams (20 mg/kg) was on average twice the
amount (1.17 ± 0.05) received by the GD18
FD
group (maintained at 0.6 mg).
Despite this large difference in the amount of CIT administered, we found similar
dose-normalized C
0
and AUC parameters between groups (Figure 3.2, Table
3.2). However, GD18 dams receiving a CIT dose adjusted to body weight
showed a higher V
D
and lower C
L
than the GD18
FD
group. Given that mice
received CIT at the same gestational stage, these results suggest that a weight-
adjusted dosage leads to saturating CIT serum concentrations at GD18. This is
also suggested by the significant increase in AUC measured in the GD18 group
(141% increase) despite the CIT dose being increased by only 100% (2-fold)
over the GD18
FD
dams. The increase in AUC is not linearly correlated to the
administered dose, as expected from systemic drug saturation and consistent
with reduced C
L
rates.
Similar results were obtained when we investigated the influence of
gestational stage on maternal CIT pharmacokinetics with GD14 and GD18 dams
receiving CIT adjusted to body weight. We found similar C
0
values following
dose-normalization, although AUC remained higher at GD18 (Figure 3.3, Table
3.3). In spite of higher serum CIT concentrations at GD18, the terminal half-life
3.4 DISCUSSION

74
was similar at both ages. These results suggest that maternal CIT metabolism is
not affected by pregnancy stage. Interestingly, pregnant mice had a lower V
D
at
GD14 than GD18; this likely results from significant physical changes taking
place throughout gestation, in particular the increase of blood volume (estimated
to be 8% of body weight and thus corresponding to approximately 3.5 ± 0.1 mL at
GD14 vs 4.7 ± 0.2 mL at GD18), and also an increase in overall body lipoprotein
content as reflected by weight gain (Shuster et al., 2014). In addition, the rapid
growth of fetuses from E14 to E18 (average fetal weight increases by 300%) may
increase the contribution of the fetal compartment to the apparent V
D
.
Changes in CIT maternal pharmacokinetics between GD14 and GD18 may
lead to differential exposure of the fetus to the drug over time. Investigation of
fetal drug disposition revealed that maternally administered CIT rapidly reaches
the fetal circulation and brain (Figure 3.4). Disposition of CIT in the fetal serum
was delayed when compared to the maternal profiles, with fetal serum CIT
concentrations peaking 15 min following maternal administration at both E14 and
E18. Despite the differences observed in the maternal pharmacokinetics of CIT,
fetal to maternal serum concentration ratios were not different between
gestational stages, and fetal serum CIT concentrations exceeded maternal
concentrations 2 hours after maternal administration. These data suggest that
there are no major differences in placental CIT permeability between ages and
that the kinetics of maternal-fetal transfer through the placenta are faster than
efflux back to the maternal compartment.
MATERNAL PHARMACOKINETICS AND FETAL
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75
The SSRI CIT has a high affinity for SERT (K
i
= 2.6 nM), which is highly
expressed in the placenta, particularly in late gestation (GD18).
44–49
We
investigated whether CIT binding to placental SERT could potentially limit
transfer to the fetus using heterozygous crossings to generate wildtype, HET,
and KO embryos and placentas. Results show that maternal-fetal CIT transport
and fetal disposition are completely independent of fetal or placental SERT
expression (Figure 3.5). This result is not unexpected given the multitude of non-
specific drug transporters expressed in the placenta, some of which interact with
CIT (e.g., P-gp, BCRP, MDR/ABCB). Interestingly, the results suggest that SERT
genetic polymorphisms, although studied in clinical populations and correlated to
the severity of developmental effects, may not quantitatively affect fetal exposure
to CIT during pregnancy(Oberlander et al., 2008; Mrazek et al., 2009).
Fetal exposure to biologically active metabolites is another important aspect
to consider in the investigation of therapeutic drug use during pregnancy. In
humans, SSRIs are metabolized to demethylated forms by cytochrome P450
(CYP) 2D6 and 2C19 enzymes, with large inter-individual variation in their
respective activities. This leads to important differences in parent drug and
metabolite concentrations among individuals. In contrast, rodents are generally
assumed to correspond to ‘extensive-metabolizers’ for most drugs,(Hiemke and
Härtter, 2000; Urquhart et al., 2007; Shuster et al., 2013) which constitutes a bias
for in vivo pharmacodynamics and drug disposition studies. Additionally, the
mean plasma S/R enantiomer ratio of racemic CIT is 0.56 and that of DCIT is 0.7
3.4 DISCUSSION

76
in patients, indicating stereoselective metabolism of CIT (Rochat et al., 1995).
This is possibly due to a higher affinity of S-CIT and S-DCIT to particular
metabolizing isoenzymes, suggesting that the most active enantiomer (S-) is also
preferentially metabolized. The primary metabolite of CIT, DCIT, is biologically
active and also displays a prolonged half-life (Hiemke and Härtter, 2000).
Although the inhibitory constant (K
i
) of SERT-mediated 5-HT uptake is less
potent than its parent drug (DCIT = 14 nM; CIT = 2.6 nM) it is comparable to that
of the commonly used SSRI fluoxetine (Ki = 14 nM) (Hiemke and Härtter, 2000).
In this light, maternal use of CIT during pregnancy could result in long-term fetal
exposure to a potent SERT-inhibiting metabolite, besides the parent drug itself.
Our results show that DCIT reaches the fetal circulation and brain following
CIT biotransformation (Figure 3.6). In vivo observations were consistent with ex
vivo placental perfusion studies showing that DCIT crosses the placenta (Figure
3.7). Furthermore, our data show that F:M ratios of CIT (Fig. 3.4B) are
consistently higher than DCIT (Fig. 3.6B), suggesting that DCIT does not cross
the placenta as efficiently as CIT. Ex vivo measures also showed that DCIT is
transferred transplacentally ~2 fold less efficiently than CIT (Figure 3.7A,C),
although different rates of efflux back to the maternal compartment may also
contribute to these differences. These observations are consistent with the
physicochemical properties of CIT vs. DCIT, namely its higher lipophilicity (CIT
partition coefficient = 0.48; DCIT = 0.28), and intermediate protein binding that
facilitate membrane permeability of CIT over DCIT (Heikkine et al., 2002;
MATERNAL PHARMACOKINETICS AND FETAL
DISPOSITION OF CIT DURING MOUSE PREGNANC Y

77
Heikkinen et al., 2002). Additionally, we did not measure any detectable
biotransformation when placentas were infused with CIT or in placental
microsomal incubations at E14 and E18 (Figure 3.7). These results are
consistent with previous studies showing an absence of placental expression of
CYP isoenzymes involved in CIT metabolism and suggest that maternal drug
metabolism and placental drug transport are the major determinants of fetal DCIT
exposure(Ejiri et al., 2001, 2005; Storvik et al., 2014). Importantly, CIT metabolic
capacity was detected in the fetal liver at E18 but not E14 (Figure 3.7E), leading
to higher fetal serum DCIT concentrations at E18 compared to earlier in
pregnancy (E14). These results parallel observations made in humans where
mRNA and protein expression of CIT metabolizing CYP enzymes were not
detected in the human fetal liver until 20 weeks of gestation(Hines and McCarver,
2002). The differences in fetal exposure to CIT metabolites between E14 and
E18 may thus directly reflect changes in fetal CYP expression.
In summary, we have demonstrated that pregnancy affects the
pharmacokinetics of CIT. Our findings indicate that drug metabolism and
clearance change in a gestational stage-dependent manner and that fetal
metabolism may become a significant contributor to these changes during late
gestation. These results suggest that in order to account for significant physical
and physiological changes that occur throughout pregnancy and to maintain
therapeutic efficacy, the CIT doses administered to pregnant mice may need to
be adjusted to the maternal pregnant weight. However, the possibility that a non-
3.4 DISCUSSION

78
weight-adjusted dose of CIT administered to pregnant mice still provides an
antidepressant effect remains to be tested. Importantly, although we found no
differences in fetal drug disposition between E14 and E18, our data show that
fetal CIT serum concentrations exceed maternal concentrations 1.5 to 2 hours
after maternal administration at both ages (Figure 3.6B). Combined with elevated
DCIT levels resulting from fetal metabolism at E18, results are suggestive of
extensive fetal exposures to biologically active compounds during late gestation.
The capacity of DCIT for blocking SERT function may have neurodevelopmental
consequences. Hence, SERT expression starts around mid-gestation in the fetal
brain, a time when serotonin signaling exerts critical trophic influences, such as
the modulation of thalamocortical axons pathfinding(Lebrand et al., 1996a, 1998;
Brüning and Liangos, 1997; Brüning et al., 1997; Bonnin et al., 2007a, 2012;
Narboux-Nême et al., 2008). In particular, if CIT-mediated inhibition of SERT
function in the fetal brain leads to increased extracellular serotonin levels, the
consequent increase in serotonin signaling in thalamic neurons could lead to a
dorso-medial shift in the trajectory of thalamocortical axons as they navigate
toward the cortex (Bonnin et al., 2007a). The potential impact of CIT and DCIT-
mediated inhibition of SERT function on the development of these neuronal
pathways is currently the subject of investigation. The results provide novel
insights into the pregnancy-specific pharmacokinetics of a common SSRI, and
demonstrate the importance of fetal metabolism in overall fetal drug exposure.
Although the relevance to human pregnancy will need confirmation, the data
MATERNAL PHARMACOKINETICS AND FETAL
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79
suggest that developmental toxicology studies in mice should take into account
not only maternal-fetal drug transport but also maternal, placental and fetal drug
metabolism.

3.5 MATERIALS AND METHOD S

3.5.1 Animals
Non-pregnant female and timed-pregnant CD-1 mice were obtained from Charles
River Laboratories (Wilmington, Massachusetts), the latter at GD11 (plug date
was considered GD1). The B6.129(Cg)-Slc6a4
tm1Kpl
/J serotonin transporter
knockout (KO) female mice and C57BL/6J male mice were purchased from
Jackson Laboratories (Bar Harbor, Maine) to generate non-sibling heterozygous
(HET) breeder mice, which were then bred to generate wildtype (WT), HET, and
knockout (KO) fetal genotypes. All genotyping of serotonin transporter (SERT)-
deficient mice was conducted by Laragen, Inc. (Culver City, California). Mice
were age-matched (8-10 weeks of age) and pregnancy stage was confirmed by
maternal body weights measured at GD14 and GD18 prior to drug
administration, and fetal body weights measured at time of harvesting. The GD14
and GD18 stages of mouse pregnancy (roughly equivalent to the first and second
trimesters in humans)(Cox et al., 2009) take place after the placenta has
acquired its definitive structure. These are also periods of active neurogenesis
and axonal pathway formation in the fetal brain, processes that are modulated by
3.5 MATERIALS AND METHOD S

80
5-HT and could therefore be impacted directly by maternal-fetal transfer of SSRIs
(Rossant and Cross, 2001; Kepser and Homberg, 2015) Additionally, the mouse
placenta is functional at these time points, which enabled us to assess potential
differences in transplacental drug transfer. All mice were housed in groups of 2-3
dams in standard animal facility cages, maintained under 12h:12h light-dark
cycles, and with food and water provided ad libitum. All procedures using mice
were approved by the Institutional Animal Care and Use Committee at the
University of Southern California and Azusa Pacific University (SERT study) and
conformed to NIH guidelines.

3.5.2 Citalopram Administration
Racemic citalopram (CIT) hydrobromide (TCI; C2370) was dissolved in 0.9%
saline (BD Biosciences) in a volume of 0.01 mL/g. Non-pregnant and pregnant
mice at GD14 and GD18 received a single weight adjusted intraperitoneal (ip)
injection of 20 mg/kg of body weight CIT. We chose ip administration to evaluate
the acute pharmacokinetic parameters following a single CIT dose at controlled
time intervals. The 20mg/kg dose was selected in order to quantify the
pharmacokinetic parameters of the CIT dose most-commonly used in
neuropharmacological studies and which provided an antidepressant effect in
mice (Keeney and Hogg, 1999; El Yacoubi et al., 2003; Crowley et al., 2006;
Warner-Schmidt et al., 2011). The mean drug amount administered to non-
pregnant mice (0.6 mg CIT) was also injected to GD18 mice (GD18
FD
). The
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81
weight adjustment in the administered dose allowed us to take into account the
significant maternal physical changes (i.e., weight gain) that occurs during
pregnancy, as pregnant CD-1 mice show a ~2-fold increase in body weight
compared to non-pregnant mice by late gestation (Suppl. Table 3.2). Dosage of
SSRIs during pregnancy is not usually adjusted to changes in body weight in the
clinic (Anderson, 2005; Cohen et al., 2006; David E Abel, 2013). Therefore, we
performed dose maintenance studies where the same CIT amount received by
non-pregnant mice was administered to heavier dams in late pregnancy
(GD18
FD
).
Following CIT administration (N=3 dams per time point), animals were
euthanized under isofluorane anesthesia (Western Medical Supply) by cervical
dislocation followed by cardiac puncture at various time intervals (3.5, 5, 7.5, 15,
30 min; 1, 1.5, 2, 2.5, 3, 3.5 h). Non-pregnant and maternal blood was collected
in heparinized tubes (BD Biosciences; 367812) and centrifuged at 2000 x g for
20 min at 4°C for serum isolation. The uterus was carefully dissected and the
embryos were placed in ice-cold phosphate buffered saline (PBS). Fetal blood
was collected through the carotid and jugular vasculature at E18 and through the
umbilical cord at E14 (N=5-8 pooled samples per dam). Fetal blood was
centrifuged at 2000 x g for 10 min at 4°C for serum isolation. Fetal brain samples
at E14 and E18 were also dissected at every time point (N=3-5 per dam). All
samples were flash frozen in liquid nitrogen and stored at -80°C until analysis.

3.5 MATERIALS AND METHOD S

82
3.5.3 Ex vivo Transplacental Transfer of CIT and DCIT
Untreated dams were euthanized as above, and a single placenta from each
dam was transferred to a thermostated incubation chamber receiving a flow of
oxygenated phosphate-buffered saline (PBS; Mediatech) at 37 °C. The uterine
artery (maternal input) was cannulated with a 150-μm diameter catheter, and
perfused at 20 μl/min with maternal perfusion media (M199 medium without
phenol red (Gibco), 2.9 g/dL bovine serum albumin (Amresco), 20 IU USP/mL
Heparin, 7.5 g/L Dextran40, 1 g/L glucose, 2.2 g/L sodium bicarbonate, 100 mg/L
L-glutamine (Alfa Aesar), 0.001% fast-green dye (Harleco), pH 7.3; all media
components obtained from Sigma-Aldrich unless noted otherwise) containing 500
ng/mL CIT or its primary metabolite desmethylcitalopram (DCIT; Cerilliant; D-
047). The uterine vein was connected to 355-μm inner diameter (I.D.)
microrenathane tubing (Braintree Scientific; MRE-033) to collect the maternal
output. On the fetal side, the umbilical artery was cannulated with a 105-μm I.D.
catheter and perfused at 5 μl/min with medium without drugs (modified from
above: 30 g/L Dextran40, 0.5 g/L glucose). The umbilical vein was connected to
a 305-μm I.D. microrenathane tubing to collect the fetal output. The eluate was
collected on both the maternal and fetal sides at 10 min intervals for 120 min and
stored at -80°C until analysis. The ex vivo placental perfusion system and
protocol are detailed in(Goeden and Bonnin, 2013, 2014; Velasquez and Bonnin,
2015).

3.5.4 CIT Metabolism in Microsomal Preparations
MATERNAL PHARMACOKINETICS AND FETAL
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83
Liver microsomes were prepared from individual non-pregnant and maternal
livers (N=2-4 per group), placentas, and fetal livers at GD/E14 and GD/E18
(N=10-14 per dam) by ultracentrifugation as previously described(Suski et al.,
2014). Briefly, tissues were dissected and flash-frozen in liquid nitrogen and
stored at -80 °C prior to microsomal isolation. Microsomes were prepared by
mincing and cleaning tissues in wash buffer to remove blood (225 mM mannitol,
75 mM sucrose, 30 mM Tris-HCl, pH 7.4) and isolated in homogenization buffer
(2 mL per 1 g of tissue) with a Teflon pestle (wash buffer + 0.5% (wt/vol) BSA +
0.1 mM EDTA + Roche complete protease inhibitor (Roche; #04693116001, 1
tablet / 10 mL). The crude plasma membrane fraction of the homogenate was
obtained by two centrifugation steps at 800 x g for 5 min (discarding the pellet
each time) and an additional centrifugation at 10,000 x g for 10-min. Microsomal
and cytosolic proteins were isolated by centrifugation of the supernatant at
25,000 x g for 20 min followed by 95,000 x g for 2.5 h. Microsomal proteins
present in the pellet fraction were resuspended in wash buffer and centrifuged at
95,000 x g for 2.5 h. The pellet containing microsomal proteins was resuspended
in 1 mL of wash buffer with protease inhibitor. All buffers and centrifugation steps
were carried out at 4 °C. Microsomal protein concentrations were measured
using a Bradford Assay Kit (BioRad; #500-0207). The quality of the isolated CYP
enzymes was assessed by UV spectrum measures at 450 nm and 420 nm. A
peak shifted from 450 nm to 420 nm indicated that the CYP had undergone
degradation(Jia et al., 1996).
3.5 MATERIALS AND METHOD S

84
The CIT metabolic reactions in placental and liver microsomes were prepared
as previously described with some modifications(Olesen and Linnet, 1999; Jia
and Liu, 2007; Volotinen et al., 2010). Briefly, reactions contained 0.5 mg/mL
microsomal protein and 500 ng/mL CIT in wash buffer. The CIT concentration
used encompasses maternal blood serum levels measured 5 to 60 min after 20
mg/kg CIT i.p. injections (see Fig. 3.1 to 3.3). After preincubation for 2 min in an
incubator shaker set at 37 °C, reactions were initiated by addition of NADPH
(Sigma-Aldrich; #N1630) to a final concentration of 0.85 mg/mL (final reaction
volume 500 μL). Incubations without NADPH served as negative controls and
drug stability throughout the assay was assessed by reactions without liver
microsomal proteins. A 25 μL sample was taken from each incubation in 10 min
intervals from 0-120 min. Reactions were terminated with the addition of 25 μL
ice-cold HPLC extraction buffer (see sample preparation for HPLC).

3.5.5 Quantification of CIT and DCIT in biological samples
Sample preparation for HPLC
A liquid-liquid extraction was used to prepare samples for analysis as previously
described(Velasquez and Bonnin, 2015). Samples were thawed on ice and
extracted with ice-cold extraction buffer (0.2 M perchloric acid + 500 mM D-
mannitol + 100 μM EDTA) with isoproterenol as internal standard. Extraction
buffer was added to fetal serum and microsomal incubations (1:1 v/v) and to
maternal serum samples (1:3 v/v). Brain samples were extracted by addition of
buffer (E14 = 300 μL; E18 = 325 μL) followed by sonication (Qsonica; 35%
MATERNAL PHARMACOKINETICS AND FETAL
DISPOSITION OF CIT DURING MOUSE PREGNANC Y

85
amplification, 15 sec). The samples were kept on ice for 30 min before
centrifugation 20,000 x g for 15 min at 4°C. The supernatant volume was
measured and a 10-μL aliquot was injected per sample for HPLC analysis. The
resulting pellets were used to measure protein concentrations in brain samples
using a Bradford assay.

Perfusion sample preparation
Maternal and fetal perfusion samples were thawed on ice. Individual sample
volumes were measured (approx. 200 μL for maternal output samples and 50 μL
for fetal output samples). Acetonitrile was added to each sample (1:1 v/v)
followed by incubation at room temperature for 10 min. Samples were
centrifuged at 5,000 x g for 10 min at room temperature. The supernatant volume
was measured and evaporated in a SpeedVac concentrator at room temperature.
Evaporated samples were resuspended in extraction buffer to the original sample
volume. Following HPLC-FLD analysis, the transplacental transfer percentage
(TPT) of each drug and associated metabolite was calculated using the following
equation: TPT = (C
f
x S
f
x 100) / (C
m
x S
m
); where C
f
is the concentration in fetal
venous outflow, S
f
is the fetal flow rate (5 μL/min), C
m
is the SSRI concentration
in maternal arterial inflow and S
m
is the maternal flow rate (20 μL/min). The
transplacental transfer index (TI) (i.e., the ratio of transfer between SSRI and
antipyrine - used as internal standard) was calculated by dividing the TPT
(SSRI)
by
the TPT
(antipyrine).

3.5 MATERIALS AND METHOD S

86
HPLC chromatographic conditions
The measurement of CIT and DCIT in all samples was carried out by high
performance liquid chromatography coupled to a fluorescence detector (HPLC-
FLD). The analysis was performed using an Eicom 700 system (Eicom
Corporation, Kyoto, Japan) consisting of a Shimatzu RF-20AX fluorescence
detector (Shimadzu, Kyoto, Japan), an Eicom 700 Insight autosampler, and
Envision integration software. An Eicompak SC-30DS C
18
reversed-phase
column packed with 3-μm silica particles (3.0 mm I.D. x 100-mm) was used as
the analytical column. Chromatographic conditions were set as previously
described(Meng and Gauthier, 2005; Velasquez and Bonnin, 2015). Briefly,
10-μL aliquots of each extracted sample were injected into the column and eluted
with a mobile phase consisting of 10 mM KH
2
PO
4
/acetonitrile (3:1 v/v; EMD
Millipore, AX0145P1) (pH 4.0 adjusted with 1 M phosphoric acid), at a flow rate
of 500 μL/min. Detection wavelengths were set at 250 nm (excitation) and 325
nm (emission). The retention times were 7.15 min and 7.5 min for DCIT and CIT,
respectively. The limit of detection for CIT was 1.5 ng/mL and 500 pg/mL for
DCIT.

3.5.6 Pharmacokinetic Analysis of CIT and DCIT
Serum drug concentrations-time profiles were fitted to a two-compartment model
described by the following equation: C
(t)
= Ae
-αt
+ Be
-βt
(where C(t): serum
concentration at time t after dosing, A: fast exponential term, B: slow exponential
term, a: distribution rate constant, b: elimination rate constant (Supp. Table 3.1))
MATERNAL PHARMACOKINETICS AND FETAL
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87
using the compartmental module of Saam II software (Univ. of Washington,
Seattle, WA, USA). The software-generated system of differential equations was
modeled after the in vivo serum data using a two-stage population PK
analysis(Heatherington et al., 1998; Finkelstein et al., 2009). Equation term
values were obtained from fitting the original serum CIT concentrations obtained
in vivo followed by the introduction of a Bayesian (population) term. Once the
data were fit, an additional set of population terms were introduced and fitted into
the model until 50 iterations that cycled through the data were reached. This
method converged to population parameters that reflected an appropriate fit of
the model to the data for each of the groups. The goodness of fit was evaluated
using the residual method and visual comparison of the actual serum CIT
concentration-time profiles to the estimated curve generated by the model. The
volume of distribution (V
D
), and rates of elimination, absorption, and transfer
between the central and peripheral compartments were calculated by the SAAM
II software. Other pharmacokinetic parameters were calculated manually as
follows:
§ Half-life: t½ =
!" #
$
where % =
'#, )*'), #*'+, ), '#, )*'), #*'+, ) ^#, . 0 '), # 0 '+, )
#

and where k0-2 are rate constants through the different compartments
§ Peak concentration: C
(0)
=
1234
56

§ Area under the curve: AUC =
1234
56 7 '+, )

§ Clearance: (C
L
) =
1234
89:

3.5 MATERIALS AND METHOD S

88
Dose-normalized (dn) parameters (C
0dn
, AUC
dn
) were also calculated by dividing
the measured serum CIT concentrations by injected drug amounts (E14 = 0.9 ±
0.04; E18 = 1.17 ± 0.05 mg CIT) followed by fitting to a two-compartment model.
The biexponential equation terms of the model (Supp. Table 3.1) and the
calculated mean ± SD of each pharmacokinetic parameter were transferred to
GraphPad Prism software v6.0 (La Jolla, CA, USA) to generate concentration-
time profile graphs and to perform statistical analyses.

3.5.7 Statistical analysis
In order to avoid any potential litter effect, each data point was obtained from 3
independent dams for maternal pharmacokinetic analyses, or 3 fetuses pooled
from 3 independent dams for fetal pharmacokinetics analyses. Differences in
pharmacokinetic parameters between any two groups were analyzed for
statistical significance with unpaired two-tailed Student’s t-test. Comparisons
between more than two groups or added conditions were analyzed with one-way
ANOVA and adjusted for multiple comparisons with the Bonferroni correction.
Fetal:maternal drug concentration ratios between E14 and E18 were analyzed
using two-way ANOVA and adjusted for multiple comparisons with the Bonferroni
correction. All statistically significant differences were set at a level of P<0.05.

MATERNAL PHARMACOKINETICS AND FETAL
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89

3.6 SUPPORTING INFORMATION
Supplementary Figure 3.1: Maternal serum DCIT concentration-time profiles
during pregnancy.
Supplementary Table 3.1: Biexponential equation terms for maternal CIT
concentration-time profile models.
Supplementary Table 3.2: Body weights for adult CD-1 mice (non-pregnant) and
pregnant dams at GD14 and GD18.

Figure S3.1 Maternal serum DCIT concentration-time
profiles during pregnancy
Pregnant dams were administered 20 mg/kg CIT ip. The
DCIT concentrations in maternal serum, and fetal serum
and brain were measured by HPLC. (A) Data show the
means ± SD. The DCIT concentrations were measured in
maternal serum from 3.5 min to 3.5 h (N = 3 dams per
time point) at E14 and E18.
3.6 SUPPORTING INFORMATION

90

Table S3.1 Biexponential equation terms for maternal CIT concentration-
time profile models
Serum drug concentrations-time profiles were fitted to a two-compartment model
described by the following equation: C
(t)
= Ae
-αt
+ Be
-βt
(where C(t): serum
concentration at time t after dosing, A: fast exponential term, B: slow exponential
term, a: distribution rate constant, b: elimination rate constant using the
compartmental module of SAAM II software
MATERNAL PHARMACOKINETICS AND FETAL
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91

Table S3.2: Body weights for adult CD-1
mice (non-pregnant) and pregnant dams
at GD14 and GD18

92
4

COMBINATORIAL EFFECTS OF PRENATAL
EXPOSURES TO MATERNAL STRESS AND
ANTIDEPRESSANT ( CITALOPRAM) ON FETAL
BRAIN SEROTONERGIC NEURODEVELOPMENT
IN MICE

Juan C Velasquez, Yen Chan, Ligia Galindo, Clarissa James, Irina Burd*, and
Alexandre Bonnin

Neuroscience Graduate Program and Zilkha Neurogenetic Institute, Department
of Cell and Neurobiology, Keck School of Medicine, University of Southern
California, Los Angeles, CA 90089;
*Integrative Research Center for Fetal Medicine, Department of Gynecology and
Obstetrics, Johns Hopkins University School of Medicine,
Baltimore, MD 21287

Manuscript in preparation for submission in 2017
COMBINATORIAL EFFECTS OF PRENATAL EXPOSURES TO
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93
CHAPTER 4 POINTS OF INTEREST

SSRI exposure during pregnancy has been associated with multiple adverse fetal
outcomes in epidemiological studies. Given the transfer kinetics of CIT
(described in detail in Chapter 3) and other SSRIs, these medications taken to
alleviate symptoms of depression result in considerable fetal drug exposures
during pregnancy. However, epidemiological studies are often contradictory, with
some establishing increased risks of fetal exposure to SSRIs for negative effects
on offspring development (e.g. ASD, ADHD) and others finding no risk later on.
Given the unethical aspect of a randomized SSRI trial during human pregnancy,
animal models are key to find definitive answers about the risks posed by SSRIs
in the context of maternal depression. With this public health concern in mind, the
next chapter presents work in progress that will eventually compose a meaningful
contribution to questions regarding the safety of SSRI treatment during
pregnancy with key elements:
§ Validation of a stress model of maternal depression during pregnancy in
mice
§ Delineation of the independent effects of chronic maternal SSRI treatment
in the absence of stress
§ Evaluation of effects of chronic stress with and without SSRI exposure
§ A snapshot of the effects of these various treatments on fetal brain
serotonergic neurochemistry and circuit architecture before birth
§ Innovative diffusion magnetic resonance imaging of fetal brain structures
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4.1 INTRODUCTION
Neuropsychiatric disorders such as major depression and anxiety are highly
prevalent, begin early in life, and disproportionately affect women (Burt and
Stein, 2002; Kessler et al., 2007; Le Strat et al., 2011; Ornoy, 2017). With a
prevalence of about 15% during pregnancy, depression is a common
complication during gestation, and an increasing number of pregnant women are
prescribed selective serotonin (5-HT) reuptake inhibitor (SSRI) antidepressants
to treat and manage symptoms (Cooper et al., 2007; Toohey, 2012; Epstein et
al., 2014). Depression during pregnancy presents a therapeutic dilemma, as
epidemiological studies have associated early SSRI exposures to increased risks
of adverse fetal outcomes, including autism spectrum disorder (ASD), attention-
deficit/hyperactivity disorder (ADHD), depression and anxiety disorders, and
other sequelae that span broad developmental domains during the lifetime
(Hadjikhani, 2010b; Rai et al., 2013; Velasquez et al., 2013; Harrington et al.,
2014; Kaplan et al., 2016; Brown et al., 2017a, 2017b; Man et al., 2017; Sujan et
al., 2017). However, forgoing treatment to avoid fetal risks exposes both mother
and fetus to the effects of untreated depression and stress, which have
associations with adverse physiological and cognitive outcomes that also include
ADHD, behavioral and emotional regulation, developmental delays and
psychiatric disorders later in life (Fox et al.; Diego et al., 2004; Thompson et al.,
2011; Duman and Li, 2012; Velasquez et al., 2013; Glover, 2014).

4.1 INTRODUCTION

95
4.1.1 THE CASE OF ASD AND PRENATAL SSRI EXPOSURE

A major challenge in addressing this clinical dilemma lies in the innate
methodological constraints of epidemiological studies that link (or unlink)
increased risks of adverse outcomes with exposures to either maternal
depression or its treatment with SSRI antidepressants. For instance, much
attention has been shifted to the association between ASD and prenatal
exposures to SSRIs in recent years. Studies have reported increased risks
following (1) SSRI exposure during pregnancy (Croen et al., 2011a; Rai et al.,
2013; Gidaya et al., 2014; Harrington et al., 2014) , (2) untreated gestational
depression, not SSRI exposure (Ronald et al., 2011), and (3) found no increased
risks following maternal SSRI treatment (Hviid et al., 2013; Sorensen et al., 2013;
Clements et al., 2015; Brown et al., 2017b; Sujan et al., 2017). There is a clear
lack of consensus, rendering the causality between fetal exposure to SSRIs and
ASD inconclusive.
These observations highlight the uncertainty of whether SSRIs aggravate or
ameliorate the impact of maternal illness on the developing fetal brain. A
challenge that limits our understanding of prenatal exposures lies in
distinguishing between the effects of untreated maternal depression and stress
from those that are specifically induced by SSRIs. Considering high relapse rates
in depression (40-50%) in pregnant women using SSRIs, it is likely for a patient
to take medication without achieving euthymia, a scenario few epidemiological
studies account for. Additionally, it would be unethical to administer SSRI
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96

medications to a healthy, expectant woman for the purpose of testing the effects
of drug exposure alone.

4.1.2 UNTANGLING THE EFFECTS OF DEPRESSION FROM SSRI
EFFECTS
In response to the limitations posed by epidemiological studies, the present study
has been designed with this challenge in mind, and it aims to contribute to
delineating the independent effects of stress and depression from those that are
exclusively induced by exposure to SSRIs. Using a mouse model, we capitalized
on the established link between chronic exposure to stress as a critical
predisposing factor of depression and anxiety disorders in humans (Anisman and
Zacharko, 1990; Kessler, 1997; Kendler et al., 1998; Grant et al., 2005). This
study employs the chronic unpredictable stress (CUS) paradigm in mice to
induce anxiety- and depressive-like behaviors during pregnancy. We also
examine the effects of chronic oral administration of the widely-prescribed SSRI
(±)-Citalopram (CIT) during CUS exposure. We assess the effects of CIT
exposure in the absence of stress in addition to untreated control group. After
verifying the predictive validity of the CUS paradigm as a model for depression
during pregnancy, we evaluate the effects on fetal brain 5-HT neurochemistry,
serotonergic system development, and fetal brain structure volumes during late
pregnancy. The data reveal important changes triggered by maternal depression
and/or exposure to CIT that contribute to understanding the combinatorial effects
of prenatal exposures on fetal brain development.
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97
4.2 RESULTS

4.2.1 DEPRESSION - AND ANXIETY -LIKE BEHAVIORS
FOLLOWING GESTATIONAL CUS ARE REVERSED BY CIT
ADMINISTRATION DURING PREGNANCY
To initiate this study, we sought to implement a gestational CUS protocol and
establish its predictive validity by verifying the induction of anxiety- and
depression-like behaviors reversible by chronic administration of an SSRI
antidepressant in late pregnancy (Figure 4.1). Pregnant mice were exposed to a
sequence of daily, non-habituating, non-physically painful sequence of mild
stressors from embryonic day (E)8 to E17 (Figure 4.1A) (Mueller and Bale,
2008). In parallel, a group of dams was treated with oral CIT while also
undergoing CUS (CUS+CIT; Figure 4.1B). Animals in the untreated control and
CIT alone groups did not go through the daily CUS sequence of stressors.
Figure 4.1 Gestational chronic unpredictable stress experimental schedule
(A) Timed-pregnant mice were subjected to a daily sequence of stressors from E8 to
E17. (B) Only dams in the CUS and CUS+CIT groups were exposed to stressors.
Untreated control and oral CIT pregnant mice received routine food and water check-
ups and cage changes. All animals were assessed for anxiety- and depression-like
behaviors at E15 and E16 (noted by green arrows). o/n = overnight
4.2 RESULTS

98
In order to assess the effects of CUS and treatment with CIT on depression-like
behavior, pregnant mice were tested in the forced swim test (FST) at E16. Dams
undergoing CUS had the longest immobility time (246 ± 43 sec over a 6 min
testing period), an effect that was reversed by treatment with CIT during prenatal
stress (137 ± 53 sec; Figure 4.2A). The immobility time of CUS exposed dams
was significantly higher than all the other groups in the study (F
3, 27
= 7.477; P =
0.0293 to 0.0005). Interestingly, there were no immobility time differences
between untreated controls, CUS+CIT (Untreated = 176 ± 25 sec vs CUS+CIT =
137 ± 53; P = 0.5565) and CIT alone (167 ± 59 sec; P > 0.9999; Figure 4.2A).
There is a high degree of comorbidity of depression and anxiety disorders
(Mineka et al., 1998; Morilak and Frazer, 2004; Grant et al., 2005; Bondi et al.,
2008; Hamilton et al., 2015). Therefore, we evaluated the effects of CUS and
treatment with CIT on anxiety-like behavior on the open field test (OFT) at E15.
Dams undergoing CUS spent more time around the periphery than the center of
an OFT arena (3.1 ± 2 sec over a 30 min testing period; Figure 4.2B), a
noticeable effect that is evidenced by visual inspection of representative arena
traces generated during the acquisition phase of the OFT (Figure 4.2E-H).
Indeed, the time CUS-exposed dams spent in the center of the OFT arena was
significantly lower than untreated controls (7.2 ± 2 sec; F
3, 29
= 3.775; P = 0.0405;
Figure 4.2B, E-F). There were no differences in time spent in the center between
untreated controls, CUS+CIT (6.7 ± 2 sec; P > 0.9999; Figure 4.2B, F-G) and CIT
groups (7.2 ± 5 sec; P > 0.9999; Figure 4.2B, H).
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99

Figure 4.2 Gestational CUS induces depression- and anxiety-like behaviors on
pregnant mice
Pregnant mice were exposed to CUS and/or oral CIT from E8 to E17. (A) In the forced
swim test, CUS exposed dams had the longest immobility times compared to every other
study group. This effect was reversed by exposure to CIT during gestational CUS. Dams in
the untreated control group were not different than those receiving CIT. Data show the
mean ± SD time spent immobile in the FST at E16 (N = 7-9 dams per condition). (B) CUS
induced a significant decrease of time spent in the center during the OFT when compared
to untreated controls. There was no effect on CIT exposed pregnant mice. Data show the
mean ± SD time spent in the arena center during OFT at E15 (N = 6-9 dams per condition).
(C) There were no differences in activity levels as measured by total distance traveled
during OFT, regardless of exposure to CUS and/or CIT. Data show the mean ± SD
distance traveled during OFT at E15 (N = 6-9 dams per condition). (E-H) Representative
activity traces acquired during OFT sessions. The blue squares denote the boundary of the
center area quantified in (B). (D) Maternal serum corticosterone levels following CUS
and/or CIT exposures during pregnancy. There were no differences observed in basal
serum corticosterone concentrations among groups. Data represent the mean ± SD serum
corticosterone concentration at E17 (N = 5-8 dams per condition). Statistical differences for
all outcomes were analyzed by one-way ANOVA followed by Bonferroni adjustment for
multiple comparisons. *P < 0.05; **P < 0.01; ****P < 0.0001. Only statistically significant
differences between groups are indicated.
4.2 RESULTS

100
To ensure that time reduction in the OFT arena center and the immobility
changes in the FST were not due to nonspecific effects on general activity levels,
we also assessed locomotor activity during OFT. None of the groups, regardless
of gestational CUS or CIT exposure, showed an effect on overall locomotion as
measured by total distance traveled (Figure 4.2C; F
3, 27
= 0.5996; P > 0.9999).
To further investigate the effects of CUS on inducing a depressive-like state
during pregnancy, we also investigated whether the established association
between depression and increased corticosterone levels was consistent during
the mouse pregnancy (McEwen, 2002; Nestler et al., 2002; Anisman and Hayley,
2012; Fardet et al., 2012; Otte et al., 2016). Basal corticosterone concentrations
were determined from maternal serum collected approximately 3 hours after the
last stressor at E17. We found that serum corticosterone levels following CUS
and/or CIT exposure were not different compared to untreated controls (Figure
4.2D; F
3, 24
= 0.2944; P > 0.9999).
The behavioral results above are consistent with previously published
observations made in non-pregnant mice. (Richardson et al., 2006; Yang et al.,
2006; Bourke et al., 2013, 2014b; Sarro et al., 2014; Van den Hove et al., 2014).
In the FST, pregnant mice exposed to CUS showed increased immobility,
whereas mice exposed to CUS+CIT did not show elevated immobility, consistent
with results obtained in non-pregnant mice (Rygula et al., 2006; Warner-Schmidt
et al., 2011).

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101
4.2. 2 EXPOSURE TO MATERNAL CUS AND CIT
DIFFERENTIALLY AFFECT NEUROCHEMISTRY AND
NEURAL ARCHITECTURE OF FETAL FOREBRAIN 5 -HT
Animal studies have shown that developmental exposure to SSRIs affects
serotonergic innervation and results in anxiety- and depression-like behaviors in
adulthood (Ansorge et al., 2004a, 2008a; Bairy et al., 2007; Shanahan et al.,
2009; Altieri et al., 2015). The mechanisms by which SSRI exposures result in
these postnatal outcomes are unknown, particularly in the context of underlying
maternal stress conditions, also one of the earliest adverse experiences with
long-term effects on the offspring (Peters, 1990; Hayashi et al., 1998; Pilowsky et
al., 2006; Talge et al., 2007; Deave et al., 2008; Paulson et al., 2009; Bick and
Nelson, 2015). We have previously shown that CIT readily crosses the placenta
into the fetal circulation, where it could potentially target the development of the
serotonergic system (see Chapter 3).

To investigate the potential effects of maternal exposure to CUS and/or CIT,
we measured fetal forebrain tissue concentrations of 5-HT at E17. Fetal forebrain
serotonin levels in embryos exposed to maternal CUS were significantly higher
than all other groups in the study (3.0 ± 0.34 ng/mg; Figure 4.3A; F
3, 28
= 53.04; P
< 0.0001). In stark contrast, maternal CIT exposure in the absence of CUS led to
the significantly lowest levels of forebrain 5-HT in the study (1.7 ± 0.20 ng/mg;
Figure 3A; P = 0.0062 to < 0.0001).
4.2 RESULTS

102
Figure 4.3 Fetal forebrain 5-HT concentration and serotonergic outgrowth are
differentially affected by gestational exposure to CUS and CIT
Pregnant mice were exposed to CUS and/or oral CIT from E8 to E17. (A) Maternal CUS induced
a significant elevation of tissue 5-HT concentration in the fetal forebrain, in contrast to the
decrease observed following exposure to maternal CIT. Forebrain serotonin levels following co-
exposure to CUS+CIT were not different from untreated controls. Data show the mean ± SD
forebrain 5-HT tissue concentrations at E17. Serotonin was quantified by HPLC and measures
were normalized to total forebrain protein concentrations (N = 7-9 dams per condition, 3-4 fetal
brain samples per dam). Statistical differences were analyzed by one-way ANOVA followed by
Bonferroni adjustment for multiple comparisons. *P < 0.05; **P < 0.01; ****P < 0.0001. Only
statistically significant differences between groups are indicated. (E) Serotonergic axons were
immunostained on coronal E17 brain sections at different rostro-caudal levels following
gestational exposures to CUS and CIT. Serotonin immunostaining in the dorsal thalamus (DT)
region appeared to be affected in the ventroposteriolateral thalamic nuclei (vpl; E, top panels) but
not the lateral hypothalamic area (Hyp; E bottom panel). (B-D) Quantifications of 5-HT+ axon
density (normalized fluorescence intensity) confirmed an increase in serotonin immunoreactivity
following CUS and a decrease after CIT exposures in the vpl (B) but not the Hyp (C) nor the
medial forebrain bundle (mfb) (D). (F) 5-HT immunofluorescence quantification along
thalamocortical axons (TCA) pathway in the cortex. CUS increased 5-HT immunofluorescence
intensity in TCAs. Data show the mean ± SD normalized pixel intensity in each region of interest
(N = 4 dams per condition, 1 fetal brain per dam). Statistical differences were analyzed by one-
way ANOVA followed by Bonferroni adjustment for multiple comparisons. *P < 0.05; **P < 0.01;
****P < 0.0001.
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Interestingly, in forebrains of embryos exposed to maternal CUS+CIT 5-HT
concentrations were not different from levels measured in untreated controls
(Untreated = 2.2 ± 0.12 ng/mg vs CUS+CIT = 2.1 ± 0.11; Figure 4.3A; P >
0.9999).
The observed disruption of normal serotonin levels following CUS or CIT
exposures could originate from changes in serotonergic axon outgrowth into the
forebrain. We explored this possibility by performing 5-HT immunostaining
through the rostrocaudal axis of the forebrain at E17. We compared coronal
sections from untreated control brains with brains exposed to CUS, CUS+CIT,
and CIT at matching level and brain regions. Following visual inspection, we
performed densitometric analysis of 5-HT immunofluorescence intensity along
the caudal-to-rostral extent of the forebrain, in selected regions of interest. We
found that CUS led to the largest increase in 5-HT immunofluorescent intensity in
cell bodies located in the ventroposteriolateral thalamic nucleus (vpl) of the
dorsal thalamus (DT) when compared to all other conditions (Figure 4.3B, E; F
3,
12
= 17 ; P = 0.0419 to < 0.0001). Also consistent with HPLC 5-HT forebrain
measures, brains exposed to maternal CIT without CUS had significantly
decreased 5-HT immunoreactivity in the vpl when compared to untreated
controls (Figure 4.3B,E top panels; P < 0.0001). Additionally, overall 5-HT
immunofluorescence intensity in the vpl of CUS+CIT brains were not different
than controls (P > 0.9999). We also evaluated the lateral hypothalamic area and
found no differences in 5-HT immunofluorescence intensity between groups
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104
(Figure 4.3C, E top panels; F
3, 12
= 1.429 ; P = 0.6229 to > 0.9999). When we
evaluated the immunofluorescence of 5-HT+ axons that grow ventrally into the
forebrain through the rostral part of the medial forebrain bundle (mfb), we did not
find any differences across groups (Figure 4.3D, E bottom panels; F
3, 11
=
0.0611 ; P > 0.9999). Since DT neurons located in the vpl send axonal
projections into the cortex, we next assessed if 5-HT immunofluorescence
intensity was affected along the 5-HT-uptaking thalamocortical axons (TCAs).
Although the overall pathway of TCAs was similar across all groups (that’s were
a panel with NetG1 staining would be useful), we found that TCAs in CUS brains
had significantly higher 5-HT immunofluorescence intensity when compared to
the other groups in the study (Figure 4.3F; F
3, 200
= 44.81 ; P < 0.0001) and that
TCAs from CIT exposed brains had the lowest 5-HT immunoreactivity (Figure
4.3E; P = 0.0005 to < 0.0001). These changes revealed by densitometric
analysis of 5-HT immunofluorescence in the DT and TCA projections appear
overall consistent with the effects observed in tissue concentrations of 5-HT in
the fetal forebrain.

4.2. 3 DEVELOPMENTAL EFFECTS OF CUS EXPOSURE ON
HINDBRAIN 5 -HT ARE REVERSED BY CIT
ADMINISTRATION

Serotonergic neurons and axons compose one of the most ubiquitous circuits in
the brain. 5-HT cell bodies emerge early in development (~E10 in mice) and
cluster in multiple nuclei, including the dorsal raphe nucleus (DR) in the fetal
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hindbrain. The DR serotonergic neuron population forms vast axonal projections
that innervate the forebrain in a caudal-to-rostral gradient (Azmitia and Segal,
1978; Hawthorne et al., 2010; Pollak Dorocic et al., 2014). After noting the
distinct effects that CUS and CIT exposures individually have on fetal forebrain
serotonergic development, we wondered if those changes had origins that traced
back to the hindbrain and the DR. We measured tissue levels of hindbrain 5-HT
and noted that, on average, CUS exposure led to a 19% increase in
concentrations over untreated control hindbrains (Figure 4.4A; CUS = 5.285 ±
0.92 ng/mg; Untreated = 4.372 ± 0.49 ng/mg). Although this effect did not
represent a statistical significant difference between these 2 groups (F
3, 28
=
6.453 ; P = 0.0806), both CUS+CIT and CIT exposed hindbrains had significantly
reduced serotonin concentrations compared to the CUS group (P = 0.0092 to
0.0028). Both of these groups exposed to CIT were no different than untreated
controls either (Figure 4.4A; P > 0.9999). However, coronal brain sections
immunostained with an anti-5-HT antibody in the DR region revealed a greater
number of serotonergic cell bodies in the lateral wing of the DR in CUS
hindbrains compared to untreated controls (Figure 4.4B-D; F
3,12
= 4.903; P =
0.0271). Maternal administration of CIT during CUS reversed this increase in 5-
HT cell body number (Figure 4.4B, D-E; P = 0.0474). There was no difference
between both groups exposed to CIT and untreated control hindbrains (Figure
4.4C,E,F; P > 0.9999). Together, these results suggest that CUS leads to a
significantly greater number of serotonergic cell bodies in the lateral wing of the
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106
DR and a modest increase in overall tissue 5-HT concentration in the hindbrain.
This effect is reversed to normal untreated levels by oral CIT treatment during
gestation. CIT in the absence of gestational CUS does not affect hindbrain 5-HT
tissue concentration or 5-HT neuron number.
Figure 4.4 Effects on fetal hindbrain 5-HT following gestational exposure to
CUS and CIT
Pregnant mice were exposed to CUS and/or oral CIT from E8 to E17. (A) Maternal
CUS increased the tissue concentration of 5-HT in the fetal hindbrain only when
compared to CIT exposed groups. None of the groups differed from untreated
controls. Data show the mean ± SD hindbrain 5-HT tissue concentrations at E17.
Serotonin was quantified by HPLC and measures were normalized to total
hindbrain protein concentrations (N = 7-9 dams per condition, 3-4 fetal brain
samples per dam). (B) Quantification of 5-HT+ cell bodies in the lateral wing
region of the DR immunolabeled with an anti-5-HT antibody on coronal brain
sections at E17 (C-F). Gestational exposure to CUS induced a significant increase
of 5-HT+ cell body counts (D). There were no differences between the control (C)
and CIT exposed groups (E, F). Data show the mean ± SD number of 5-HT+ cell
bodies in the DR. (N = 3-4 dams per condition, 1 fetal brain per dam). Statistical
differences in serotonin concentrations (A) and number of 5-HT+ cell bodies (B)
were analyzed by one-way ANOVA followed by Bonferroni adjustment for multiple
comparisons. *P < 0.05; **P < 0.01; ****P < 0.0001. Only statistically significant
differences between groups are indicated. DR = dorsal raphe
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4.2. 4 PRELIMINARY DIFFUSION MAGNETIC RESONANCE
IMAGING OF THE EMBRYONIC MOUSE BRAIN
FOLLOWING GESTATIONAL EXPOSURE TO CUS AND CIT

After gaining insight into the 5-HT neurochemistry and circuitry architectural
changes following exposures to CUS and CIT, we explored the feasibility of in
utero dMRI of the embryonic mouse brain to delineate more globally specific fetal
brain structures that may be affected. This novel technique provides superb
tissue contrast and allows for the clear identification of the major gray and white
matter structures in post mortem embryonic brains (Figure 4.5A) (Wu et al.,
2015; Wu and Zhang, 2016). We observed profound effects of exposures to CUS
and to CIT in our 5-HT densitometric analysis of the DT and TCAs at E17 (Figure
4.3B, E-F). Since DT regions send distinct axonal projections to the cortex,
amygdala, and the hippocampal formation, we focused on delineating those
structures in addition to the thalamus and the brain overall and assessing
differences in structural volume.
Our preliminary analysis of male fetal brain samples showed no significant
differences in volume in any of those structures across our treatment groups
(Figure 4.5B-F; overall P = 0.2410 to > 0.9999). This portion of our study may,
however, reveal important findings when pending analyses increase the sample
size and female brain samples are added.

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Figure 4.5 Diffusion magnetic resonance imaging (dMRI) in the E17
embryonic brain following maternal CUS and/or CIT.
A) dMRI and dMRI-based tractography clearly delineate the major gray matter
and white matter structures, e.g., the cortical plate (CP), intermediate zone (IZ),
cerebral peduncle (cp), optic tract (opt). (B-F) Preliminary results indicate that
exposure to maternal CUS and/or CIT does not affect the volume of specific fetal
brain regions. Brain region volumes were measured at E17 by dMRI. Data show
the mean ± SD volume of specific fetal brain structures. (N = 2-3 dams per
condition, 1-2 fetal brain per dam). Statistical differences were analyzed by one-
way ANOVA followed by Bonferroni adjustment for multiple comparisons. *P <
0.05; **P < 0.01; ****P < 0.0001. Only statistically significant differences between
groups are indicated.
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4. 3 DISCUSSION

The purpose of the present study was to verify the predictive validity of the CUS
paradigm as a model of gestational depression in mice and to evaluate the
combinatorial impact of prenatal stress and exposure to CIT on fetal brain
development. The development of animal models of depression has capitalized
on the observations that stressful life events often predict and precede the onset
of depressive episodes. The CUS paradigm has been shown to produce anxiety-
and depressive-like behaviors in non-pregnant rodents, and numerous studies
have highlighted that chronic administration of antidepressant drugs prevents
behaviors induced by stress (Katz, 1982; Stamford et al., 1991; Monleon et al.,
1995; Mineur et al., 2006; Bairy et al., 2007; Bondi et al., 2008; Mueller and Bale,
2008; Cox et al., 2011; Zhu et al., 2014).
Several studies have demonstrated that CUS leads to anxiety- and
depression-like behaviors in non-pregnant, male rodents, highlighting that
chronic administration of antidepressant drugs prevents behaviors induced by
stressed rodents (Katz, 1982; Stamford et al., 1991; Monleon et al., 1995; Cryan
and Holmes, 2005; Mueller and Bale, 2008; Richardson et al., 2006; Yang et al.,
2006; Mineur et al., 2006; Mueller and Bale, 2006; Bairy et al., 2007; Bondi et al.,
2008; Cox et al., 2011; Bourke et al., 2013, 2014c; Sarro et al., 2014; Van den
Hove et al., 2014; Zhu et al., 2014). Although some animal studies have
employed the use of chronic stress procedures during pregnancy, these have
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110
only shown the induction of depression-like behaviors in the postpartum period
(Hillerer et al., 2011; Leuner et al., 2014; Haim et al., 2016) and others are
exclusively focused on outcomes for the offspring later in life (Alonso et al., 1991;
Mueller and Bale, 2006, 2008; Benoit et al., 2015). To our knowledge, and as of
this writing, only recently did Salari et al. assess the CUS paradigm for its
efficacy in producing depression-like behaviors reversed by the SSRI fluoxetine
in pregnant mice (Salari et al., 2016).
Consistent with this recent report, we also found that pregnant mice exposed
to CUS from E8 to E17 display anxiety- and depressive-like behaviors as
revealed by OFT and FST, respectively (Figure 4.2). Notably, CUS-exposed mice
spent significantly less time in the center of an open field compared to mice in all
other conditions. There were no effects on locomotion behavior. These results
are consistent with previously published observations made in non-pregnant
mice (Richardson et al., 2006; Yang et al., 2006; Bourke et al., 2013, 2014a; Van
den Hove et al., 2014; Salari et al., 2016). In the forced swim test (FST),
pregnant mice exposed to CUS showed increased immobility, whereas mice
exposed to CUS + CIT delivered through the drinking water from E8 to E17 did
not show elevated immobility. Consistent with results obtained in non-pregnant
mice (Rygula and Abumaria, 2006; Warner-Schmidt et al., 2011), and therefore
validating the behavioral effects of CIT in pregnant mice. Interestingly, CIT
exposure in the absence of CUS had no effects on maternal behavior, as there
were no differences between untreated controls and CIT alone groups. As a
stress measure of the CUS treatment paradigm, maternal corticosteroid
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concentrations were also measured at baseline. Despite the effects of CUS and CIT
on maternal behaviors, we did not find any differences across conditions. This
indicates that CUS does not affect the steady-state baseline corticosterone
concentration in the maternal serum.
Once we had established that CUS induced depression-like behaviors during
pregnancy, we investigated the effects on the fetal brain. Animal studies have
shown that pre- and postnatal SSRI exposure reduces adult serotonergic
innervation and elicits depressive- and anxiety-like behaviors in adulthood (Bairy
et al., 2007; Ansorge et al., 2008a; Shanahan et al., 2009; Altieri et al., 2015).
However, the mechanisms by which prenatal SSRI exposure alters circuit
formation and behaviors in adult offspring are unknown, particularly in the context
of an underlying maternal stress condition. We used the widely prescribed SSRI
antidepressant CIT. We have intricately detailed the pharmacokinetics of CIT
during pregnancy and found that it readily crosses the placenta to reach the fetal
bloodstream and brain. More importantly, Desmethylcitalopram (DCIT), the
primary metabolite of CIT, also crosses the placenta, and with a powerful binding
affinity for its primary target, the serotonin transporter (SERT), it also acts as an
SSRI (Velasquez et al., 2016). This combination of CIT and DCIT could directly
affect brain development of the offspring because these molecules specifically
block the 5-HT transporter (SERT) and therefore affect 5-HT signaling, which
plays important modulatory roles in neurotrophic processes involved in
serotonergic and thalamocortical circuit wiring (Gaspar et al., 2003) in the fetal
brain. Because these circuits underlie social, emotional, and cognitive higher
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112
function, disruption of normal 5-HT levels by SSRIs and/or maternal stress can
lead to life-long changes in brain circuitry and thus, function (Bonnin et al., 2011).
With our experimental design, we were able to distinguish between the effects
of CUS from those of CIT in addition to the combination of both CUS+CIT.
Our research into the serotonin neurochemistry of the fetal brain revealed
differential effects between CUS and CIT. In the forebrain, there is a consistent,
significant trend in our data, namely that CUS increases serotonin levels and that
CIT lowers them below untreated control levels. Like balancing between two
opposing forces, the effect of exposure to both CUS+CIT resulted in normal 5-HT
levels that were no different than controls. Importantly, these observations
contradict the common assumption that SSRIs increase 5-HT in the fetal brain,
when in actuality, the opposite occurs.
Given that CUS+CIT forebrains have normal 5-HT levels, it would be easy
to conclude that there are no effects or risks in taking SSRIs during pregnancy in
the context of depression for fetal brain development (at least in terms of
serotonin-dependent neurodevelopmental processes). However,
immunohistochemical analysis in the forebrain shows a more complex picture.
Both CUS+CIT and CIT groups have significantly different localization of
serotonin immunoreactivity, that is markedly reduced in the DT region and
extends to the cortex through TCAs. Contrasting this effect, CUS exposed brains
have significant increases in 5-HT immunofluorescence in these regions.
Together, these observations are suggestive that increased 5-HT concentrations
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are the result of increased 5-HT uptake in thalamic neurons/axons and possibly
increased serotonin neuron/axon densities. The latter however is unlikely given
that 5-HT immunofluorescence in the mfb (which contains the bulk of
serotonergic axons growing into the forebrain) was not affected by any of the
treatment. In contrast, prenatal exposure to the uptake blocker CIT reduced 5-HT
immunofluorescence intensity in thalamic neurons and axons. This observation is
consistent with the possibility that changes in 5-HT uptake into TCAs and DT
neurons are primarily driving the changes in 5-HT tissue concentrations
measured by HPLC. Although the effects on the hindbrain are not as significant,
we still noted an increase in the number of serotonergic cell bodies in the DR
despite modest increases in tissue concentration of 5-HT.
Taken together, the findings analyzed here indicate that both gestational
stress and exposure to CIT result in abnormal tissue concentration or distribution
of 5-HT during a critical time in brain development. While the use of a CIT
antidepressant had generally positive results for the maternal behaviors,
exposures to CUS, CIT, and both CUS+CIT result in an imbalance of serotonin
neurochemistry or tissue distribution that potentially alters fetal brain
development, having a lasting impact. Important future directions include the
evaluation of long term effects on offspring brain neurochemistry, circuit
architecture and behavior. Inclusion of other biogenic monoamines such as
dopamine (DA) and metabolites, which are also associated with depression and
other psychiatric conditions, would be an important assessment as mesolimbic DA
pathways(Cabib and Puglisi-Allegra, 2012), DA transporter levels(Isovich et al.,
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114
2000; Lucas et al., 2004), and sensitization of mesocortical DA responses(Chrapusta
et al., 1997; Cuadra et al., 1999) has been shown to be affected following exposure
to stress in adult rodents. Given the effects on 5-HT in TCAs following prenatal
exposures to CUS and CIT, abnormal levels of 5-HT in these axons(Lebrand et al.,
1996b) could alter the postnatal formation of barrel field structures in the somatosensory
cortex (Alvarez et al., 2002; Rebsam et al., 2002; van Kleef et al., 2012).
However, a lack of 5-HT in TCAs appears more likely to lead to a delay in
somatosensory cortical barrel formation, rather than over altered development (see Van
Kleef et al., 2012). Although an effect of delayed innervation might only be transient
(Persico et al., 2000, 2001) it could potentially be aggravated by re-occurring insults (e.g.
stress) during the early postnatal period. It will be important to test this possibility when
assessing the long-term effects of these prenatal exposures on offspring brain function.
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4.4 MATERIALS AND METHOD S
Animals
Timed-pregnant CD-1 mice were obtained from Charles River Laboratories at E7
(Wilmington, Massachusetts). Mice were age-matched (8-10 weeks of age) and
were assigned to one of the following groups at E8: untreated control, CUS,
CIT+CIT, and oral CIT. All mice were housed in groups of 2-3 dams in standard
animal facility cages, maintained under 12h:12h light-dark cycles, and with food
and water provided ad libitum. All procedures using mice were approved by the
Institutional Animal Care and Use Committee at the University of Southern
California and the Johns Hopkins University (dMRI study) and conformed to NIH
guidelines. At the end of the study at E17, animals were euthanized under
isofluorane anesthesia (Western Medical Supply) by cervical dislocation followed
by cardiac puncture. Maternal blood was collected in heparinized tubes (BD
Biosciences; 367812) and centrifuged at 2000 x g for 20 min at 4°C for serum
isolation. The uterus was carefully dissected and the embryos were placed in ice-
cold phosphate buffered saline (PBS). Fetal brain samples were also separated
(precollicular coronal bisection to separate the forebrain+midbrain (termed
forebrain) from the hindbrain). All samples were flash frozen in liquid nitrogen
and stored at -80°C until analysis

Chronic Unpredictable Stress
Following arrival of timed-pregnant mice at E7, they were allowed to acclimate for
24 hours prior to experimental group assignment at E8 and starting the sequence
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116
of CUS stressors. Pregnant mice in the CUS and the CUS+CIT groups were
subjected to the CUS series of stressors during mid- to late-gestation (E8-17;
Figure 4.1). The stressors included: 36 hour of constant light exposure, 15
minutes of fox odor (1:5000; 2,4,5-trimethylthiazole; Sigma) during light cycle, 3-
hour social isolation, intra-peritoneal saline injection, stress during light cycle,
white noise exposure (70 dB) overnight, 45-degree angle cage tilt overnight, no
bedding overnight, and saturated bedding overnight. The CUS paradigm is non-
habituating, not-physically painful, and does not affect maternal food intake or
weight gain (Mueller and Bale, 2008). The exposure period between E8 and E17
of mouse pregnancy are roughly equivalent to the first and second trimesters in
humans (Cox et al., 2009) and take place after the placenta has acquired its
definitive structure. These are also periods of active neurogenesis and axonal
pathway formation in the fetal brain, processes that are modulated by 5-HT and
could therefore be impacted directly by CUS and maternal-fetal transfer of CIT
(Rossant and Cross, 2001; Kepser and Homberg, 2015).

Citalopram administration
Dams assigned to the CUS+CIT and CIT groups were administered CIT in the
drinking water (260 mg/L) from E8 through E17 supplemented with 1.5% sucrose
to mask adverse taste of drug. The concentration is based on values that
provide efficacious antidepressant effects in mice (Jiao et al., 2011; Warner-
Schmidt et al., 2011). We confirmed steady-state levels of CIT in the maternal
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serum after 5 days of dam exposure through drinking water (steady state level of
117.9 ng/mL at E17 – data not shown), which falls within the therapeutic drug
concentration range measured in humans (30-200 ng/mL (Meng and Gauthier,
2005), and is also consistent with previously published values (Rygula and
Abumaria, 2006; Jiao et al., 2011). CIT water consumption was assessed and
replaced every 4 days.

Quantification of Fetal Brain 5-HT
Sample preparation for HPLC
A liquid-liquid extraction was used to prepare samples for analysis as previously
described (Velasquez and Bonnin, 2015; Goeden et al., 2016; Velasquez et al.,
2016). Briefly, samples were thawed on ice and extracted with ice-cold extraction
buffer (0.2 M perchloric acid + 500 mM D-mannitol + 100 μM EDTA) with
isoproterenol as internal standard. The extraction buffer was added to fetal
serum and microsomal incubations (1:1 v/v) and to maternal serum samples (1:3
v/v). Brain samples were extracted by addition of buffer (forebrain = 175 μL;
hindbrain = 150 μL) followed by sonication (Qsonica; 35% amplification, 15 sec).
The samples were kept on ice for 30 min before centrifugation 20,000 x g for 15
min at 4°C. The supernatant volume was measured and a 10-μL aliquot was
injected per sample for HPLC analysis. The resulting pellets were used to
measure protein concentrations in brain samples using a Bradford assay.

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118
HPLC Chromatographic Conditions
Neurochemical measures of all samples were done with High Pressure Liquid
Chromatography coupled to an Electrochemical Detector (HPLC-ECD). The
analysis was performed using an Eicom 700 system (Eicom Corpotation, Kyoto,
Japan) consisting of an Eicom ECD-700 detector and column oven, an Eicom
700 Insight autosampler, and Envision integration software. An Eicompak SC-
30DS C
18
reversed-phase column packed with 3-μm silica particles (3.0 x 100-
mm I.D.) was used as the analytical column. Chromatographic conditions were
set as previously described (Velasquez and Bonnin, 2015; Goeden et al., 2016).
Briefly, a 10 μL aliquot of extracted sample was injected into the column
maintained at 25°C and eluted with a mobile phase consisting of 0.1 M citric
acetate buffer, 18.5% methanol, 150 mg/L sodium octyl sulfate (SOS), and 5
mg/L EDTA-2Na. The flow rate was set at 400 μL/min. The retention time and
limit of detection of 5-HT was 16.5 min and 100 pg/mL.

Open Field Test
The dams (E16) were each introduced into an open field test arena, and their
behavior was monitored and recorded for 30 minutes. The open field arena
(40cm x 40cm x 30 cm) is a standard behavioral model that assesses anxiety
states in rodents in which anxious mice behavior is to avoid open, unprotected
areas, with preference for peripheral areas. The floor space was divided into
distinct regions with the peripheral arena defined as a distance of 2.5 cm from
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edge of apparatus, the middle arena as 15cm x 15cm, and the center arena as
2cm x 2cm. Motor activity and distance traveled were recorded with a video
camera. The amount of time in which the mouse stayed in the center of the
arena in the OFT and the total distance traveled were measured, recorded and
analyzed by a video tracking system (Smart v3.0 software, Harvard Apparatus,
Holliston, MA, USA) and confirmed by a trained and blinded observer. All
analyses were performed blind.

Forced Swim Test
The dams (E17) were each introduced into a water-filled glass cylinder (30cm
height x 20cm diameter) containing 15cm of water filled with 24 ± 1 C water) for 6
minutes. The water glass cylinder test also known as the “behavioral despair
test” is a standard behavioral model that assesses “despair” and “depressive”
states in rodents in which depressed mice behavior is to attempt to escape
initially, but eventually the mice take on a posture of immobility; shorter time to
and duration of immobility is interpreted as greater despair. Swimming activity
was recorded with 2 video cameras, one located in front of and the other one
above the cylinder. Immediately after the swim test, the mice were dried with
towels and placed under a heating lamp until they were completely dry. Water in
the cylinder was replaced between subjects. The total duration of swimming and
immobility was recorded and measured and analyzed by a software activity
tracking system (Smart v3.0 software, Harvard Apparatus USA, Holliston, MA,
4.4 MATERIALS AND METHODS

120
USA) and confirmed by trained and blinded observers. The duration of immobility
was measured as the time spent without any motion except for a single limb
paddling to keep the nose above the water surface and maintain floating. All
analyses were performed blind.

Immunohistochemistry
Fetal brains were immersion-fixed and cryosectioned. Serotonergic neurons and
axon projections were identified by IHC and measured in series of 20 μm-thick
coronal sections encompassing the whole fetal brain, using quantitative density
measures as previously described (Eagleson et al., 2005; Luellen et al., 2006,
2007, Bonnin et al., 2007a, 2011; Goeden et al., 2016). Briefly, 5-HT
immunofluorescence intensity at E17 was measured in coronal planes on at least
3 sections per embryo that encompass caudal to rostral regions of the fetal
forebrain. Three regions were delimited for quantification: 1) from the medial
cortex to the claustrum (for TCA analysis), and 2) from the hypothalamus midline
to the internal capsule, including the medial forebrain bundle and 3) the dorsal
thalamus region at the ventroposteriolateral nuclei and the laternal hypothalamic
region. Pixel intensity was be normalized and expressed as a percentage of the
maximal value measured inside the region of interest (ROI). The ROI was be
straightened with ImageJ software and immuno-fluorescence intensity measured
along the rectangular selection as ‘gray value’ such that the x-axis represented
the relative distance between the two landmarks used to define the ROI and the
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y-axis the vertically averaged pixel intensity. Pixel intensity was then normalized
by subtracting the average background intensity (measured from an unlabeled
area) and expressed as a percentage of the maximal value measured inside the
ROI.

Determination of Corticosterone Concentration
Maternal and fetal serum corticosterone concentrations were measured using the
corticosterone mouse Enzyme-linked immunosorbent assay (ELISA) Kit (Enzo
Life Sciences, Inc., Farmingdale, NY, USA) with a sensitivity of 27 pg/mL and
compared between the groups. The optical density (OD) of the sample was
determined at 450 nm using the SpectraMax i3 Multi-Mode microplate reader
(Molecular Devices, Sunnyvale, CA, USA) after the reader was zeroed using a
blank well. The concentration of each sample was extrapolated from a standard
curve. Each determination was performed in duplicate. The variation between
duplicate values was all less than 5%.

Diffusion Magnetic Resonance Imaging
Ex vivo dMRI-based tractography was performed with an 11.7 Tesla vertical bore
magnet to reveal white matter tracts in the E17 mouse brain. dMRI was
performed on post mortem whole male embryos fixed overnight in 4%
paraformaldehyde and shipped to Johns Hopkins University for analysis in
collaboration with Dr. Burd.
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122

Statistical Analysis
The number of dams required was calculated based on power analysis assuming
a similar degree of variability observed in preliminary and other related
studies,(Bonnin et al., 2007a, 2011) with the aim of detecting differences
between groups that are 1-2 standard deviations from the mean and the
probability of type I error α = 0.05. All data are expressed as the mean ±
standard deviation (S.D.). Significant differences were determined using one-
way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test for
multiple comparisons or unpaired two-tailed Student’s t-test for comparisons
between two groups. P < 0.05 was considered to indicate a statistically
significant difference. All analyses were performed using GraphPad Prism 6 (La
Jolla, CA, USA).

124
5

CONCLUSION AND FUTURE DIRECTIONS

Nevertheless the difference in mind between man and the higher animals, great
as it is, certainly is one of degree and not of kind. We have seen that the senses
and intuitions, the various emotions and faculties, such as love, memory,
attention, curiosity, imitation, reason, etc., of which man boasts, may be found in
an incipient, or even sometimes in a well-developed condition, in the lower
animals.

— Charles Darwin, The Descent of Man, page 86 (1871).

5.1 OVERVIEW

Depression is known to have powerful effects, both for those directly afflicted and
the people around them. This common mental disorder affects 350 million people
around the world, the majority being women who face the highest risk of a
depressive episode during childbearing years (Kessler et al., 2007; Olivier et al.,
2015). About 15-18% of pregnant women experience symptoms (Gavin et al.,
2005; Biaggi et al., 2016), a majority of whom are prescribed selective serotonin
reuptake inhibitors (SSRIs) antidepressants (Cooper et al., 2007). Although not
CONCLUSION AND FUTURE DIRECTIONS

125
without controversy, the use of SSRIs during pregnancy has increased
significantly in the last decades (Cooper et al., 2007; Bourke et al., 2014a; Zoega
et al., 2015). As discussed in Chapter 1, epidemiological studies linking
gestational SSRI exposures to increased risks for a constellation of congenital,
developmental, cognitive and physiological outcomes abound. Nonetheless,
depression left untreated is far from harmless, as it is also associated with
multiple increased risks for consequences that span the lifetime.
SSRIs target the serotonin (5-HT) transporter and block its function, thereby
increasing extracellular 5-HT concentration. During pregnancy, serotonin is
involved in multiple complex and dynamic processes that are crucial for the
development of the brain. Critical histogenic processes such as neuronal cell
proliferation, migration, and brain circuit wiring take place in specific temporal
and spatial patterns, and 5-HT is a key player in modulating these developmental
milestones. Early in development, the placenta is the primary source of 5-HT to
the fetal forebrain. It does so by synthesizing 5-HT from maternally derived
tryptophan (TRP). It is possible that perturbations of placental physiology and
TRP metabolism may also affect 5-HT concentrations in the fetal brain. It follows
that anything that alters 5-HT concentration may impact the processes that result
in proper brain development and function.
Indeed, whether to treat depression with SSRIs or to leave it untreated
comprise a major therapeutic dilemma in obstetrics today. This is obscured
further by epidemiological studies, as they are innately unable to disentangle the
5.2 SUMMARY OF FINDINGS

126
associations between SSRI exposure from those exclusively linked to maternal
depression. Methodological and ethical constraints prevent us from randomizing
exposure to SSRIs during pregnancy. In response, this dissertation turned to
animal studies to evaluate the effects that SSRIs have on fetal brain
development independently of maternal stress/depression.
Chapter 2 details methodological approaches to explore these questions,
namely ex vivo dual perfusion of the mouse placenta for SSRI transplacental
transfer studies, designing in vivo studies of maternal to fetal SSRI transfer,
determination of SSRI drug concentrations and biogenic monoamine analyses in
biological samples by HPLC, and IHC of fetal brains to study serotonergic system
circuit development. These methods were carefully applied throughout this
dissertation to begin to elucidate how early developmental exposures to SSRIs
and maternal stress affect the fetal brain. As more mechanistic influences of 5-
HT signaling on various aspects of fetal brain development and their long-term
consequences are being uncovered, this signaling pathway will undoubtedly
become a central tenet of the developmental programming of adult mental
disorders.

5.2 SUMMARY OF FINDINGS

The first question I posed in this dissertation was whether SSRIs cross the
placenta and reach the fetal compartment. Using a mouse animal model, I
explored the detailed pharmacokinetics of the SSRI Citalopram during
CONCLUSION AND FUTURE DIRECTIONS

127
pregnancy. This subject matter is of particular importance, as it provides insight
into answering questions posed earlier in chapters 1 and 2: how might fetal SSRI
exposures affect 5-HT development through the highly dynamic time of
pregnancy?
I showed that CIT and its metabolite desmethylcitalopram (DCIT) cross the
placenta using ex vivo perfusion systems in both mouse and human.
Interestingly, I found that the transfer index of CIT was not different between
these two species, thereby raising the translational value of the mouse placental
perfusion system as it pertains to placental drug transfer. I also demonstrated in
vivo that CIT and DCIT are rapidly transferred from the maternal to the fetal
circulation and reach the fetal brain during both early and late gestation following
maternal intraperitoneal administration of the parent drug. The transfer kinetics of
CIT and the extent of fetal exposure were dependent on pregnancy stage. An
important difference between developmental stages was the significant
accumulation of DCIT in fetal blood sera and brain in late gestation. I examined
the 3 possible sources of DCIT by performing in vitro metabolic assays in
placental extracts and maternal and fetal liver microsomes. While the placenta
showed no metabolic capacity of conversion of CIT to DCIT, there was no
difference in maternal metabolism of CIT between ages. I detected that CIT
metabolic capacity in the fetus was efficient in late gestation at E18 but not E14,
leading to higher fetal serum and brain DCIT concentrations in late pregnancy.
The importance of this finding is that it supports the notion that extensive fetal
5.2 SUMMARY OF FINDINGS

128
exposures occur not only from the parent drug, but also from the metabolite
DCIT. DCIT is a biologically active compound, with an affinity for SERT as high
as the SSRI fluoxetine (Prozac), and a longer half-life than that of CIT. The
results I reported provide novel insights into the pregnancy-specific
pharmacokinetics of CIT, and demonstrate the importance of fetal metabolism in
determining overall fetal drug exposures.
Given that extensive fetal exposures occur from maternal CIT treatment,
Chapter 4 of this dissertation warranted a thorough investigation of the potential
impact of CIT and DCIT-mediated inhibition of SERT on the development of the
brain’s serotonergic system. In tandem, I also aimed my investigation to the
independent effects of maternal stress on the fetal brain and the effects of oral
CIT administration in this context. The starting point for this project was the
verification that the current chronic unpredictable stress (CUS) model of
depression was valid in pregnant animals. For decades, the CUS paradigm has
been used to induce a plethora of behavioral responses relating to anxiety and
depression in non-pregnant animals. Some studies have examined the effects of
prenatal CUS on offspring outcomes, but have not reported results pertaining to
classic depression-like behaviors during pregnancy. The question of how
depression and treatment with SSRIs affects the fetal brain ought to be
addressed in a model system shown to induce depression-like behaviors during
pregnancy.
CONCLUSION AND FUTURE DIRECTIONS

129
When pregnant mice were chronically exposed to constant unpredictable
micro-stressors starting at E8, they had developed anxiety-like behaviors by E15
and depression-like behaviors by E16 as assessed by the open field (OFT) and
forced swim tests (FST), respectively. In the OFT, I reported that CUS mice
spent significantly less time in the center of the arena compared to untreated
dams. Mice exposed to CUS+CIT prevented this effect, and dams receiving CIT
were no different than controls. In the FST assessing behavioral despair, CUS
dams spent significantly longer amount of time immobile, an effect also
prevented by oral CIT administration.
In a 1997 review, Willner argued that “the only symptoms of depression that
have not been demonstrated in animals exposed to (CUS) are those uniquely
human symptoms that are only accessible to verbal enquiry” and that by applying
the DSM diagnostic rules, “a rat exposed to CUS could, in principle, legitimately
attract a DSM-IV diagnosis of either major depressive disorder or major
depressive disorder with melancholic features.” The results above match
observations from other CUS studies conducted in non pregnant animals, but a
lot more studies need to be done in pregnancy before reassuring the assertion by
Willner.
In the fetal brain at E17, I found that CUS exposed embryos had significantly
higher levels of tissue concentration of 5-HT in the fetal forebrain when
compared to untreated controls. Exposure to CIT during CUS prevented these
effects, noting no difference from the untreated forebrains. Interestingly, CIT
5.2 SUMMARY OF FINDINGS

130
exposure significantly lowered forebrain tissue 5-HT. This observation contradicts
the widespread notion that “SSRIs increase 5-HT concentration” in the brain. The
work presented in my dissertation is supportive of the possibility that blocking
uptake of 5-HT is part of early feedback regulation that results in SSRIs actually
decreasing tissue 5-HT. When I looked at antibody stained brain sections at
different brain levels, I noted a very similar pattern along thalamocortical axons
and the dorsal thalamus, where their cell bodies are found. CUS exposed brains
had higher immunofluorescence intensity than untreated controls, an effect
prevented by CIT exposure during CUS, and further reduced to significantly
lower levels by oral CIT. While tissue concentrations of 5-HT in the hindbrain
were not significantly affected, there was a modest increase following CUS. IHC
analysis revealed that CUS exposed brains had the highest counts of
serotonergic cell bodies along the lateral wing of the dorsal raphe. There were no
differences between untreated, CUS+CIT and CIT groups.
Overall, these observations support the idea that the differences in tissue 5-
HT concentration in the forebrain may be modulated by the increased uptake of
5-HT in the DT and along TCAs during CUS. Since uptake is blocked by CIT, it
leads to normal tissue concentrations in the CUS+CIT group and further
reductions in the CIT exposed forebrains. But even though CUS+CIT exposed
forebrains have normal tissue concentration, the distribution of 5-HT is not
normal.
CONCLUSION AND FUTURE DIRECTIONS

131
5 .3 FUTURE DIRECTIONS

The findings above underscore the need for additional research into the effects of
depression and SSRI exposures during pregnancy. An important next step is to
determine how CUS leads to increased 5-HT uptake in the forebrain and higher
5-HT cell body count in the hindbrain. Important future directions include the
evaluation of long term effects on offspring brain neurochemistry, circuit
architecture and behavior. Inclusion of other biogenic monoamines such as
dopamine (DA) and metabolites, which are also associated with depression and
other psychiatric conditions, would be an important assessment as mesolimbic
DA pathways (Cabib and Puglisi-Allegra, 2012), DA transporter levels(Isovich et
al., 2000; Lucas et al., 2004), and sensitization of mesocortical DA responses
(Chrapusta et al., 1997; Cuadra et al., 1999) has been shown to be affected
following exposure to stress in adult rodents. Given the effects on 5-HT in TCAs
following prenatal exposures to CUS and CIT, abnormal levels of 5-HT in these
axons(Lebrand et al., 1996b) could alter the postnatal formation of barrel field
structures in the somatosensory cortex (Alvarez et al., 2002; Rebsam et al.,
2002; van Kleef et al., 2012).
However, a lack of 5-HT in TCAs appears more likely to lead to a delay in
somatosensory cortical barrel formation, rather than over altered development
(see Van Kleef et al., 2012). Although an effect of delayed innervation might only
be transient (Persico et al., 2000, 2001) it could potentially be aggravated by re-
occurring insults (e.g. stress) during the early postnatal period. It will be important
5.3 FUTURE DIRECTIONS

132
to test this possibility when assessing the long-term effects of these prenatal
exposures on offspring brain function throughout the lifespan.

133
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Abstract (if available)

Abstract Despite extensive research efforts, the neurobiology and pathophysiology of Major Depression Disorder (MDD) remains poorly understood. On a global scale, mental disorders are among the leading causes of illness-induced disability throughout adulthood, of which depression is the most prevalent, especially in women of reproductive age (Global Burden of Disease Report & World Health Organization 2013). Considering the chronic nature of depression and its substantial impact, there is great need for improved understanding of the etiology and pathophysiology of MDD. ❧ Drugs like selective serotonin reuptake inhibitors (SSRIs) that are indicated for the treatment of neuropsychiatric conditions such as depression and anxiety target serotonergic mechanisms. During pregnancy, a time when the prevalence rates of depression are ~15%, treatment with SSRIs is of major concern due to the potential effects on the development of the fetal serotonergic system. Since 5-HT is implicated in multiple processes that orchestrate critical functions during brain development, altering serotonin levels or serotonergic system development could impact this delicate process. Consistent with a potential role in the fetal programming of adult mental disorders, basic and epidemiological findings have linked developmental disruption of 5-HT signaling to diverse functional disorders in adulthood. ❧ The work described in this dissertation aims to investigate how prenatal exposures affect serotonin and fetal brain development. Of special interest is the role of the placenta, which synthesizes 5-HT reaching the developing fetal forebrain early in gestation. Additionally, it takes a focused look into the pharmacokinetics of SSRIs in pregnancy, uncovering a pathway by which fetal drug exposures are regulated during development. Finally, this thesis also begins to distinguish between the independent effects induced by maternal stress from those that are pharmacologically induced, a much needed approach to start to address the clinical dilemma of SSRI treatment during pregnancy.

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Asset Metadata

Creator Huezo Velasquez, Juan C. (author)

Core Title Pharmacokinetics and developmental effects of exposures to selective serotonin reuptake inhibitor (SSRI) antidepressants

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 01/26/2018

Defense Date 07/26/2017

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

Tag OAI-PMH Harvest,serotonin, SSRI, placenta, fetal brain, 5-HT, pregnancy, citalopram

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chang, Karen (committee chair), Bonnin, Alexandre (committee member), Campbell, Daniel (committee member), MacKay, Andrew (committee member)

Creator Email Juan.Velasquez@med.usc.edu,juanhvelasquez@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-418538

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Legacy Identifier etd-HuezoVelas-5647.pdf

Dmrecord 418538

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Rights Huezo Velasquez, Juan C.

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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