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REGULATION OF THE MATERNAL HYPOTHALAMIC-PITUITARY-ADRENAL AXIS DURING PREGNANCY IN THE EWE: RELATIVE ROLES OF THE

MINERALOCORTICOID RECEPTOR AND THE SEROTONERGIC SYSTEM

By

MELISSA DAWN LINGIS

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2009

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© 2009 Melissa Dawn Lingis

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To my husband, Rob, for his constant support and encouragement To my parents, Dave and Pam Landen, for their unconditional love

And to EACH of them for always believing in me For my son, Matthew, may even your dreams exceed your expectations

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ACKNOWLEDGMENTS

I would first like to thank all of the members of my committee: Dr. Charles Wood, Dr.

Joanna Peris, Dr. Dorette Ellis, and Dr. Colin Sumners for their suggestions and guidance both in

my pursuit of this degree and on aspects of this research. A special thank you goes to Dr.

Maureen Keller-Wood, my committee chairperson, for her mentorship and unwavering

confidence in my abilities. For me, Dr. Keller-Wood has served as a wonderful role model as a

woman in the field of scientific research who still maintains the ability to be an involved mother

in the lives of her three children. As a future mentor myself, I hope to have inherited her energy,

enthusiasm, and encouraging attitude.

Within the College of Pharmacy, there are several people I would like to acknowledge.

Special thanks and congratulations go to my classmate and colleague, Dr. Chinki Bhatia. I

always enjoyed our chats over lunch and I am truly grateful for her support and friendship,

especially as we struggled through our qualifying exams together. I would also like to thank Dr.

Elaine Sumners for her mentorship, contributions to this research, and friendship. Through her

impeccable research ethic and technical abilities, she has served as an invaluable resource. Many

thanks also go to Dr. Yun-Ju He for his skillful work in various plasma hormone assays which

contributed to the data within this manuscript. Additionally, I would like to thank past members

of the department who played key roles in shaping me as a scientist. Thanks go to Dr. Marcela

von Reitzenstein, a former post-doctoral associate in our lab and Mrs. Krista Koehler, a former

technician, for encouraging me to pursue this degree. Special thanks go to Ms. Monique

Sutherland, who volunteered in the lab as an undergraduate student, for her friendship and

contributions to this research. I would also like to thank all of the undergraduate and pharmacy

students who have volunteered and contributed in any way to the data presented here.

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Most importantly, I would like to express my appreciation for my wonderfully supportive

family. My husband, Rob, always supported me even through the most difficult times and

provided gentle, loving nudges to keep me from giving up. His outstanding academic and career

accomplishments continue to encourage me every day. I cannot thank him enough for the

sacrifices he has made in order to provide for our family and to enable me to finish this

dissertation in a timely manner. Finally, I would like to thank my parents for their never-ending

encouragement and confidence in me. It is impossible to put into words exactly how

appreciative I am for all they have given me. I know that I never would have accomplished this

much without their guidance, love, and understanding throughout my entire career at the

University of Florida.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF TABLES ...........................................................................................................................9

LIST OF FIGURES .......................................................................................................................10

ABSTRACT ...................................................................................................................................12

CHAPTER

1 INTRODUCTION ..................................................................................................................14

Specific Aim 1: Relative Role of the Mineralocorticoid Receptor in Regulation of HPA Axis Activation During Pregnancy in the Ewe ...................................................................15

Specific Aim 2: Relative Role of Serotonergic System Responsivity in Regulation of HPA Axis Activation During Pregnancy in the Ewe ..........................................................16

Specific Aim 3: Relative Hypothalamic Expression of Genes Related to HPA Axis Regulation and the Serotonergic System ............................................................................17

2 REVIEW OF LITERATURE .................................................................................................18

Hypothalamic-Pituitary-Adrenal (HPA) Axis ........................................................................18 Role of Corticosteroids in Normal Physiology ...............................................................19 Negative Feedback Regulation ........................................................................................23 Role of Corticosteroids in Pregnancy and Parturition .....................................................25

Serotonergic System ...............................................................................................................28 Impact of the Serotonin System on HPA Axis Activity ..................................................31 Serotonergic System on Regulation of Food Intake ........................................................33

Evidence for Influence of Ovarian Hormones ........................................................................35

3 ROLE OF MINERALOCORTICOID RECEPTORS IN REGULATION OF CORTISOL, ALDOSTERONE, ELECTROLYTES, AND BLOOD PRESSURE IN PREGNANCY ........................................................................................................................40

Introduction .............................................................................................................................40 Materials and Methods ...........................................................................................................43

Animals ............................................................................................................................43 Surgical Protocol .............................................................................................................43 Experimental Protocol .....................................................................................................44 Plasma Hormone Determination .....................................................................................45 Plasma Volume Determination ........................................................................................45 Data Analysis ...................................................................................................................46

Results .....................................................................................................................................46 Plasma Hormone Levels ..................................................................................................46

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Adrenocorticotropic hormone (ACTH) ....................................................................46 Cortisol .....................................................................................................................47 Aldosterone ..............................................................................................................48 Angiotensin II ...........................................................................................................48

Mean Arterial Blood Pressure .........................................................................................48 Hematocrit (%) and Plasma Solute (Total Protein, Potassium, and Sodium)

Concentrations .............................................................................................................49 Plasma Volume ................................................................................................................50

Discussion ...............................................................................................................................50

4 RELATIVE SEROTONERGIC ACTIVITY/RESPONSIVITY DURING PREGNANCY ........................................................................................................................64

Introduction .............................................................................................................................64 Materials and Methods ...........................................................................................................66

Animals ............................................................................................................................66 Surgical Protocol .............................................................................................................67 Experimental Protocol .....................................................................................................68 Daily Food Intake ............................................................................................................70 Plasma Hormone Determination .....................................................................................70 Euthanasia and Tissue Recovery .....................................................................................70 Data Analysis ...................................................................................................................71

Results .....................................................................................................................................71 Study I: HPA Axis Responses to Acute, Icv Fluoxetine .................................................71

Plasma ACTH ..........................................................................................................71 Plasma cortisol .........................................................................................................72

Study II: HPA Axis Responses to Subchronic, Icv Fluoxetine .......................................73 Plasma ACTH ..........................................................................................................73 Plasma cortisol .........................................................................................................73 Mean arterial pressure, hematocrit (%) and plasma solute (total protein,

potassium, and sodium) concentrations ................................................................74 Daily food intake ......................................................................................................74

Discussion ...............................................................................................................................75 Study I: HPA Axis Responses to Acute, Icv Fluoxetine .................................................75 Study II: HPA Axis Responses to Subchronic, Icv Fluoxetine .......................................77 Summary ..........................................................................................................................80

5 HYPOTHALAMIC EXPRESSION OF GENES RELATED TO HPA AXIS REGULATION AND THE SEROTONERGIC SYSTEM IN EWES ...................................90

Introduction .............................................................................................................................90 Materials and Methods ...........................................................................................................92

Euthanasia and Tissue Recovery .....................................................................................92 RNA Extraction and Quantification ................................................................................93 Reverse Transcription and Real-Time Quantitative PCR ...............................................93 Data Analysis ...................................................................................................................94

Results .....................................................................................................................................95

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Glucocorticoid Receptor (GR) ........................................................................................95 Mineralocorticoid Receptor (MR) ...................................................................................95 Corticotropin-Releasing Hormone (CRH) ......................................................................95 Arginine Vasopressin (AVP) ...........................................................................................96 5-HT1A Receptor ..............................................................................................................96 5-HT2A Receptor ..............................................................................................................96 Serotonin Reuptake Transporter (SERT) ........................................................................96 Proopiomelanocortin (POMC) ........................................................................................97

Discussion ...............................................................................................................................97

6 SUMMARY ..........................................................................................................................110

LIST OF REFERENCES .............................................................................................................116

BIOGRAPHICAL SKETCH .......................................................................................................133

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LIST OF TABLES

Table page 5-1 Probe and primer sequences for qPCR. ...........................................................................101

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LIST OF FIGURES

Figure page 2-1 A general model of HPA axis regulation. ..........................................................................38

2-2 Basic regulatory circuitry for food intake ..........................................................................39

3-1 Plasma ACTH concentrations during canrenoate or vehicle infusion ...............................56

3-2 Plasma cortisol concentrations during canrenoate or vehicle infusion ..............................57

3-3 Plasma aldosterone concentrations during canrenoate or vehicle infusion .......................58

3-4 Plasma angiotensin II concentrations during canrenoate or vehicle infusion ....................59

3-5 Mean arterial pressure during canrenoate or vehicle infusion. ..........................................60

3-6 Hematocrit and plasma solute concentrations ....................................................................61

3-7 Linear regression of plasma potassium and plasma aldosterone during canrenoate infusion ..............................................................................................................................62

3-8 Plasma volume after 4 hours of vehicle or canrenoate infusion ........................................63

4-1 Study I: Plasma ACTH following acute icv fluoxetine or vehicle ....................................83

4-2 Study I: Plasma cortisol following acute icv fluoxetine or vehicle ...................................84

4-3 Study II: Plasma ACTH response to subchronic icv fluoxetine or vehicle .......................85

4-4 Study II: Plasma cortisol response to subchronic icv fluoxetine or vehicle ......................86

4-5 Study II: Mean arterial pressure during subchronic icv fluoxetine or vehicle. ..................87

4-6 Study II: Hematocrit and plasma solute concentrations during subchronic icv fluoxetine or vehicle. .........................................................................................................88

4-7 Study II: Daily food intake during subchronic icv fluoxetine or vehicle ..........................89

5-1 Relative hypothalamic glucocorticoid receptor (GR) mRNA expression ........................102

5-2 Relative hypothalamic mineralocorticoid receptor (MR) mRNA expression .................103

5-3 Relative hypothalamic corticotropin-releasing hormone (CRH) mRNA expression .......104

5-4 Relative hypothalamic arginine vasopressin (AVP) mRNA expression ..........................105

5-5 Relative hypothalamic 5-HT1A receptor mRNA expression ............................................106

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5-6 Relative hypothalamic 5-HT2A receptor mRNA expression ............................................107

5-7 Relative hypothalamic serotonin reuptake transporter (SERT) mRNA expression .........108

5-8 Relative hypothalamic proopiomelanocortin (POMC) mRNA expression ......................109

6-1 Proposed circuitry for regulation of HPA axis ................................................................115

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

REGULATION OF THE MATERNAL HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

DURING PREGNANCY IN THE EWE: RELATIVE ROLES OF THE MINERALOCORTICOID RECEPTOR AND THE SEROTONERGIC SYSTEM

By

Melissa Dawn Lingis

December 2009 Chair: Maureen Keller-Wood Major: Pharmaceutical Sciences

Studies in our laboratory and others have shown that cortisol, a corticosteroid important for

maternal hemodynamic changes and fetal homeostasis is basally regulated at higher levels in the

pregnant state than in the non-pregnant state. Our lab is testing two broad hypotheses to, at least

in part, explain this increase in activity during pregnancy: (1) alterations to the axis’ basal

negative feedback via mineralocorticoid receptors (MR) or (2) alterations in the serotonergic

system, a stimulatory central input to the axis.

To study the relative role of MR during pregnancy, I studied the effect of an acute

intravenous (iv) infusion of the MR antagonist, canrenoate on plasma adrenocorticotropic

hormone (ACTH), cortisol, aldosterone and angiotensin II as well as electrolyte balance and

blood pressure in pregnant and non-pregnant ewes. These two groups demonstrated a

differential time course for the stimulation of ACTH and cortisol in response to intravenous

mineralocorticoid blockade, suggestive of alterations in central MR-mediated regulation of the

axis while also supporting a role for MR in electrolyte balance and volume expansion in

pregnancy. To study possible alterations in the serotonergic system, I compared ACTH and

cortisol responses to a high-dose, acute intracerebroventricular (icv) injection of the selective

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serotonin reuptake inhibitor (SSRI), fluoxetine (FLX) and the responses to low-dose, subchronic

icv infusion of fluoxetine between nonpregnant and pregnant ewes. Opposite to our hypothesis,

serotonergic responsivity may be blunted in pregnancy providing evidence that an upregulation

of this system may not be responsible for the increases in basal plasma ACTH and cortisol that

occur during pregnancy. The differential HPA axis responses to both mineralocorticoid receptor

blockade and serotonergic system stimulation were however suggestive of alterations in the roles

of these two systems in basal HPA axis activity regulation during pregnancy. Analyses of

relative hypothalamic mRNA expression of pertinent HPA axis and serotonergic system genes

did not demonstrate any differences at the gene level. Continued research to completely

characterize regulation of the HPA axis will prove beneficial to the clinical sector, both in its

importance to pregnancy and to nearly all aspects of human physiology and homeostasis.

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CHAPTER 1 INTRODUCTION

The primary role of the hypothalamic-pituitary-adrenal (HPA) axis is to aid in the

adaptation to a variety of stressors through the production of the adrenal corticosteroid

hormones. One of the main focuses of our lab is to investigate alterations in the regulation of

basal maternal HPA axis activity that arise during pregnancy. Studies in humans and other

animal models such as the sheep have demonstrated upregulation of the maternal HPA axis

activity and therefore cortisol, a key corticosteroid, in pregnancy (Carr et al. 1981, Bell et al.

1991, Keller-Wood 1998, Erickson et al. 2001, Sandman et al. 2006, Kirschbaum et al. 2009).

Our lab has shown that corticosteroids are important for maternal volume expansion, uterine

blood flow, and fetal homeostasis and that clamping cortisol to levels observed in the

nonpregnant ewe has dramatic impact on these factors and the growth rate of the fetus (Jensen et

al. 2002, Jensen et al. 2003, Jensen et al. 2005). Our lab is interested in understanding the

mechanisms controlling basal HPA axis activity during pregnancy using sheep as an alternative

model to human pregnancy (Keller-Wood et al. 1998). This research focuses on an

understudied, basic adaptive process of successful pregnancy. It is important that we fully

characterize the regulation of the HPA axis to ultimately provide beneficial information for

future investigation of innovative approaches to treat dysregulation of such processes and

therefore prevent the development of an adverse environment for the fetus and to protect the

health of the mother.

The following studies are aimed at comparing: (1) the relative role of the mineralocorticoid

receptor and (2) relative serotonergic responsivity between pregnant and non-pregnant sheep as

they relate to negative feedback maintenance of basal HPA axis activity. The end-points of the

following studies are plasma levels of relevant hormones, such as adrenocorticotropic hormone

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(ACTH), cortisol, aldosterone, angiotensin II and progesterone as well as hemodynamic

parameters including blood pressure, electrolyte concentration, plasma protein, and hematocrit.

Additionally, I have compared relative hypothalamic mRNA expression of relevant HPA axis

related and serotonergic system-associated genes between pregnant, nonpregnant, and

postpartum ewes, including corticotropin-releasing hormone (CRH), arginine vasopressin

(AVP), mineralocorticoid receptor (MR), glucocorticoid receptor (GR), serotonin receptors (5-

HT1A and 5-HT2A), and the serotonin reuptake transporter (SERT). An additional investigation

of relative hypothalamic expression of proopiomelanocortin (POMC) mRNA was also

performed.

Specific Aim 1: Relative Role of the Mineralocorticoid Receptor in Regulation of HPA Axis Activation During Pregnancy in the Ewe

Based on a number of studies in both sheep and humans, our lab and others have theorized

that there is an alteration in ‘set-point’ of the negative feedback regulation of specifically basal

HPA axis activity that may at least in part explain the elevations of maternal plasma ACTH and

cortisol seen in pregnancy (Nolten & Rueckert 1981, Charnvises et al. 1985, Tropper et al. 1987,

Odagiri et al. 1988, Keller-Wood 1996, Keller-Wood 1998). Cortisol acts at two corticosteroid

receptors located at sites upstream and along the axis in order to inhibit its activity. Of these two

receptors, the higher affinity mineralocorticoid receptor (MR) is thought to be important for

regulating the axis when cortisol levels are low, at basal, non-stressed levels.

In order to test our lab’s overall hypothesis that progesterone, which is elevated during

pregnancy, may be influencing cortisol’s ability to act at MR along negative feedback sites of the

axis, I have administered the MR antagonist, canrenoate to both pregnant and nonpregnant ewes

in order to examine the relative role of MR during pregnancy on HPA axis activation, blood

pressure, and plasma solute regulation.

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Specific Aim 2: Relative Role of Serotonergic System Responsivity in Regulation of HPA Axis Activation During Pregnancy in the Ewe

The serotonergic system is thought to be one of the stimulatory afferent systems to the

HPA axis (Calogero et al. 1990, Fuller 1996). Upon intravenous or oral administration of

serotonin, its precursors, or 5-HT receptor agonists, significant increases in plasma ACTH and

cortisol are observed in human subjects and rodent models (Kile & Turner 1985, Calogero et al.

1990, Contesse et al. 2000, Heisler et al. 2007). In the synapse, cessation of the serotonergic

signal is achieved mainly by the removal of serotonin from the synaptic cleft through an active

membrane transporter encoded for by the serotonin transporter (SERT) gene. Administration of

selective serotonin reuptake inhibitors (SSRI’s), which act on this transporter, is also associated

with a rise in both portal CRH and plasma ACTH (Bevan & Scanlon 1998). Meanwhile, several

studies have indicated a relationship between estrogen and progesterone with serotonin receptor

activity (for review see, Bethea et al 2002). In this way, it becomes increasingly apparent that

there may be changes in serotonergic responsivity during pregnancy and these changes might be

in part responsible for altered basal HPA axis activity.

The objective of this study was to determine if there are alterations in the serotonergic

component of basal HPA axis regulation during pregnancy. There were two parts to this

objective: (1) to compare HPA axis responses to an acute intracerebroventricular (icv) injection

of a selective serotonin reuptake inhibitor, fluoxetine (FLX) in pregnant ewes to the response in

the same ewes post-partum; and (2) to compare responses to icv administration of a lower, but

more chronic dose of fluoxetine in pregnant ewes to non-pregnant ewes. The use of a selective

serotonin reuptake inhibitor in these studies was designed to exploit the inherent serotonergic

activity in these ewes and allow us to observe any differences that might exist relating to

differential regulation of the HPA axis during pregnancy.

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Specific Aim 3: Relative Hypothalamic Expression of Genes Related to HPA Axis Regulation and the Serotonergic System

For this portion of the dissertation work, we sought to determine whether changes at the

genes level in the hypothalamus might at least in part explain the elevations in ACTH and

cortisol during pregnancy in the ewe. We chose the hypothalamus as it is the point of integration

for all upstream inputs driving or inhibiting HPA axis activity. Therefore, the objective of this

study was to characterize relative mRNA expression levels of HPA axis- and serotonergic

system-relevant genes in the hypothalamus between nonpregnant, pregnant, and post-partum

ewes. Any difference found here might additionally provide insight for any differential HPA

axis responses that might occur between pregnant and nonpregnant or postpartum ewes in

response to MR antagonism or selective serotonin reuptake transporter inhibition discussed in the

preceding specific aims of this dissertation.

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CHAPTER 2 REVIEW OF LITERATURE

Hypothalamic-Pituitary-Adrenal (HPA) Axis

As the name implies, the hypothalamic-pituitary-adrenal axis comprises the pathway of

communication that exists between certain areas of the hypothalamus, the anterior lobe of the

pituitary gland, and the cortices of the adrenal glands (Figure 2-1). The ‘messages’ that are sent

between these components are in the form of hormones. Within the paraventricular nucleus

(PVN) of the hypothalamus, a subset of neurons called parvocellular neurons synthesize and

secrete both corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) (Chrousos

& Gold 1992, Chrousos 1992, Tsigos & Chrousos 1994). The axons of these neurons project to

the median eminence and synapse onto the hypophyseal portal system. It is through this path

that CRH is carried to the anterior pituitary where it acts primarily through CRH1 receptors

located on the plasma membrane of pituitary corticotropes. Within the pituitary,

adrenocorticotropic hormone (ACTH) is synthesized as it is cleaved from its pre-cursor

proopiomelanocortin (POMC), whose transcription is stimulated by CRH binding (Fukuda et al.

2003). ACTH is stored intracellularly and is secreted by elevation of cytosolic Ca2+

concentration which is induced by activation of voltage-gated calcium channels or intracellular

calcium release (Luini et al. 1985). Once released, the ACTH travels through the systemic

circulation toward its main target organs, the adrenal glands, or more specifically, the adrenal

cortices. The adrenal cortex is then stimulated to synthesize and secrete corticosteroids. The

two classes of corticosteroids, glucocorticoids and mineralocorticoids, are synthesized and

released from the zona fasciculata and zona glomerulosa of the adrenal cortex, respectively.

Increased corticosteroid synthesis occurs as ACTH binding causes an increase in cholesterol

processing, such as movement into the mitochondria and subsequent conversion to

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pregnenolone. Pregnenolone ultimately gets shuttled back to the endoplasmic reticulum,

converted to progesterone or 17-hydroxypregnenolone, and after multiple hydroxylation steps

gets converted to aldosterone or cortisol, respectively (Nussey & Whitehead 2001b). In addition

to ACTH stimulation of the adrenal cortex, corticosteroid synthesis and release may also be

regulated by cytokines, angiotensin II, plasma potassium concentration, lipid mediators of

inflammation, and through autonomic innervation of the adrenal glands themselves.

Role of Corticosteroids in Normal Physiology

The primary role of the hypothalamic-pituitary-adrenal (HPA) axis is to aid in the

adaptation to a variety of stressors as a means to maintain homeostasis. This process is called

allostasis or re-establishing “stability through change” (Sterling & Eyer 1988). In the case of the

HPA axis, the process of adaptation is achieved as hormones, acting through receptors, cause

changes at the cellular level. The stressors which evoke such changes can come in many forms

but are mainly classified as either neurogenic (psychological) or systemic (physiological).

Examples of animal models for neurogenic stress are environmental stimuli such as restraint,

electrical footshock, and maternal separation. Systemic stressor models include immunological

challenges or disease states, hemorrhage, and pregnancy. Interestingly, various studies have

uncovered the fact that the stress response circuitry up until activation of the PVN of the

hypothalamus appears to be somewhat specific to the type of stressor the individual is

encountering.

The end result upon activation of the HPA axis is the production and release of

corticosteroids from the adrenal cortex. These hormones are involved in the regulation of a wide

variety of physiologic systems. Two types of corticosteroids are glucocorticoids and

mineralocorticoids. Glucocorticoids and mineralocorticoids classically exert their effects

through two intracellular receptors. These receptors belong to the super-family of nuclear

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receptors and are located in the cytoplasm and are complexed with heat-shock proteins and

others while in the inactive state. Being lipophilic, the corticosteroids readily enter the cell and

bind the receptor, causing dissociation from these inactivating proteins. After subsequent

phosphorylation, the hormone-receptor complex translocates into the nucleus and dimerizes with

another corticosteroid-receptor complex. The dimer is then able to bind steroid-response

elements found in the promoter region of a variety of target genes, usually with the aid of other

transcription factors thereby stimulating or suppressing transcription of the targeted gene

(Nussey & Whitehead 2001d). Additionally, gene transcription can be influenced through

receptor-ligand complex interactions with other transcription factors (Diamond et al. 1990,

Yang-Yen et al. 1990).

Glucocorticoids come from the zona fasciculata of the adrenal cortex. It has been

established that these adrenal gland molecules are essential to life based on previous research

which showed that bilateral adrenalectomy will result in death if glucocorticoids are not

replaced. Endogenous glucocorticoids, like cortisol in humans and corticosterone in rodents, are

involved in regulating many processes that serve to mobilize energy in order to optimize the

‘fight-or-flight’ mechanism in times of stress. Glucocorticoids are therefore involved in

stimulation of gluconeogenesis, lipolysis and proteolysis to aid in production of fuel sources for

this stress response. In addition, glucocorticoids have been shown to inhibit or suppress innate

immune responses, bone and muscle growth, and reproductive function (McEwen & Stellar

1993). Cortisol is also essential for maintaining normal blood pressure by influencing

myocardial function and arteriolar sensitivity to both sympathetic nervous stimulation and to

angiotensin II. In the brain, glucocorticoids may be involved in cognition, memory and mood as

receptors have been found to be expressed in functionally pertinent regions including the

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prefrontal cortex, hippocampus and amygdala. Many groups have provided evidence that

cortisol alters neuronal excitability and can induce cell death in certain brain regions, whereas in

other regions glucocorticoids are thought to play a protective role (for review see, McEwen

1994, Nussey & Whitehead 2001a).

Aside from fluctuations in glucocorticoid levels occurring in response to stress, there exists

a circadian rhythm to their basal release. This is most likely due to the connections from the

suprachiasmatic nucleus (SCN) to the paraventricular nuclei of the hypothalamus. The SCN

consists of various cell types containing genes whose expression is responsive to light-sensitive

input signals from the optic tract. CRH is released in a circadian-dependent, pulsatile fashion

from the parvocellular cells of the PVN (Hauger & Dautzenberg 2000). Lesions of the SCN in

rats result in the loss of corticosteroid cyclicity (Moore & Eichler 1972). In humans, the pattern

of diurnal cortisol release is such that the lowest concentration of cortisol is found at about

midnight, continues to rise until it peaks around 9 am, and then gradually declines throughout the

course of the day (Nussey & Whitehead 2001c). This diurnal release of cortisol is often altered

by stress as well as changes in lighting, feeding schedules, and activity (Charmandari et al.

2005). Logically, this pattern is reversed in rats in correlation with their nocturnal habits.

Interestingly, in humans, a recent study suggests that increasing demands for glucose by the

brain over the course of sleep is the main contributor to the early morning rise in cortisol

(Benedict et al. 2009). Similarly, in dogs, there is no distinct rhythm at all but rather pulsatile

release of cortisol in response to episodic secretion of ACTH that may occur in response to

feeding patterns or possibly to the mild stress associated with human interaction (Kemppainen &

Sartin 1984). Such ultradian rhythms have been observed in lactating dairy cows and sheep, as

well (Brinklow & Forbes 1984, Lefcourt et al. 1993).

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Mineralocorticoids such as aldosterone are produced and released by the zona glomerulosa.

Aldosterone is released in response to activation of the renin-angiotensin-aldosterone system

(RAAS) which occurs in response to a fall in renal perfusion pressure, stimulation by renal

sympathetic nerve afferents, or a reduction in sodium chloride delivery to the macula densa.

Classically, these cues cause an increase in the production of the enzyme renin, which starts the

process of converting angiotensinogen to angiotensin II, a peptide known to stimulate the release

of aldosterone from the adrenal cortex. Additionally, according to most textbooks, aldosterone

release can also be stimulated by an increase in plasma potassium (K+) concentration detected

directly by the adrenal zona glomerulosa cells. Aldosterone, by binding within the cells along

the kidney tubule, can contribute to the regulation of fluid and electrolyte balance by stimulating

K+ excretion into the urine, sodium (Na+) reabsorption and the resultant water retention at both

the distal convoluted segment and collecting duct of the renal tubule. Specifically, it has been

shown that aldosterone activates the epithelial sodium channel (ENaC) by regulating the

abundance and apical distribution of the α-subunit in principal cells (Masilamani et al. 1999). As

discussed in a review by Staub and Verrey (2005), the expression of serum- and glucocorticoid-

induced kinase 1 (sgk1) is rapidly upregulated by aldosterone. This review focuses on work

done in numerous labs to provide evidence that sgk1 phosphorylates Nedd4-2, a ubiquitin-

protein ligase, and thus inhibits its ability to interact with ENaC. Without ubiquitylation, ENaC

maintains its cell surface expression and can therefore aid in Na+ reabsorption in this segment of

the renal tubule. At the same time, aldosterone has the ability to stimulate Na/K ATPase activity

and to increase the driving force for K+ excretion into the tubule lumen. Consequently,

aldosterone plays a major role in regulation of electrolytes and plasma volume and, therefore, in

regulation of blood pressure.

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Negative Feedback Regulation

The discussion of HPA axis circuitry does not end at the resultant responses of the various

effector organs to corticosteroid binding. Glucocorticoids also play an important role in

regulating the basal activity of the HPA axis and in terminating the stress response by exerting

negative feedback at several sites along the circuit. If unchecked or dysregulated, a chronically

hyper- or hypoactive HPA axis can be damaging and/or exacerbate disease states. The negative

feedback of glucocorticoids on CRH and ACTH release limits the duration of total tissue

exposure to these molecules, thereby minimizing their catabolic, antireproductive, and

immunosuppressive effects. In a review by Charmandari et al (2005), some of the adverse

effects of a hyperactive HPA axis are discussed, such as melancholic depression and insulin

resistance; while a hypoactive axis can contribute to the pathogenesis of fibromyalgia, chronic

fatigue syndromes and susceptibility to autoimmune inflammatory disease.

Likely due to varying mechanisms, elevations in glucocorticoids may cause suppression of

ACTH by a fast (within seconds to minutes), intermediate (over period of 2 to 10 hours) or slow

(which occurs over a period of hours to days in response peak cortisol concentrations) negative

feedback effect (for review see, Keller-Wood & Dallman 1984). The two receptors for

corticosteroid binding, mentioned above, are found in varying concentrations all along the circuit

to exert negative feedback effects. The low-affinity glucocorticoid receptor (GR) binds at

elevated glucocorticoid levels that occur in times of stress; while the high-affinity

mineralocorticoid receptor (MR) binds at both basal and stress concentrations (Keller-Wood &

Dallman 1984, Reul & de Kloet 1985, Reul et al. 1987, Bradbury et al. 1991, de Kloet et al.

1993). MR affinity for corticosterone, the main glucocorticoid in rodents, is 10-fold higher than

that of GR. Therefore, central MR is almost completely occupied at basal corticosterone

concentrations, while GR only becomes substantially occupied during times of stress or at the

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peak of the circadian cycle (Reul & de Kloet 1986, De Kloet et al. 1998). Central glucocorticoid

receptors have widespread distribution throughout the brain, but are most densely expressed in

areas that are have been shown to be involved with regulation of the stress response such as the

PVN of the hypothalamus, the corticotrophs of the anterior pituitary, and the hippocampus (Reul

& de Kloet 1986).

It is important to note, both aldosterone and corticosterone (in rodents) and cortisol (in

humans) bind MR with subnanomolar affinity, but the glucocorticoids circulate at 1000-fold

higher concentrations (100-fold higher even after considering the presence of corticosteroid

binding proteins in the plasma). Experiments done in vitro have shown that the aldosterone is 10

times more potent at stimulating transcriptional changes by binding to MR (Arriza et al. 1988).

However, there is marginal brain penetration of aldosterone due at least in part to multidrug

resistance protein (mdr1 or P-glycoprotein), a protein transporter at the blood-brain barrier which

pumps select substrates back into the blood (Ueda et al. 1992, Uhr et al. 2002, Parker et al.

2006). Therefore, the vast majority of central mineralocorticoid receptors are occupied by

glucocorticoids rather than aldosterone.

Mineralocorticoid receptors have been studied extensively within the central nervous

system of many rodent models (Reul & de Kloet 1985, Luttge & Rupp 1989, Funder 1996). In

the rodent brain, MR have been found primarily in the hippocampus and septum. In fact, regions

of the hippocampal formation express both receptors and this is not surprising as it is thought to

play an inhibitory role in regulation of the HPA axis through its indirect connections to the PVN,

such as those made through the septal nucleus of the stria terminalis (Sapolsky et al. 1986,

Jacobson & Sapolsky 1991, Herman & Cullinan 1997, Herman & Mueller 2006). It is thought

that hippocampal neuronal inputs activate inhibitory gamma-aminobutyric acid- (GABA-)

25

releasing neurons that project to CRH neurons in the PVN. Evidence in support of this

relationship, as discussed in a review by Carrasco and Van de Kar (2003), include the use of

synthetic mineralocorticoid receptor antagonist RU28318 to elevate baseline corticosterone

levels in rats which can be blocked by the GABAA receptor agonist alprazolam (Grottoli et al.

2002). Distribution and function of mineralocorticoid receptors at the hippocampal level has

recently been investigated in other species. Studies characterizing corticosteroid receptor

distribution in the primate brain, found that MR mRNA and protein levels were much higher in

the dentate gyrus (DG) and Cornu Ammonis (CA) of the hippocampus (Sanchez et al. 2000).

Additionally, electrical stimulation of the hippocampus in humans and cats produces a decrease

in the plasma levels of cortisol, supporting an inhibitory role of the hippocampus on HPA axis

activity in these species as well (Carrasco & Van de Kar 2003). Like the rodent and primate

models, sheep are also known to express both MR and GR within the main regulatory areas of

the HPA axis: the hypothalamus, hippocampus, and pituitary (Roesch & Keller-Wood 1999).

Role of Corticosteroids in Pregnancy and Parturition

Studies in our laboratory and others have shown that cortisol, a key corticosteroid, is

basally regulated at higher levels in the pregnant state than in the non-pregnant state (Carr et al.

1981, Bell et al. 1991, Keller-Wood 1998, Erickson et al. 2001, Sandman et al. 2006,

Kirschbaum et al. 2009). Additionally, there is a marked increase in plasma aldosterone by the

8th week of gestation in human pregnancy which continues to rise throughout pregnancy to levels

4 to 6 fold higher than nonpregnant levels (Watanabe et al. 1963). Our lab, specifically, has

shown that corticosteroids are important for maternal volume expansion, uterine blood flow, and

fetal homeostasis (Jensen et al. 2002, Jensen et al. 2003, Jensen et al. 2005). In these studies,

pregnant ewes were adrenalectomized at 112 days of gestation and were under-replaced for

either cortisol or aldosterone to levels observed in nonpregnant ewes. It was determined that

26

reducing either corticosteroid prevented the increase in maternal plasma volume that occurs from

120 days to 130 days of gestation. Several adaptive responses were observed in the fetuses of

these ewes such as reduced lung-liquid and urine production presumably to combat the effects

that maternal hypovolemia might have on their own plasma volume. Additionally, in these

pregnant ewes which were inadequately replaced after adrenalectomy, the normal increase in

uterine and placental blood flow that occurs as gestation progresses was attenuated.

Consequently, Jensen et al (2005) reported adverse effects on fetal development such as

impaired fetal somatic growth rate and arterial oxygen tension. In support of these findings, case

studies in humans have shown that pregnancies with untreated adrenal insufficiency are

associated with high maternal and fetal morbidity and mortality (Lindsay & Nieman 2006). At

the same time, a link has been proposed between rising cortisol and changes in maternal basal

metabolic rate and body weight during pregnancy (Damjanovic et al. 2009).

Also important to note, the HPA axis has been implicated for many years in fetal organ

development and in the initiation of parturition. In a review by Liggins (1994), the role of

cortisol as it relates to organ maturation in the fetus is discussed. Cortisol is responsible for

regulating important proteins in many organ systems that are necessary for adaption to the extra-

uterine environment. The fetal lung for example, must rapidly absorb the liquid that has filled

each lobe throughout gestation as well as produce phospholipid surfactant to prevent alveolar

collapse by reducing surface tension. In fact, Liggins and Howie in (1972) first introduced the

idea of giving antenatal glucocorticoids to mothers who were threatening premature labor in

order to prevent respiratory problems in the neonate. Another example of cortisol-mediated

organ maturation is increased production of tri-iodothyronine by the thyroid gland which occurs

27

to address the need for increased thermogenesis and the higher metabolic rate that is necessary

for adaption to the colder external air and increased energy expenditure of breathing.

The role of cortisol in the initiation of parturition is currently being investigated. The

placenta is known to be an extrahypothalamic cite of CRH production and is primarily

responsible for the increased plasma CRH in late gestation (Jones et al. 1989, Zoumakis et al.

1996). Plasma CRH concentrations peak during labor and immediately decline postpartum

(McLean et al. 1995). Contrary to the inhibitory effect on hypothalamic production of CRH,

cortisol is thought to stimulate placental CRH production (Gonen et al. 1992). The maturing

fetal HPA axis near term is thought to contribute to this increase in placental CRH as well and is

the basis for the ‘placental clock’ theory of the timing of parturition (McLean et al. 1995).

Additionally, norepinephrine, angiotensin II and vasopressin which may be increased during

times of stress can also stimulate CRH production by the placenta (Jones et al. 1989, Petraglia et

al. 1989, Petraglia et al. 1991). These findings have lead to the theory that maternal or fetal

stress can lead to premature delivery in humans. The role of CRH in parturition is thought to

include its ability to stimulate of prostaglandin production in placenta in vitro (Jones & Challis

1989, Jones & Challis 1990). The prostaglandins are then thought to increase myometrial

oxytocin receptor levels and gap junctions, thereby enhancing the myometrial response to

oxytocin (Neulen & Breckwoldt 1994, Grazul-Bilska et al. 1996) as well as influence

extracellular matrix remodeling in the cervix. In vitro and in isolated placental tissues,

prostaglandins stimulate CRH release which would indicate a positive feedback relationship

(Jones & Challis 1989, Jones & Challis 1990, Petraglia et al. 1991). Although controversial,

elevated plasma CRH concentration midgestation has been suggested as a marker for preterm

delivery (Wolfe et al. 1988, Warren et al. 1992, McLean et al. 1995, Lockwood et al. 1996).

28

Serotonergic System

I will now direct attention to the serotonergic system, which has previously been shown to

be associated with HPA axis activation. The interactions of these two systems are particularly

evident in pathological conditions such as major depression, which is characterized by

dysregulation of both systems (for review see, Lanfumey et al. 2008).

In the mid-nineteenth and early twentieth centuries, a substance was being isolated in

platelets that was capable of constricting vascular smooth muscle. Due to its "tonic" action and

locale in "serum," the substance was given the name serotonin. Serotonin is found in a variety of

organ systems such as the intestinal mucosa and serum. Approximately 1–2% of the body’s

serotonin is contained in the central nervous system, specifically in serotonergic neurons

(Lozeva-Thomas 2004). Within the central nervous system, serotonin serves as a

neurotransmitter which is likely involved in the regulation of feeding behavior, body weight, the

sleep–wake cycle, circadian rhythmicity, locomotion, and learning and memory (Jacobs &

Azmitia 1992, Jacobs & Fornal 1999, Cooper et al. 2003). Serotonin has also been implicated in

several pathological conditions such as migraine, obsessive-compulsive disorders, depression

and suicidal behavior, bipolar disorder, schizophrenia, narcolepsy, alcohol dependence, obesity,

and chronic fatigue syndrome (Jacobs & Azmitia 1992, Jacobs & Fornal 1999, Cooper et al.

2003).

Within the central nervous system, serotonergic cell bodies are primarily found in discrete

clusters or groups of cells along the midline of the brain stem while their axons, however,

innervate nearly every area of the central nervous system. These clusters of cell bodies were

named and classified by Olszewski and Baxter in 1954. The nuclei found in the more caudal

region of the brain stem are thought to be involved in modulating sensory and motor processing

of the spinal cord, whereas the cells of the rostral nuclei send long axons to the forebrain.

29

Immunohistochemical staining techniques for either 5-HT or tryptophan hydroxylase have been

utilized to further characterize serotonergic innervation in the various forebrain regions (Frazer

& Hensler 1999).

In the cytosol of serotonergic neurons, a two step process occurs to synthesize serotonin or

5-hydoxytryptamine (5-HT) from the precursor amino acid, l-tryptophan. The primary source of

the tryptophan is dietary protein. Facilitated transport is necessary to move l-tryptophan from

the blood into the brain. This process is regulated not only by the concentration of tryptophan,

but also by the presence of other amino acids that compete for this type of transport. Therefore,

lowering intake of tryptophan relative to these other amino acids can affect the amount of

serotonin synthesis. In fact this strategy has been used to help elucidate the importance of brain

5-HT. Once tryptophan has crossed the blood-brain barrier and entered a serotonergic cell body,

it is hydroxylated to 5-hydroxytryptophan (5-HTP) catalyzed by tryptophan hydroxylase, the

rate-limiting step in the synthesis of serotonin. Activation of this enzyme requires

phosphorylation and the accessory protein 14-3-3 (Ichimura et al. 1995, Kuhn et al. 1997). Next,

5-HTP undergoes a decarboxylation reaction which is catalyzed by l-amino acid decarboxylase

to form serotonin. As is the case for many other neurotransmitters, serotonin is then stored

predominantly in vesicles which release their contents in response to an action potential passing

down the axon of the neuron. Serotonergic activity is then determined by the processes of

serotonin synthesis, reuptake, neuronal activity, degradation by monoamine oxidase (MAO), and

pre- and post-synaptic receptor activation.

There are thought to be at least seven major families which are further classified into at

least 14 subtypes of serotonin receptors. Serotonin binding at these receptors can either directly

stimulate or inhibit the activity of the target cell, whereas the presence of these receptors on

30

GABA-ergic interneurons can allow for indirect modulation of their downstream target cells as

well (Uphouse 1997). The following information regarding the serotonin receptors investigated

in this dissertation has been summarized from a textbook chapter entitled ‘Serotonin’ by Frazer

and Hensler (1999). The 5-HT1 family consists of receptors that are negatively coupled to

adenylyl cyclase and therefore, receptor activation causes a decrease in cAMP which reduces the

amount of kinase activation. The 5-HT1A receptor is additionally coupled to the opening of K+

channels, which results in neuronal hyperpolarization. These receptors are coupled to both

effector systems at the nerve terminus, but in the dorsal raphe nucleus, 5-HT1A receptors are

coupled only to the opening of potassium channels. 5-HT1A receptors have been shown to be

present in high density in the hippocampus, septum, amygdala, hypothalamus and neocortex.

The next family, the 5-HT2 receptors are G-protein coupled receptors as well, stimulating

phosphoinositide hydrolysis through activation of phospholipase C. Contrary to 5-HT1A

receptors, activation of 5-HT2A receptors mediates neuronal depolarization due to closing of

potassium channels. This family of receptors is particularly concentrated in the frontal cortex, in

parts of the limbic system, and the PVN of the hypothalamus.

Cessation of the serotonin signal has also been characterized. The majority (80%) of the

serotonin released into the synaptic cleft is removed through an active membrane transporter on

the pre-synaptic neuron encoded for by the serotonin transporter (SERT) gene. If not actively

transporting serotonin, the SERT protein is phosphorylated and subsequently degraded

(Ramamoorthy & Blakely 1999). Deactivation of free serotonin both in the synapse and in the

nerve terminal also occurs via a reaction catalyzed by mitochondrial monoamine oxidase (MAO)

which converts 5-HT to 5-hydroxyindoleacetic acid (Lozeva-Thomas 2004).

31

Impact of the Serotonin System on HPA Axis Activity

Early studies had already established a spatial relationship between the serotonergic

systems and the HPA axis. Using immunocytochemistry techniques, researchers demonstrated

the existence of serotonergic synapses on CRH-containing neurons of the PVN (Liposits et al.

1987). The major brain stem source of serotonergic innervation to the PVN of the hypothalamus

is from the dorsal raphe nuclei (Azmitia & Segal 1978). More recently, in situ hybridization

studies have uncovered moderately dense populations of 5-HT2A/2C binding sites and 5-HT2A

transcripts within the hypothalamic PVN.

It was also shown in vivo by Tsagarakis et al (1989) that hypothalamic implants of

serotonin directly stimulate CRH release; while Calogero et al (1989) demonstrated the same

effect in hypothalamic cultures. A review by Carrasco and Van de Kar (2003) discusses a

number of studies in rodents have shown the stimulatory effects of serotonergic agents of plasma

ACTH and corticosterone (the primary glucocorticoid in these species). Additionally, upon

intravenous injection of serotonin or oral administration of serotonin or its precursors, tryptophan

or 5-HTP, a significant increase in plasma cortisol is observed in human subjects and other

animal models such as sheep (Kile & Turner 1985, Calogero et al. 1990, Fuller 1996, Broadbear

et al. 2004, Broadbear et al. 2005, Heisler et al. 2007). Drugs that are designed to potentiate the

actions of serotonin, such as selective serotonin reuptake inhibitors (SSRIs), are also associated

with a rise in both portal CRH and plasma ACTH (Bevan & Scanlon 1998). The association of

these two systems is even being explored in fish. One group of researchers in Sweden has been

characterizing the hypothalamic-pituitary-interrenal axis, which is analogous to the HPA axis.

The end-product of this axis is also cortisol; and a recent study showed that tryptophan-

supplemented fish without exposure to a stressor exhibit elevated cortisol levels (Lepage et al.

2002).

32

Several studies have focused on elucidating exactly which 5-HT receptor is responsible for

stimulation of the HPA axis. Gartside and Cowen (1990) found the serotonin precursor 5-HTP

given intraperitoneally (ip) to dose dependently increase plasma ACTH in the male rat. These

responses were attenuated by pretreatment with the non-selective 5-HT receptor antagonist and

also by a variety of selective 5-HT2 receptor antagonists. The 5-HT1 receptor antagonists used in

those studies failed to antagonize these responses suggesting that the increases in ACTH with ip

injection of 5-HTP in the male rat are mediated by 5-HT2 receptors. Another study which

utilized systemic administration of 5-HTP in conjunction with the 5-HT reuptake inhibitor

fluoxetine, found a 64% increase in CRH mRNA expression within the PVN and a 17% increase

in POMC mRNA within the anterior pituitary. In agreement with the previous study, this group

also found ACTH secretion to be elevated five-fold. Through their use of specific 5-HT receptor

subtype agonists and anti-CRH antiserum, this group have indicated a number of serotonin

receptors might be responsible for increases in CRH and POMC synthesis including 5-HT1A and

5-HT2A and that these effects are mediated by CRH (Jorgensen et al. 2002).

The hippocampus, which is thought to be a site for HPA axis regulation as previously

mentioned, receives dense serotonergic innervation from the raphe nuclei as well and the

involvement of this line of communication in HPA axis regulation is currently being explored.

In studies utilizing parachlorophenylalanine, a specific 5-HT synthesis inhibitor, Semont et al

(1999) detected a significant increase in the number of hippocampal MR-binding sites. Upon

injection of the precursor, 5-HTP, MR-binding site levels were restored down to control levels.

No change in number of GR-binding sites was detected; however, hippocampal GR mRNA

levels were reduced. These researchers propose that hippocampal MR synthesis is inhibited by

5-HT and that this effect is not mediated by changes in hormone secretion of HPA axis. In

33

contrast, the expression of both glucocorticoid and mineralocorticoid receptor mRNA in

hippocampal cells has been reported to be upregulated upon activation of 5-HT receptors in vitro

(Seckl & Fink 1991, Lai et al. 2003). Moreover, in studies using reserpine, a substance which

depletes all monoamines, a reduction in both MR and GR levels is seen in hippocampal cytosol

(Lowy 1990). Additionally, lesions of the serotonergic projections to the hippocampus of

adrenalectomized rats also results in decreased levels of MR and GR mRNA in sub-regions of

the hippocampus (Seckl et al. 1990).

Finally, it is important to note that the complexity of the relationship between the

serotonergic system and the HPA axis is further complicated by the fact that the serotonergic

neurons themselves are known to express corticosteroid receptors. This would therefore imply

that they have the potential to be regulated by circulating glucocorticoid, directly (Harfstrand et

al. 1986, Fuxe et al. 1987, Morimoto et al. 1996).

Serotonergic System on Regulation of Food Intake

Serotonin is thought to play a role in food intake regulation as evidenced by the

anorexogenic effects of serotonergic agents, such as selective serotonin reuptake inhibitors and

serotonin releasing agents (Heisler et al. 2003). A brief overview on basic central food intake

circuitry is necessary to understand the proposed involvement of serotonin in these pathways. A

vast array of gastrointestinal, pancreatic, and adipocytic hormones which are responsive to

physical and chemical cues regarding meal size and energy stores are known to centrally regulate

food intake (Kelley & Berridge 2002, Dhillo & Bloom 2004, Woods et al. 2006, Naslund &

Hellstrom 2007). These hormones act via the nucleus of the solitary tract (NTS) or directly at

brain stem as well as hypothalamic and corticolimbic nuclei that are known to regulate feeding

(Swanson 2000, Browning & Travagli 2006). One such area of the hypothalamus, the arcuate

nucleus (ARC), consists of two neuronal populations, proopiomelanocortin (POMC) neurons and

34

those which co-express agouti-related protein (AgRP) and neuropeptide Y (NPY). These

neurons are known to be responsive to the peripheral hormones discussed above; therefore the

ARC can subsequently integrate an array of signals governing food intake (for review see,

Cummings & Overduin 2007). Figure 2-2 depicts basic signaling circuitry for inhibiting food

intake mediated by the peripheral hormones ghrelin (from the stomach) and leptin (from

adipocytes) acting at the neuronal systems. Briefly, leptin stimulates POMC neurons to release

melanocortins (cleavage products of the precursor POMC) which then act on melanocortin

receptors and ultimately inhibit feeding (Cone 2006). Meanwhile, ghrelin activates the

AgRP/NPY neuronal population. AgRP is thought to be an inverse agonist at melanocortin

receptors and therefore its effects are orexogenic (Chen et al. 2004).

It has been postulated that serotonergic agents which decrease body weight and food intake

are doing so by acting directly on the neurons of the arcuate nucleus. Heisler and colleagues

(2002) demonstrated fos-like immunoreactivity (FOS-IR) induction, a marker of neuronal

activation, in ARC POMC neurons of rats that have been given anorectic doses of fenfluramine

which blocks 5-HT reuptake while also stimulating its release. Additionally, they reported

consistent depolarization of ARC POMC neurons upon application of fenfluramine to coronal

hypothalamic slices from transgenic mice with POMC promoter-controlled green fluorescent

protein expression. Furthermore, this group found that in melanocortin receptor (MCR)

knockout mice and in those that have been given the MCR antagonist SHU9119, fenfluramine

exhibited limited efficacy at reducing food intake and body weight.

However, the mechanisms of serotonergic involvement in feeding regulation are likely to

be more complicated than simply its action at the arcuate nucleus of the hypothalamus. As

discussed earlier, it is known that in mammals, divergent serotonergic axons arise from the raphe

35

nuclei, thereby innervating a large proportion of the forebrain structures, as well as other regions

of the brain stem and spinal cord. In fact, all brain nuclei implicated in energy balance

regulation receive serotonergic afferents (for review see, Tecott 2007).

Evidence for Influence of Ovarian Hormones

In addition to the connections and receptor populations discussed above, the hypothalamus

is also densely populated with receptors for the ovarian hormones, estrogen and progesterone

(Bethea et al. 1996). Additionally, other brain regions upstream of the hypothalamus that are

known to regulate HPA axis activity such as the hippocampus and dorsal raphe nuclei express

these receptors as well. Several studies have demonstrated elevations in basal HPA axis activity

during periods of the estrous cycle that are defined by higher levels of progesterone and estrogen

(Raps et al. 1971, Pollard et al. 1975, Ogle & Kitay 1977, Buckingham et al. 1978, Phillips &

Poolsanguan 1978, Carey et al. 1995). The rise in CRH mRNA expression in the PVN in the

afternoon of pro-estrus in the rat has been attributed to estradiol (Bohler et al. 1990), however

when estrogen is administered in a chronic low dose, CRH expression has been shown to

decrease (Dayas et al. 2000). Another group has shown that in primates, estrogen given in a

manner that mimics the preovulatory surge increases CRH expression in the PVN (Roy et al.

1999), but if given chronically, estrogen or progesterone or both will decrease CRH mRNA and

protein (Bethea & Centeno 2008). Estrogen and progesterone are both known to increase

throughout the course of gestation as well and therefore might be at least partially responsible for

changes in HPA axis regulatory mechanisms during pregnancy.

Numerous reports indicate that progesterone can act as an MR antagonist (Rupprecht et al.

1993) and that in the presence of progesterone (Carey et al. 1995 and unpublished data from our

lab) or during pregnancy (Roesch & Keller-Wood 1999), cytosolic availability of hippocampal

MR is increased. Taken together, these findings suggest that progesterone might interfere with

36

cortisol’s ability to negative feedback at hippocampal MR. Contradictory effects of estrogen on

MR have been found as well which include estradiol-induced decreases in hypothalamic and

hippocampal MR binding capacity and mRNA levels (Carey et al. 1995, Castren et al. 1995)

while other groups reported increases in these same regions (Ferrini & De Nicola 1991) or no

change to hippocampal binding capacity in response to longer exposure to estradiol (Burgess &

Handa 1992). Several groups have demonstrated that progesterone will attenuate the estradiol-

induced decreases in hippocampal MR binding capacity, but progesterone will have no effect on

hippocampal mRNA when given alone (Carey et al. 1995, Castren et al. 1995).

Estrogen has been shown to reduce GR binding and mRNA in the anterior pituitary,

hypothalamus, and hippocampus (Peiffer & Barden 1987, Turner 1990, Turner 1992, Burgess &

Handa 1993), but no changes in GR mRNA expression have been found across the estrous cycle

(Sliwowska et al. 2008). It has been proposed that downregulation requires long term exposure

to estrogen (Burgess & Handa 1993, Redei et al. 1994). Our lab has reported no effect of

pregnancy on hippocampal expression of MR or GR in the ewe (unpublished data by Yi Hua).

Additional work by Carey et al (1995) suggests that neither estrogen nor progesterone influences

GR binding in the hippocampus. However, progesterone has also been proposed to be a partial

agonist at GR since binding causes translocation, however it is not entirely clear whether the

necessary conformational change occurs that would allow for transcription factor loading

(Rupprecht et al. 1993, Nordeen et al. 1995).

Meanwhile, ovarian hormones have been reported to cause changes in several components

of the serotonergic system. A review by Bethea et al (2002) describes the large variability in

ovarian hormone influence on serotonin synthesis across species. Estrogen was reported to

increase SERT binding in the rat hypothalamus (Mendelson et al. 1993, McQueen et al. 1997)

37

but decrease binding in the hippocampus (Mendelson et al. 1993). In contrast, Pecins-Thompson

et al (1998) found that estrogen decreased SERT mRNA in the dorsal raphe in nonhuman

primates. Additionally, the same group found that long-term exposure to estrogen decreases

mRNA expression of the 5-HT1A autoreceptor in the dorsal raphe (Pecins-Thompson et al. 1998,

Pecins-Thompson & Bethea 1999). At the level of the hypothalamus, estrogen has been shown

to attenuate 5-HT1A-stimulated increases in ACTH and corticosterone, and this effect appears to

be related to estrogen’s ability to reduce levels of G-proteins that are known to mediate the

actions of this receptor (Raap et al. 2000), but according to Frankfurt et al (1994) the density of

5-HT1A receptors in the hypothalamus is not altered by estrogen. Estrogen has previously been

reported to increase 5-HT2A receptors several non-hypothalamic regions in rats (Biegon et al.

1983, Sumner & Fink 1995, Sumner & Fink 1997, Cyr et al. 1998, Osterlund & Hurd 1998), but

more recently was shown to have no effect on mRNA levels in the hypothalamus of nonhuman

primates (Gundlah et al. 1999). Undoubtedly, there is strong evidence for estrogen and

progesterone modulation of several components which mediate the actions of serotonin in many

brain regions and across many species; however, the directionality and sensitivity of the

responses are quite variable. This is likely due the variability in the receptor milieu and/or

intracellular machinery present in these systems.

38

Figure 2-1. A general model of HPA axis regulation. Corticosteroids negatively feedback at

each level of the axis via mineralocorticoid (MR) and glucocorticoid (GR) receptors; while other systems, including the hippocampus and serotonergic nuclei in the brain stem are also thought to regulate its activity.

39

Figure 2-2. Basic regulatory circuitry for food intake mediated by the arcuate nucleus (ARC) of

the hypothalamus. Leptin from adipocytes stimulates proopiomelanocortin (POMC) neurons which release melanocortins such as α-melanocyte stimulating hormone (α-MSH, a cleavage product of POMC) which then act on melanocortin receptors to ultimately inhibit feeding by stimulating anorexogenic peptides in the paraventricular nuclei (PVN) and inhibiting orexogenic peptides in the lateral hypothalamic area (LHA). Ghrelin from the stomach activates the agouti-related protein (AgRP)/neuropeptide Y (NPY) neuronal population. AgRP is thought to be an inverse agonist at melanocortin receptors and therefore its effects are orexogenic.

40

CHAPTER 3 ROLE OF MINERALOCORTICOID RECEPTORS IN REGULATION OF CORTISOL,

ALDOSTERONE, ELECTROLYTES, AND BLOOD PRESSURE IN PREGNANCY

Introduction

One of the main focuses of our lab is to investigate alterations in the regulation of basal,

maternal hypothalamic-pituitary-adrenal (HPA) axis activity that arise during pregnancy. In both

human (Carr et al. 1981, Erickson et al. 2001, Sandman et al. 2006, Kirschbaum et al. 2009) and

sheep (Bell et al. 1991) studies, basal plasma adrenocorticotropic hormone (ACTH) and cortisol

have been shown to increase during pregnancy. From previous studies in our lab, we understand

that this increase in corticosteroid secretion in pregnancy contributes to the maternal volume

expansion and increased uterine blood flow that must occur to protect the health of the mother

and to insure a supportive environment for the developing fetus (Jensen et al. 2002, Jensen et al.

2003, Jensen et al. 2005). Additional studies in our lab in adrenalectomized sheep have shown

that the concentration for intravenous cortisol replacement required to normalize basal plasma

ACTH is increased for the pregnant ewes and that supplementation with nonpregnant cortisol

levels increases hypotension-stimulated ACTH release (Keller-Wood 1998, Keller-Wood &

Wood 2008). On the other hand, in studies using adrenal intact ewes, ACTH feedback

suppression by raising plasma cortisol above resting levels is not different during pregnancy

(Keller-Wood 1996). It has therefore been theorized by this lab and others that there is an

alteration in ‘set-point’ of the negative feedback regulation of basal HPA axis activity that may

at least in part explain the elevations of maternal plasma ACTH and cortisol seen in pregnancy.

There are a variety of endogenous mechanisms that control the overall activity of the HPA

axis, both basally and in times of stress, none of which have been completely characterized. It is

now known that corticosteroids exert their actions, such as feedback inhibition, through binding

to intracellular receptors, known as glucocorticoid receptors (GR) and mineralocorticoid

41

receptors (MR). It has been postulated through work in several species that activation of central

and pituitary GR leads to feedback inhibition of stress-induced HPA axis activation, thereby

reducing ACTH and cortisol secretion; while activation of the higher affinity MR mainly

expressed in the hippocampus inhibits the activity of the HPA axis at basal corticosteroid levels

(Keller-Wood & Dallman 1984, Reul & de Kloet 1985, Reul et al. 1987, Bradbury et al. 1991,

de Kloet et al. 1993, Reul et al. 2000). For the purposes of studying alterations in basal HPA

axis regulation during pregnancy, this study aims to characterize the relative role of MR in the

pregnant ewe.

MR have been studied extensively within the central nervous system of many rodent

models (Reul & de Kloet 1985, Luttge & Rupp 1989, Funder 1996). In the rodent brain, MR

have been found primarily in the hippocampus and septum. In fact, regions of the hippocampal

formation express both receptors and this is not surprising as it is thought to play an inhibitory

role in regulation of the HPA axis through indirect connections to the PVN, such as those made

through the septal nucleus of the stria terminalis (Sapolsky et al. 1986, Jacobson & Sapolsky

1991, Herman & Cullinan 1997, Herman & Mueller 2006). More recent studies characterizing

corticosteroid receptor distribution in the primate brain, also found that MR mRNA and protein

levels were much higher in the dentate gyrus (DG) and cornu ammonis (CA) of the hippocampus

than other brain regions (Sanchez et al. 2000). In sheep, both receptors are expressed within the

main regulatory areas of the HPA axis: the hypothalamus, hippocampus, and pituitary (Roesch &

Keller-Wood 1999). Our lab has also shown that the hippocampus has increased cytosolic MR

availability in pregnant ewes compared to nonpregnant ewes and a tendency, although not

significant, toward increased MR availability in the hypothalamus and pituitary as well. These

findings suggest reduced MR activation despite the higher plasma cortisol levels that occur with

42

pregnancy (Roesch & Keller-Wood 1999). We therefore hypothesize that the importance of MR

in feedback inhibition is altered in the pregnant state, allowing HPA activity to be increased.

Furthermore, renal MR are classically known to be important in electrolyte balance and

plasma volume regulation through its effects at the distal nephron. Specifically, when a drop in

renal perfusion pressure, stimulation by renal sympathetic nerves, or a reduction in sodium

chloride delivery is detected at the macula densa, aldosterone release from the adrenal cortex is

triggered by angiotensin II via increased activity of the renin-angiotensin-aldosterone system.

Additionally, aldosterone release can be stimulated directly when increased plasma potassium

concentration is detected by the adrenal zona glomerulosa cells. MR binding by aldosterone

starts the chain of events to increase production of proteins that are involved in active sodium

(Na+) reabsorption by principal cells along the distal tubule and collecting duct. Meanwhile,

potassium excretion is increased due to the actions of aldosterone on both Na/K ATPase activity

and on apical conductance of K+.

It has been reported previously that spironolactone has a very low affinity for the

glucocorticoid receptor (GR) relative to MR (Couette et al. 1992, Rupprecht et al. 1993).

Canrenoate potassium, the active metabolite of spironolactone, penetrates the blood-brain barrier

and in humans has a half-life of 3.7 ± 1.2 hrs (Funder et al. 1974, Rothuizen et al. 1993).

Previous reports have shown that administration of MR antagonists in animal and human studies

elevates plasma cortisol (Dodt et al. 1993, Young et al. 1998, Arvat et al. 2001, Grottoli et al.

2002). In the current study, by infusing canrenoate intravenously, we expect to effectively

antagonize both central and renal MR as a means to characterize the relative importance of MR

actions in the regulation of adrenal hormone secretion, electrolytes, and blood pressure in

pregnancy in the ewe. We therefore hypothesize that if reduced central MR action is at least in

43

part responsible for elevated cortisol during pregnancy, and if hemodynamic changes such as

plasma volume and blood pressure that occur during pregnancy are at least in part mediated by

MR, then systemic MR antagonism will result in attenuated HPA axis stimulation and greater

hemodynamic challenges in pregnant ewes compared to nonpregnant ewes.

Materials and Methods

Animals

Animals were housed in climate controlled, individual pens located in the University of

Florida Animal Care Facility; all animal use was in accordance with the rules and regulations of

the Institutional Animal Care and Use Committee (IACUC) at the University of Florida. Mixed

western breed ewes, both nonpregnant (NP: n=6) and pregnant (P: n= 6, at 136 ± 3 days of

gestation; term is approximately 147 days), were used in this study.

Surgical Protocol

Before surgery, food was withheld from the ewe for 24 hours. All surgeries were

performed in the surgery suite of the Health Science Center, Animal Resources Department.

Animals were prepared for and underwent surgery under aseptic conditions. Animals were

induced with isoflurane inhalant and maintained using either isoflurane or halothane inhalant (1-

3% in oxygen).

Sterile bilateral polyvinyl catheters (inner diameter (ID): 0.050in, Tygon® Microbore

Tubing, Saint-Gobain Performance Plastics Corp., Akron, Ohio) were placed into the femoral

arteries and veins of both nonpregnant and pregnant ewes. A trocar device was then used to

direct the catheters to an exit site near the flank of the ewe. For a five day recovery period, the

animals received twice daily intramuscular injections of ampicillin (1g); body temperature was

also monitored twice daily. Post-operative care during the recovery period and through the

remainder of the study also consisted of daily exit site cleansing with a povodine iodine solution.

44

Experimental Protocol

At the end of the recovery period, each ewe was randomly assigned to one of two

treatment regimens: (1) intravenously (iv) infused with the MR antagonist canrenoate in saline

(CAN: 4 mg/kg bolus followed by 1 mg/kg/hr infusion for 4 hours) or (2) saline (VEH) at the

same infusion rate (total volume infused for both treatment groups was 40 ml over 4 hours).

Two days later, the experiment was repeated for each ewe using the other of the two treatments.

Because this study focuses on basal HPA axis activity, access to arterial and venous

catheters was achieved using methodology previously established by this lab that minimizes

human interaction with the ewes (Bell et al. 1991). Briefly, the catheters were externalized from

the pens via a swiveling duct system, prior to each experiment. Following an acclimation period

of 1 hour, it was then possible to re-enter the room quietly in order to access the catheters

remotely from just outside the pen. Venous catheters were attached to syringes that were

positioned into syringe pumps containing either saline or canrenoate in saline. Arterial catheters

were filled with heparinized saline at the start of the experiment and after each sample; and this

‘dead-space’ was removed just prior to the subsequent sample. Plasma was acquired from

samples taken just prior to the start of the infusion and then at every hour during the infusion by

collecting 8 ml of whole blood into tubes containing 400 µL 0.3M ethylenediaminetetraacetic

acid (EDTA) followed by centrifugation at 3000 x g for 20 min at 4°C. Plasma was transferred

to a clean tube and stored at −20oC for future analysis. At the time of the experiment, separate

1.5 ml whole blood samples collected into heparinized syringes were used for determination of

hematocrit and total protein as well as for determination of plasma sodium and potassium

concentrations using ion specific electrodes (AVL 9180 Electrolyte Analyzer, AVL/Roche

Diagnostics, Roswell, Georgia). Basal mean arterial blood pressure (MAP) was recorded

continuously for the 4 hours of infusion (except during the brief sampling periods) via pressure

45

transducers connected to an analog-to-digital conversion board (LabView, National Instruments,

Austin, Texas). Blood pressures were taken at a sampling rate of 60 Hz and later averaged into

5-min bins for statistical analysis.

Plasma Hormone Determination

Plasma cortisol was determined using ELISA (Oxford Biomedical Research, Oxford,

Michigan). Plasma ACTH was determined by radioimmunoassay (RIA) as previously described

using an antibody to 1-39 ACTH (Bell et al. 1991). Plasma progesterone and aldosterone were

determined by RIA using the 125I Coat-A-Count® progesterone and aldosterone kits (Siemens

Healthcare Diagnostics Inc., Deerfield, Illinois). Plasma Angiotensin II levels were determined

by RIA after extraction from plasma using acetone, a method previously used in this laboratory

(Pecins-Thompson & Keller-Wood 1997).

Plasma Volume Determination

At the end of each experiment, the total plasma volume was determined using the Evans

blue dye dilution method (Pecins-Thompson & Keller-Wood 1997). More specifically, after the

last sample of the experiment and prior to injection of Evans blue dye, 10 ml of whole blood was

collected into EDTA-treated tubes. The plasma from this sample was used as the ‘no-dye’

sample and also as the vehicle for the standard curve of the plasma volume assay.

Approximately 2.5-4 ml (15-25 mg in saline) was injected into the venous catheter, followed by

saline (enough to cover the length of the catheter dead-space). Starting at 10 min post-Evans

blue injection, 2 ml of whole arterial blood was collected into EDTA-treated tubes at 5 min

intervals until 45 min. Following centrifugation, the plasma was transferred to clean tubes.

Evans blue concentration in the plasma was determined by measuring the absorbance at 620 nm

in a Synergy HT Multi-Mode Microplate reader (Bio Tek Instruments, Inc., Winooski, Vermont)

in triplicate and extrapolating the concentration from the standard curve. Using the

46

concentration of Evans blue at each time-point for each animal, the plasma volume was then

determined using the Indicator Dilution Principle. This method utilizes the formula: plasma

volume (PV) = Vd/BW, where BW is body weight and Vd (volume of distribution in liters) is

defined as: [volume of dye injected X concentration of injected dye X 1000]/C0. C0 is the

extrapolated concentration of dye in plasma at Time 0.

Data Analysis

The effects of pregnancy and MR blockade on blood pressure and plasma levels of

cortisol, ACTH, aldosterone, angiotensin II, sodium, potassium, hematocrit, and total protein

over time were analyzed using a three-way repeated measures analysis of variance (RM

ANOVA) of the between-subjects effect of pregnancy status and the within-subjects effects of

treatment and time. Separate two-way RM ANOVA’s were then performed, where indicated, to

identify differences within a given treatment or group. The effects of pregnancy and MR

blockade on plasma volume were analyzed using a two-way RM ANOVA of the between-

subjects effect of pregnancy status and the within-subjects effect of treatment. Duncan’s

multiple range test was performed to determine the source(s) of significance for each two-way

RM ANOVA. Plasma progesterone was compared between pregnant and nonpregnant ewes

using the Student’s t-test. Data are expressed as mean ± standard error of the mean (SEM). A

value of p < 0.05 was considered significant.

Results

Plasma Hormone Levels

Adrenocorticotropic hormone (ACTH)

As expected, there was an overall effect of pregnancy on the plasma ACTH concentration

(Figure 3-1). Three way RM ANOVA indicated that ACTH concentrations in the pregnant ewes

were significantly greater than in the nonpregnant ewes during (p<0.05). The mean plasma

47

ACTH collapsed over the four hours of vehicle infusion was 55 ± 2 pg/ml in the nonpregnant

ewes and 76 ± 5 in the pregnant ewes. Three-way RM ANOVA revealed that the MR antagonist

significantly increased plasma ACTH concentrations, but the effect of treatment varied by group

(main effect of treatment, interaction of treatment and group, p<0.05). Two-way RM ANOVA

performed within nonpregnant ewes followed by Duncan’s Multiple range test suggested that

canrenoate infusion resulted in transiently increased plasma ACTH at the 1 hour time-point in

the nonpregnant ewes compared to 1 hour levels during saline infusion, however this was not

significant. Two-way RM ANOVA performed within pregnant ewes revealed a significant

effect of treatment and Duncan’s Multiple Range test demonstrated a significant increase at 2

and 4 hours of canrenoate infusion compared to the corresponding time points during saline

infusion.

Cortisol

Three-way RM ANOVA on plasma cortisol concentrations in these ewes, indicated

significant main effects of treatment and time and significant time by group, treatment by time,

and treatment by time by group interactions. Separate two-way RM ANOVA followed by

Duncan’s multiple range test performed on nonpregnant and pregnant ewes revealed that in

nonpregnant ewes, cortisol exhibited an initial, transient rise at 1 hour and then declined to levels

that were not different from levels measured at the corresponding time point during infusion of

saline (Figure 3-2). In contrast, during infusion of canrenoate in pregnant ewes, there was still

no increase in plasma cortisol by 1 hour, but cortisol was significantly increased compared to

saline by 2 hours of canrenoate infusion and continued to be elevated over the next two time-

points.

48

Aldosterone

The plasma aldosterone response to canrenoate infusion over time varied between pregnant

and nonpregnant ewes (Figure 3-3). Aldosterone levels in nonpregnant ewes either prior to

infusion of canrenoate or throughout the vehicle infusion were either at or below the lower limit

of detection for this assay (12.5 pg/ml). Three-way RM ANOVA demonstrated that there were

main effects of time, treatment, and pregnancy status on plasma levels of aldosterone (p<0.05).

There were also significant two- and three-way interactions (treatment by pregnancy status, time

by treatment, pregnancy status by time, and time by treatment by pregnancy status; p<0.05).

Upon separate two-way RM ANOVA followed by Duncan’s multiple range test within each

group, the effect of canrenoate infusion in nonpregnant ewes was determined to be significantly

increased by 3 hours; whereas canrenoate effects in pregnant ewes are significantly increased by

2 hours. Additionally, a separate analysis within the canrenoate-treated ewes revealed that the

aldosterone response to MR blockade was significantly greater for pregnant ewes than for

nonpregnant ewes from the 2 hour time point onward.

Angiotensin II

Three-way RM ANOVA revealed significant main effects of time and pregnancy status on

plasma angiotensin concentration (Figure 3-4). Overall, angiotensin II was significantly greater

in pregnant compared to nonpregnant ewes. Additionally, plasma angiotensin concentration

increases over time (three-way RM ANOVA: main effect of time, p<0.05).

Mean Arterial Blood Pressure

As expected, mean arterial blood pressure (MAP) was significantly lower in the pregnant

ewes during infusion of either saline or MR antagonist (Figure 3-5, p<0.05). The MAP values

collapsed over time for the vehicle-infused nonpregnant and pregnant ewes were 116 ± 4 and

102 ± 3 mmHg, respectively. Based on three-way RM ANOVA, the decrease in MAP in

49

response to canrenoate infusion was not significant for either group, however, there were time by

group as well as treatment by group by time interactions (p<0.05).

Hematocrit (%) and Plasma Solute (Total Protein, Potassium, and Sodium) Concentrations

There was a significant effect of infusion of either vehicle or canrenoate on hematocrit as it

significantly decreased in both groups over time (Figure 3-6A, main effect of time based on

three-way RM ANOVA, p<0.05). This effect was attenuated by infusion with canrenoate

(interaction of time and treatment, p<0.05). Plasma protein concentration was significantly

lower overall in the pregnant ewes (Figure 3-6B, main effect of group based on three-way RM

ANOVA, p<0.05). There was no effect of canrenoate on plasma protein concentrations in either

the pregnant or the nonpregnant ewes. There were no overall differences in plasma potassium or

sodium levels between pregnant and nonpregnant ewes. Plasma potassium (K+) increased in

both groups during canrenoate infusion (Figure 3-6C, three-way RM ANOVA: main effects of

time and treatment, interaction of time and group, p<0.05). Two-way RM ANOVA followed by

Duncan’s Multiple Range test performed on each group separately revealed that elevations in

potassium concentration due to canrenoate infusion were significantly greater than vehicle by 2

hours in the pregnant ewes versus 3 hours in the nonpregnant ewes. There was no effect of

canrenoate infusion on plasma sodium (Na+) concentration in ewes in either group (Figure 3-

6D).

When backward stepwise linear regression was performed to assess the role of angiotensin

II and K+ in driving plasma aldosterone during infusion of canrenoate, both were found to be

significant (p=0.009 and P<0.001, respectively). However, when the relationship between

plasma angiotensin II and plasma aldosterone was determined for nonpregnant and pregnant

ewes separately, no significant correlation was found in either group (NP: r=0.147, p=0.437; P:

r=0.305, p=0.101). However, when the relationship between plasma K+ and plasma aldosterone

50

was compared between pregnant and nonpregnant ewes, there appeared to be a difference in this

relationship between the two groups (Figure 3-7). Two-way ANOVA of the between-subjects

effects of pregnancy status and 0.3 mEq/L bins of plasma K+ on plasma aldosterone

concentration during canrenoate infusion revealed a significant effect of pregnancy status on the

aldosterone values at plasma K+ of greater than 4.8 mEq/L (main effects of group and binned K+

concentration as well as a two-way interaction, p<0.05).

Plasma Volume

Plasma volume tended to be higher in the pregnant ewes (44.3 ± 2.3 ml/kg in P ewes

versus 34.6 ±2.3 ml/kg in the NP ewes, p=0.062). Although the differences in plasma volume

measured at the end of canrenoate infusion did not reach significance, there appeared to be a

trend for MR blockade to decrease plasma volume in both groups with an apparent tendency

toward greater decreases in the pregnant ewes (Figure 3-8). The reduction in plasma volume

after treatment with the MR antagonist in the pregnant and nonpregnant ewes was 3.7 ml/kg and

1.8 ml/kg, respectively.

Discussion

The nonpregnant responses in our study are consistent with those found in human and rat

studies in which anti-mineralocorticoid challenge using MR antagonists such as spironolactone

or its active metabolite, canrenoate, causes elevated plasma cortisol concentrations. In humans,

as in the nonpregnant ewes in this study, plasma cortisol concentration was increased as soon as

one hour after administration of MR blockade using spironolactone or canrenoate (Deuschle et

al. 1998, Young et al. 1998, Arvat et al. 2001, Kellner et al. 2002, Wellhoener et al. 2004,

(Grottoli et al. 2002). The plasma ACTH concentrations in these ewes follow a similar time

course, having a tendency to be transiently elevated one hour after the start of treatment with the

51

MR antagonist canrenoate. It is possible that after this time point, we may be observing the

negative feedback effect of elevated cortisol at GR, the lower affinity receptor.

Interestingly, the response pattern of the plasma cortisol and plasma ACTH measured

hourly during infusion with the MR antagonist, canrenoate, differed between pregnant and

nonpregnant ewes, which supports our hypothesis that mineralocorticoid receptor regulation of

the hypothalamic-pituitary-adrenal axis differs between the pregnant and nonpregnant states. I

hypothesize that the differential early (0-2 hours) response pattern between pregnant and

nonpregnant ewes is due, at least in part, to differences in relative importance of central and/or

pituitary MR in negative feedback control of the axis because the ACTH response to canrenoate

infusion appears to exhibit a similar temporal pattern to the cortisol response. The hippocampus

is a prime suspect for future investigations of the mechanism(s) for this differential response.

Based on previous evidence, hippocampal and/or septal MR are thought to be the mediators of

HPA axis responses to canrenoate infusion as they are most densely expressed in the dentate

gyrus and cornu ammonis of the hippocampus as well as the septum and thought to confer the

inhibitory function of the hippocampus on HPA axis regulation (Reul et al. 2000). Further

evidence of the importance of hippocampal and septal MR in regulation of the HPA axis is

provided by the fact that MR blockade-induced HPA axis activation by canrenoate can be

blocked by the GABAA receptor agonist alprazolam in humans (Grottoli et al. 2002).

It is also known that throughout gestation in humans and sheep, plasma progesterone is

much higher than it would be in the nonpregnant state (Rosenthal et al. 1969, Johansson &

Jonasson 1971, Bell et al. 1991). Our current study supports this pattern since the mean plasma

progesterone levels for nonpregnant and pregnant ewes were, 5.3 ± 1.6 and 25.4 ± 2.6 pg/ml,

respectively (p<0.001). Previous reports from this lab showed that progesterone is a

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physiological ligand for MR and that prior exposure to progesterone reduces the Kd of MR for

cortisol, suggesting that progesterone’s effects are antagonistic (Richards et al. 2003). Studies in

other labs have also shown that progesterone shows a high affinity for MR with only weak

transactivation activity (Rafestin-Oblin et al. 1992, Carey & de Kloet 1994, Funder & Myles

1996, Turner 1997). This suggests progesterone effectively antagonizes binding and subsequent

activation by cortisol, but may also serve as a weak agonist when cortisol levels are relatively

low (Keller-Wood & Wood 2008). It has therefore been theorized that the increase in

hippocampal cytosolic MR availability in pregnancy in the ewe may be due to binding by

progesterone which is thought to prevent transactivation of the receptor and localization of the

receptor to the nucleus as would occur with cortisol or aldosterone as ligands (Roesch & Keller-

Wood 1999). In fact, unpublished data from our lab supports this theory as hippocampal

cytosolic MR availability was also increased in nonpregnant ewes treated with progesterone. In

humans, prior MR antagonism in arginine vasopressin- (AVP-), corticotropin-releasing

hormone- (CRH-), and exercise-induced HPA activation exacerbated the elevation in plasma

cortisol, suggesting that prior blockade of MR activity can alter the set point for HPA axis

activation (Heuser et al. 2000, Arvat et al. 2001, Wellhoener et al. 2004). I theorize that the

differential response to MR blockade in pregnancy reflects the presence of the endogenous

antagonist, progesterone, specifically at MR in the hippocampus. This could explain the absence

of the early stimulatory effect in the pregnant ewes during canrenoate infusion. I hypothesize

that because more MR are available in the nonpregnant ewes due to relatively low progesterone

levels, MR blockade with canrenoate allows for greater increases in HPA axis activity in these

ewes. Whereas in the pregnant animals, the presence of high plasma progesterone could allow

endogenously-derived HPA axis disinhibition and therefore addition of an MR antagonist has

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relatively no direct effect on HPA axis activity. In this way, it is therefore possible that

progesterone is at least in part responsible for an alteration in HPA axis negative feedback set

point, much like the pre-treatment with MR blockade did in the human studies.

The differential late (2-4 hours) response pattern for ACTH and cortisol between pregnant

and nonpregnant ewes likely reflects differences in relative importance of MR and the HPA axis

in regulation of blood pressure and volume that must occur with pregnancy in order to support

the health of the mother and fetus. From Figure 3-5, although not significant, there appears to be

a trend for mean arterial pressure to be reduced by canrenoate infusion for both groups.

Additionally, there were tendencies for differential patterns of responses in plasma volume,

protein, potassium, and hematocrit with either with vehicle infusion or with MR blockade.

These findings may not have reached significance simply due to the need for more animals in

each group; or because the dose we used caused changes in these endpoints that were subtle

enough to be overcome by other mechanisms. I propose that the increases in cortisol over time

with canrenoate infusion in the pregnant ewes may be an indirect response to decreases (although

not significant by our methods of detection) in blood pressure and plasma volume. Additionally,

the enhanced plasma aldosterone response to anti-mineralocorticoid challenge in pregnant ewes

further supports a role for corticosteroids in the hormonal regulation of volume expansion in the

pregnant state. Angiotensin II concentrations in the pregnant ewes were greater than those

detected in the nonpregnant ewes, but this was not influenced by canrenoate infusion.

Interestingly, linear regression analyses of either plasma potassium or angiotensin II versus

plasma aldosterone concentrations with canrenoate infusion suggested that although both factors

are significant determinants of plasma aldosterone, that a difference in responsiveness to plasma

potassium was likely to be responsible for the greater plasma aldosterone concentrations between

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the pregnant ewes during canrenoate infusion. The increase in plasma potassium was higher in

pregnant ewes compared to nonpregnant ewes at the 3 hr time point based on two-way RM

ANOVA followed by Duncan’s Multiple Range test on canrenoate-treated ewes. This suggests

that the relative importance of MR in regulation of plasma potassium might be shifted during

pregnancy. The tendencies toward differences in these hemodynamic endpoints are however

consistent with the proposed theory that pregnancy is perceived by homeostatic mechanisms

which regulate plasma volume as an “underfilled” state (for review, see: Schrier & Durr 1987).

Likely, the greater increases in plasma ACTH, cortisol and aldosterone that we observed in the

pregnant ewes during the second half of the canrenoate infusion, reflect activation of such

mechanisms necessary to combat any decreases in plasma volume and MAP that might have

otherwise occurred in response to blockade of MR. These results also support previous findings

in our lab that the elevated basal corticosteroid concentration of pregnancy contributes to

maternal volume expansion and further suggests that these effects may be mediated at least in

part by the mineralocorticoid receptor, presumably at the kidney. However, direct action of

mineralocorticoids in the brain and the resulting cardiovascular effects have also been reported

(Chen et al. 1989, Gomez-Sanchez 1997). Small amounts of aldosterone delivered

intracerebroventricularly in rats produce a significant increase in arterial blood pressure; while

chronic icv infusion of an MR antagonist inhibited mineralocorticoid-induced hypertension in

rats (Chen et al. 1989, Gomez-Sanchez et al. 1990, Janiak et al. 1990).

Overall, these results support my hypothesis that plasma ACTH and cortisol would be

elevated in response to intravenous MR antagonism and that direct HPA axis responses would be

blunted in pregnant ewes relative to nonpregnant ewes. The corresponding differential response

pattern for both ACTH and cortisol release suggests differences in relative importance of central

55

and/or pituitary MR in negative feedback control of the axis. The differential HPA axis

responses during the second half of the infusion are likely reflective of the increase in relative

importance of corticosteroids in homeostatic mechanisms for maintenance of hemodynamic

endpoints such as maternal plasma volume and blood pressure.

This research was supported by an R01 grant from the NIH (DK38114) to Maureen Keller-

Wood. Some of this data was presented at the 89th Annual Meeting of the Endocrine Society,

2007.

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Figure 3-1. Plasma ACTH concentrations during canrenoate or vehicle infusion. Three-way RM

ANOVA indicated main effects of treatment and group as well as an interaction between treatment and group (p<0.05). * Significantly different from values obtained in the same group during saline infusion at the corresponding time point, based on separate two-way RM ANOVA followed by Duncan’s multiple range test for each group (p<0.05). Data are represented as means ± SEM.

57

Figure 3-2. Plasma cortisol concentrations during canrenoate or vehicle infusion. Three-way

RM ANOVA on plasma cortisol concentrations indicated significant main effects of treatment and time and significant time by group, treatment by time, and treatment by time by group interactions. * Significantly different from values obtained in the same group during saline infusion at the corresponding time point, based on separate two-way RM ANOVA followed by Duncan’s multiple range test for each group (p<0.05). Data are represented as means for plasma cortisol concentration ± SEM.

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Figure 3-3. Plasma aldosterone (pg/ml) concentrations during iv administration of saline (VEH)

or canrenoate (CAN) in (A) nonpregnant and (B) pregnant ewes. Overall, pregnant aldosterone levels were significantly greater than nonpregnant levels. * Indicates levels significantly different from those obtained from the same group during saline infusion at corresponding time point. ** Indicates significantly different from nonpregnant response to canrenoate infusion at corresponding time point. Data are represented as means ± SEM and considered significant at p<0.05. The dotted line indicates the lower limit of detection for the aldosterone assay at 12.5 pg/ml.

59

Figure 3-4. Plasma angiotensin II (pg/ml) during iv administration of saline (VEH) or

canrenoate (CAN). Three-way RM ANOVA revealed that angiotensin II concentrations were greater in pregnant ewes compared to nonpregnant ewes and overall, there was a significant increase over time (*p<0.05). There was no significant effect of canrenoate treatment in either group. Data are represented as means ± SEM.

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Figure 3-5. Mean arterial pressure during canrenoate or vehicle infusion in (A) nonpregnant and

(B) pregnant ewes. Overall, MAP in pregnant ewes was significantly lower than in nonpregnant ewes (p<0.05). Values are means ± SEM of 5-minute bins of MAP data.

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Figure 3-6. Hematocrit and plasma solute concentrations. The effect of intravenous vehicle

(VEH) infusion on percent hematocrit (A) was attenuated by canrenoate (CAN) infusion in both groups. Plasma protein (B) was significantly lower in pregnant (P) ewes with no effect of canrenoate in either group. Canrenoate infusion increased plasma potassium (C) in both nonpregnant (NP) and pregnant ewes. There were no significant differences in plasma sodium (D) in response to canrenoate infusion or due to pregnancy. Values are means ± SEM and considered significant at p<0.05.

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Figure 3-7. Linear regression of plasma potassium and plasma aldosterone during canrenoate

infusion. The adrenal responsiveness to plasma potassium concentration in nonpregnant (NP, open circles) and pregnant (P, filled circles) ewes during canrenoate infusion appears to differ (slope of 36.4 ± 7.8 in NP compared to 140.1 ± 25.4 in P). For both groups, plasma aldosterone was significantly and positively correlated with plasma potassium concentration (NP: r=0.660, p<0.001; P: r=0.722, p<0.001).

63

Figure 3-8. Plasma volume measured at the end of 4 hr infusion of canrenoate (CAN) or vehicle

(VEH). Plasma volume tended to be higher in pregnant ewes (P: 44.3 ± 2.3 ml/kg versus NP: 34.6 ±2.3 kg/ml, three-way RM ANOVA, p=0.062). There is also an apparent trend for MR blockade to decrease plasma volume in both groups, however this was not significant. Values are means ± SEM.

64

CHAPTER 4 RELATIVE SEROTONERGIC ACTIVITY/RESPONSIVITY DURING PREGNANCY

Introduction

Regulation of the HPA axis is indeed multifaceted and therefore it is not likely that

alterations in any one control mechanism are solely responsible for its increased activity during

pregnancy. I will now switch the attention to a system that has been shown to be associated with

the HPA axis based on physical proximity, pharmacological characterization and in disease

states, the serotonergic system.

The serotonergic system is thought to be one of the upstream stimulatory inputs to the

HPA axis (Calogero et al. 1990, Fuller 1996). Serotonergic cell bodies are found in discrete

clusters or groups of cells along the midline of the brain stem while their axons innervate nearly

every area of the central nervous system. Serotonin, or 5-hydroxytryptamine (5-HT),

synthesized from tryptophan within the cytosol of these neurons, serves as a neurotransmitter

involved in the regulation of mood, feeding behavior, circadian rhythmicity, learning and

memory (Jacobs & Azmitia 1992, Jacobs & Fornal 1999, Cooper et al. 2003). Dysfunction of

the serotonergic system is thought to be a key factor in the pathogenesis of many types of

depression in humans, many of which are also thought to be associated with a dysregulation of

the HPA axis.

Histologically, it has been demonstrated that serotonergic axons synapse on CRH-

containing neurons in the rat PVN and more recently, several 5-HT receptor subtypes have been

shown to moderately populate key areas for HPA axis regulation (Liposits et al. 1987, Petrov et

al. 1994, Wright et al. 1995, Li et al. 1997). Calogero et al (1989) demonstrated in vitro that

serotonin directly stimulates CRH release in hypothalamic cultures. Tsagarakis et al (1989) also

demonstrated this effect in vivo in rats using hypothalamic implants of serotonin. In fact, an

65

extensive review by Carrasco and Van de Kar (2003) discusses numerous studies in rodents that

provide evidence that serotonergic agents have a stimulatory effect on the HPA axis hormones.

In humans and animal models including sheep, intravenous injection or oral administration of

serotonin, its precursors, or 5-HT receptor agonists significantly increase plasma ACTH and

cortisol (Kile & Turner 1985, Calogero et al. 1990, Fuller 1996, Broadbear et al. 2004,

Broadbear et al. 2005, Heisler et al. 2007). Cessation of the serotonergic signal is achieved

primarily (80%) by the removal of serotonin from the synaptic cleft through an active membrane

transporter encoded for by the serotonin transporter (SERT) gene. It is therefore not surprising

that selective serotonin reuptake inhibitors (SSRI’s), which act on this transporter, are associated

with a rise in both portal CRH and plasma ACTH (Bevan & Scanlon 1998). Recently, another

group utilized 5-HT receptor subtype agonists along with anti-CRH antiserum to propose that

HPA axis activation likely occurs via 5-HT1A, 5-HT2A, 5-HT2C and/or 5-HT1B receptors and that

the response is mediated by CRH in rats (Jorgensen et al. 2002).

The goal of this study was to determine whether there are alterations in the serotonergic

component of basal HPA axis regulation during pregnancy that may at least in part explain the

elevation in plasma ACTH and cortisol that must occur to support the health of the mother and

developing fetus. We hypothesized that pregnant ewes would demonstrate an increase in

serotonergic tone or serotonergic responsivity by exhibiting a more robust HPA axis response to

fluoxetine. There were two parts to this objective: (1) to compare the response to an acute

intracerebroventricular (icv) injection of a selective serotonin reuptake inhibitor, fluoxetine

(FLX) in pregnant ewes to the response in the same ewes postpartum; and (2) to compare the

response to subchronic icv administration of a sub-maximal (and more clinically relevant) dose

of fluoxetine in pregnant ewes to nonpregnant ewes. The selective serotonin reuptake inhibitor,

66

fluoxetine has little affinity for muscarinic, histaminic, serotonergic, or noradrenergic receptors

and is selective for serotonin reuptake without affecting norepinephrine reuptake (Stark et al.

1985). Use of fluoxetine will serve to exploit the inherent serotonergic activity in these ewes by

keeping the neurotransmitter in the synaptic cleft longer in order for it to exert its effects as well

as provide information about relative responsivity of the HPA axis to serotonin between pregnant

and nonpregnant or postpartum ewes. If our findings support our hypothesis, this alteration in

serotonergic tone during pregnancy could explain, at least in part, the increase in basal HPA axis

activity that is necessary for maternal and fetal health.

Materials and Methods

Animals

Animals were housed in climate controlled, individual pens located in the University of

Florida Animal Care Facility; all animal use was in accordance with the rules and regulations of

the Institutional Animal Care and Use Committee (IACUC) at the University of Florida.

For Study I - HPA axis responses to acute, icv fluoxetine:

Pregnant (n=6, between 134 and 137 days of gestation; term is approximately 147 days)

mixed, western breed ewes were used for this study. Animals were then allowed to deliver and

were studied again in the postpartum state (8 ± 3 days postpartum). Lambs were removed

promptly after parturition in order to avoid any interference from changes in hormones

associated with lactation.

For Study II - HPA axis responses to “subchronic” icv fluoxetine:

Pregnant (P, between 117 and 126 days of gestation) and nonpregnant (NP) mixed western

breed ewes were used for this study. Prior to surgery, animals were randomly assigned to

receive either fluoxetine (FLX), a selective serotonin reuptake inhibitor (NP FLX: n=6, P FLX:

n=7) or vehicle (NP VEH: n=5, P VEH: n=5). The duration of infusion utilized in this study is

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referred to as ‘subchronic’ as the fluoxetine is infused for only 6 consecutive days. This duration

is shorter than that which produces changes in MR, GR, or CRH expression observed with more

chronic dosing (at least 2-4 weeks) as in therapeutic treatment for clinical depression (Brady et

al. 1992, Seckl & Fink 1992, Lai et al. 2003).

Surgical Protocol

Before surgery, food was withheld from the ewe for 24 hours. All surgeries were

performed in the surgery suite of the Health Science Center Animal Resources Department.

Animals were prepared for and underwent surgery under aseptic conditions. Animals were

induced with isoflurane and maintained using isoflurane or halothane inhalant (1-3% in oxygen).

For infusion of drug or vehicle, a sterile polyvinyl catheter (ID: 0.030 inch at icv tip

affixed to ID: 0.040 inch catheter prior to sterilization, Tygon® Microbore Tubing, Saint-Gobain

Performance Plastics Corp., Akron, Ohio) was placed into the lateral ventricle of the ewe.

Briefly, a small hole was placed in the skull using a sterilized portable rotary Dremel® (Robert

Bosch Tool Corp, Racine, Wisconsin) approximately 3 mm to the right of bregma, and a 21g

needle was lowered until cerebrospinal fluid flowed into the hub. The needle was then replaced

with the catheter, the hole in the skull around the catheter was filled with bone wax (CP Medical,

Portland, Oregon) and the catheter was secured to the skull using VetbondTM Tissue Adhesive

(3M™, St. Paul, Minnesota).

For Study I:

The free-end of the icv catheter was externalized, sutured in place and plugged using a

sterile 16 g brass nail. The external portion of the catheter was protected under Vetrap™ (3M™,

St. Paul, Minnesota) until icv access was needed on the day of the experiment. Additionally, at

the time of experiment, a jugular venous catheter was placed for blood sampling for hormone

levels.

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For Study II:

The free-end (ID: 0.040 in) of the icv catheter was affixed to a subcutaneous Alzet®

osmotic pump (model 2ML2; 5 µl/h; Durect Corporation, Cupertino, California) which was filled

at the time of surgery with either vehicle (50:50, dimethyl sulfoxide (DMSO): saline or H2O) or

fluoxetine (5 mg/ml in vehicle) and positioned in a subcutaneous pocket created near the base of

the skull. The Alzet® pump is designed to pump at a rate of 120 ul/day (or 0.6 mg FLX/24 hrs).

The 0.040 inch portion of the catheter in this study was 28 cm in length in order to provide two

days of ‘vehicle dead-space’ before the contents from the Alzet® pump would begin reaching the

lateral ventricle. Sterile bilateral femoral arterial and venous polyvinyl catheters (ID: 0.050 in,

Tygon® Microbore Tubing, Saint-Gobain Performance Plastics Corp., Akron, Ohio) were also

placed as previously described for blood sampling and direct mean arterial pressure recording

(Bell et al. 1991). A trocar device was then used to direct the catheters to an exit site on left side

of the ewe.

In both studies, during the five days immediately following surgery, the animals received

twice daily intramuscular injections of ampicillin (1g); body temperature was also monitored

twice daily. Post-operative care also consisted of daily exit site cleansing with a povodine iodine

solution during the recovery period and for the duration of the study.

Experimental Protocol

Access to arterial and venous catheters was achieved using a method previously

established by this lab (Bell et al. 1991). Briefly, in order to minimize the stress of human

contact to properly measure basal HPA axis activity, the femoral catheters are externalized from

the pens via a swiveling duct system prior to each experiment, followed by an acclimation period

of about 1 hour. Arterial catheters were filled with heparinized saline at the start of the

experiment and after each sample; and this ‘dead-space’ was removed just prior to the

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subsequent sample. Plasma was acquired by collecting 8 ml of whole blood into tubes

containing 400 µL 0.3M ethylenediaminetetraacetic acid (EDTA) followed by centrifugation at

3000g for 20 min at 4°C. Plasma was then removed and stored at −20oC for future analysis.

For Study I:

After 5 days of post-operative recovery, morning experiments were conducted. For each

experiment, baseline plasma was collected, followed by an injection of either 3.3 mg fluoxetine

or saline (total volume injected = 1 ml) directly into the icv catheter. Blood samples were

collected as described above in 10 minute intervals until 60 minutes post-injection. The

experiment was then repeated at least 2 days later, using the other of the two treatments. The

animals were then allowed to deliver and the experiments were repeated in the postpartum state.

For Study II:

On the morning of the second post-operative day, just before the contents of the Alzet®

pump have begun infusing into the brain (labeled as Day 0), baseline plasma samples were taken

via one of the arterial catheters for all groups. Additionally, separate 1.5 ml whole blood

samples were collected into heparinized syringes and used for determination of sodium and

potassium concentrations using ion specific electrodes (AVL 9180 Electrolyte Analyzer,

AVL/Roche Diagnostics, Roswell, Georgia), as well as for determination of hematocrit and total

protein. Basal mean arterial blood pressure (MAP) was recorded continuously for 40-60 minutes

after the hour long acclimation period via pressure transducers connected to an analog-to-digital

conversion board (LabView, National Instruments, Austin, Texas). Arterial pressure values were

collected at 30 Hz and mean arterial pressure values were analyzed from time periods with

minimal interference from the investigators or from changes in body position relative to the fixed

transducer height. Plasma samples were collected from the arterial catheter at the end of the

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recording period for replication in the hormone assays. This protocol was repeated every other

day through the 8th post-operative day (or 6 continuous days of drug infusion, labeled as Day 6).

Daily Food Intake

For Study II only, daily food intake (g) was also measured by subtracting the weight of

remaining feed from the amount provided on the previous day in order to assess any differential

effects of fluoxetine and/or pregnancy on feeding habits. In order to avoid any acute effects of

surgery on feeding, food intake was not measured until Day 0 of infusion (or 2 post-operative

days) and was not measured on Day 6 of the infusion due to the time of euthanasia.

Plasma Hormone Determination

All plasma samples were analyzed for ACTH and cortisol concentrations. ACTH levels

for both studies were determined using a radioimmunoassay (RIA) previously described using an

antibody to 1-39 ACTH (Bell et al. 1991). For Study I, cortisol levels were determined by RIA

using the 125I Coat-A-Count® cortisol kit (Siemens Healthcare Diagnostics Inc., Deerfield,

Illinois). For Study II, cortisol was determined using an RIA method previously used in this

laboratory (Wood et al. 1993). Plasma progesterone was determined for both studies by RIA

using the 125I Coat-A-Count® progesterone kit (Siemens Healthcare Diagnostics Inc., Deerfield,

Illinois).

Euthanasia and Tissue Recovery

For Study II, all animals were euthanized with an intravenous injection of a pentobarbital/

phenytoin solution (15-20 ml; Euthasol®, Virbac AH, Fort Worth, Texas) using an indwelling

venous catheter. Ewes were sacrificed in their home pens to minimize stress during this

procedure. Immediately after the ewes were moved to the necropsy room, the carotid arteries

were catheterized for perfusion with ice-cold 10% DMSO/0.9% saline solution in order to

rapidly cool the forebrain and pituitary and slow the enzymatic degradation of mRNA and

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protein that might occur during removal and dissection of the brain. Using RNase-free

instruments and collection vials, relevant brain structures (hippocampus and hypothalamus) and

other peripheral tissues including pituitary were quickly removed, immediately snap-frozen in

liquid nitrogen and stored at −80oC for future protein and mRNA quantification.

Data Analysis

For Study I, the effects of pregnancy and acute icv fluoxetine administration on plasma

levels of ACTH and cortisol were determined using three-way repeated measures analysis of

variance (RM ANOVA) with repeated measures over drug and time. For Study II, the effects of

pregnancy and subchronic, icv fluoxetine administration on blood pressure and plasma levels of

cortisol, ACTH, sodium, potassium, hematocrit, and total protein over time were analyzed using

a three-way RM ANOVA of the between-subjects effect of group and treatment and the within-

subjects effects of time. Pairwise multiple comparisons were performed using Duncan’s

Multiple Range test to determine the source(s) of significance for each RM ANOVA. Data are

expressed as mean ± standard error of the mean (SEM). A value of P < 0.05 was considered

significant.

Results

Study I: HPA Axis Responses to Acute, Icv Fluoxetine

Plasma ACTH

For both pregnant and postpartum ewes, there were significant increases in plasma ACTH

in response to the icv fluoxetine injection as well as a significant effect of time (main effects of

time and treatment; interaction of treatment and time; Figure 4-1, p<0.05). Three-way RM

ANOVA was unable to detect a significant difference between responses of the ewes during

pregnancy and during the postpartum period. Based on an apparent trend for postpartum

responses to be greater than during pregnancy, two-way RM ANOVA was performed followed

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by Duncan’s multiple range test on either each group separately or within fluoxetine treated

ewes. These tests suggested differences in the time course of the ACTH response between the

pregnant and postpartum state. Within the pregnant ewes, the plasma ACTH concentration was

significantly elevated from 10-40 minutes after injection compared to saline infusion at the

corresponding time point and the peak ACTH response to icv fluoxetine occurred 20 min post-

injection. On the other hand, in the same ewes studied during the postpartum period, plasma

ACTH was increased at all post-injection time points and the peak plasma ACTH response was

reached 30 min post-injection. Additionally, after two-way RM ANOVA within fluoxetine

treated ewes, the Duncan’s test revealed that within both 30 min and 40 min post-injection, the

postpartum plasma ACTH responses were significantly greater than those produced during

pregnancy. Two-way RM ANOVA within vehicle-treated ewes only showed that there was a

tendency for ACTH concentrations to be greater during pregnancy (p=0.097), although this was

not significant.

Plasma cortisol

Plasma cortisol was statistically greater for these ewes during pregnancy (main effect by

three way RM ANOVA; p<0.05). For both pregnant and postpartum ewes, there was a

significant increase in plasma cortisol in response to the icv fluoxetine injection (main effects of

treatment and time; interaction of treatment and time; Figure 4-2, p<0.05) and cortisol

concentrations at or near maximum adrenal secretion for this species were achieved. There were

no significant differences in the cortisol responses to acute icv fluoxetine between ewes during

pregnancy or during the postpartum period. Based on ACTH concentrations, plasma cortisol

levels were not quantified in samples from 40 and 50 minutes post-injection as the cortisol

response was expected to remain at maximal levels across these time points.

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Study II: HPA Axis Responses to Subchronic, Icv Fluoxetine

Plasma ACTH

Plasma ACTH was not different between pregnant and nonpregnant ewes, although the

mean plasma ACTH during vehicle infusion was 111 ± 22 pg/ml in the pregnant ewes and 88 ±

10 pg/ml in the nonpregnant ewes. Three-way RM ANOVA revealed an effect of fluoxetine

treatment over time (interaction of treatment and time, p<0.05), and the time course of the

plasma ACTH response to icv infusion of fluoxetine tended to differ between nonpregnant and

pregnant ewes (interaction of time, group, and treatment; Figure 4-3, p= 0.057). Two-way RM

ANOVA followed by Duncan’s Multiple Range tests performed separately on each group

suggested plasma ACTH on Day 2 of the fluoxetine infusion was transiently increased in the

pregnant ewes compared to vehicle-infused pregnant ewes on the same day; whereas,

nonpregnant ewes remained unchanged until Day 6 compared to earlier time points (Days 0, 2,

and 4) for the same ewes or compared to vehicle-treated ewes on the Day 6.

Plasma cortisol

Figure 4-4 illustrates the differential time course of the plasma cortisol response to icv

infusion of fluoxetine between nonpregnant and pregnant ewes (three-way RM ANOVA:

interaction of time, group, and treatment, p<0.05). Subsequent two-way RM ANOVA followed

by Duncan’s Multiple Range test within fluoxetine-treated ewes, revealed that plasma cortisol

concentrations in the pregnant ewes were significantly increased on Day 2 compared to Day 6 of

infusion (p<0.05), whereas plasma cortisol remain unchanged until Day 6 of fluoxetine infusion

in nonpregnant ewes. These results reflect the pattern observed for ACTH release. Plasma

cortisol was not different between pregnant and nonpregnant ewes, although the mean plasma

cortisol levels for vehicle-infused pregnant and nonpregnant ewes were 3.3 ± 0.5 ng/ml and 2.4 ±

0.8 ng/ml, respectively.

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Mean arterial pressure, hematocrit (%) and plasma solute (total protein, potassium, and sodium) concentrations

Three-way RM ANOVA revealed no significant effects of fluoxetine infusion on mean

arterial pressure (Figure 4-5). There was no main effect of group on mean arterial pressure,

contrary to findings reported in Chapter 3 and throughout the literature. Figure 4-6 illustrates the

hematocrit, plasma protein and electrolyte (sodium and potassium) responses to subchronic icv

infusion of fluoxetine or vehicle. Three-way RM ANOVA revealed that hematocrit (panel A)

was not altered by treatment with fluoxetine, but that in nonpregnant ewes, hematocrit decreased

over time in both vehicle- and fluoxetine-treated ewes (main effect of time, interaction of time

and group, p<0.05). Plasma protein (panel B) concentrations were significantly altered by

fluoxetine over time and were lower in pregnant ewes (main effects of treatment and group,

interaction of time and treatment). There were no significant effects on plasma sodium

concentrations (panel C). Plasma potassium (panel D) concentrations were significantly altered

by fluoxetine, but this effect varied between groups over time (main effect of treatment,

interaction of time, treatment, and group, p<0.05), with a trend for decreasing plasma K+ over

time in the nonpregnant ewes and increasing plasma K+ in the pregnant ewes.

Daily food intake

Figure 4-7 illustrates daily food consumption from the first day of the experiment until one

day prior to euthanasia. Three-way RM ANOVA was unable to detect significant effects of

fluoxetine treatment in either group. Post-hoc Duncan’s Multiple Range test after two-way RM

ANOVA revealed significant increases in food intake in both groups of vehicle-treated ewes on

Days 3 and 5 as compared to Day 0 (two-way RM ANOVA followed by Duncan’s Multiple

Range test performed on vehicle-treated ewes; p<0.05). Additionally, similar analyses within in

each group suggested that in the fluoxetine-treated nonpregnant ewes, food intake was lower on

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Day 2 and Day 4 relative to Day 0 of the infusion (p<0.05), whereas there were no significant

differences in food intake in the pregnant ewes during fluoxetine infusion.

Discussion

Study I: HPA Axis Responses to Acute, Icv Fluoxetine

The ACTH responses to infusion of acute selective serotonin reuptake inhibitor during

pregnancy and postpartum were not significantly different by three-way RM ANOVA. These

findings disprove our hypothesis that pregnant ewes would have greater ACTH responses to

treatment with a selective serotonin reuptake inhibitor. These results suggest that not only is

there is not an increase in serotonergic responsivity driving the elevation in basal ACTH levels

during pregnancy, but rather our results suggest that serotonergic responsivity of the HPA axis

during pregnancy may in fact be depressed, based on follow-up analysis using two-way RM

ANOVA on fluoxetine responses only.

One possible mechanism for this apparent difference might be an effect of chronically

elevated cortisol on the serotonergic system. It has been reported that manipulation of

corticosteroid levels via adrenalectomy or exogenous administration regulates the abundance of

post-synaptic 5-HT1A mRNA and protein in the hippocampus (Chalmers et al. 1993, Chalmers et

al. 1994, Kuroda et al. 1994, Briones-Aranda et al. 2008). The administration of corticosterone

in rats reverses the increases in 5-HT1A binding that occur following adrenalectomy (Mendelson

& McEwen 1992). Recently, Lee and colleagues (2009) demonstrated 5-HT2A receptors within

the PVN may be desensitized by chronic corticosterone administration. Together these results

suggest a possible mechanism for the attenuated response to acute icv fluoxetine that may occur

during pregnancy compared to the postpartum period in the ewe.

The fact that three-way RM ANOVA was unable to detect an overall difference in the

ACTH response to acute icv fluoxetine administration during pregnancy and postpartum, was

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surprising given the obvious trend shown in Figure 4-1. However, individual assessment of

ACTH responses in each subject revealed that 2 out of the 6 ewes’ responses during pregnancy

more closely resembled their respective postpartum responses to fluoxetine. As discussed in

Chapter 2, it is possible that progesterone and estrogen which become elevated during pregnancy

are impacting serotonergic responsivity of the HPA axis through their effects on pre- and post-

synaptic receptors, reuptake transporters, and intracellular coupling with other proteins which

mediate serotonin’s downstream effects (Bethea et al. 2002). We measured plasma progesterone

concentrations in these ewes and found that these 2 ewes had plasma progesterone

concentrations of 4.4 and 5.8 pg/ml (in the range of concentrations found in the nonpregnant

ewes in Chapter 3 and in Study II of this chapter), while the average plasma progesterone in the

remaining 4 ewes during pregnancy was 16.3 ± 2.3 pg/ml. Interestingly, linear regression

analysis revealed a significant inverse relationship between plasma progesterone and peak

ACTH responses to icv fluoxetine (r=0.687, p<0.05). This would suggest that serotonin-

stimulated HPA axis activation is blunted in the presence of progesterone levels typically

measured during pregnancy. Additionally, it is known that estrogen, which was not measured in

these ewes but was undoubtedly elevated in pregnancy, has been shown to attenuate 5-HT1A-

stimulated increases in ACTH and corticosterone, and this effect appears to be related to

estrogen’s ability to reduce levels of G-proteins that are known to mediate the actions of this

receptor (Raap et al. 2000).

On the other hand, the fact that there were no differences in plasma cortisol responses to

acute icv fluoxetine injection between these reproductive states was not surprising. Maximal

adrenal secretion of corticosteroid occurs in most species in response to plasma ACTH

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concentrations well below the values achieved in this study in response to fluoxetine injection

(Keller-Wood et al. 1983).

Study II: HPA Axis Responses to Subchronic, Icv Fluoxetine

Because our previous study produced near maximal secretion of ACTH and likely

maximal cortisol secretion in response to fluoxetine, we investigated central treatment with a

more chronic, but submaximal dose of fluoxetine. In this study, we also found that, contrary to

our hypothesis, there was not a sustained increase in serotonergic responsivity during pregnancy

as exploited by subchronic administration of a lower dose of fluoxetine. Although the time

course for the ACTH response to subchronic fluoxetine infusion had a tendency to differ

between the reproductive groups, more animals are needed to confirm this. However, there was

a significant difference in the time course of the plasma cortisol response to fluoxetine infusion

between the two groups, suggesting that the trends observed in the plasma ACTH responses are

not simply due to random sampling variability. The continuous presence of serotonin within the

synaptic cleft appears to affect the release of ACTH and cortisol in a different manner between

pregnant and nonpregnant ewes. Specifically, in nonpregnant ewes, subchronic, icv fluoxetine

was not effective at stimulating cortisol until the 6th day of continuous infusion, while in

pregnant ewes, stimulation occurred transiently at Day 2. I propose that pregnant ewes might

have more 5-HT2A receptors on parvocellular neurons in the PVN at the start of the study than

the nonpregnant ewes likely due to estrogen (Sumner & Fink 1995, Sumner & Fink 1997,

Osterlund & Hurd 1998), allowing for stimulation of the HPA axis on Day 2 in response to

elevations in the synaptic cleft by fluoxetine. This hypothesis was later refuted, at least at the

gene level, as discussed in Chapter 5 of this dissertation. Prolonged serotonin in the synaptic

cleft may then downregulate these receptors thereby reducing the effect of fluoxetine for the

remainder of the infusion. Meanwhile, the delayed effect observed in nonpregnant ewes, maybe

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a result of downregulation of the 5-HT1A autoreceptors, although in humans, this is not thought

to occur before 2 weeks of chronic SSRI treatment. Overall, the subchronic, icv fluoxetine

infusion had milder effects on plasma ACTH and cortisol than the acute fluoxetine injection

given in Study I, and therefore this dose of fluoxetine may have been too low to produce

measureable differences between pregnant and nonpregnant ewes.

Unexpectedly, neither plasma cortisol nor ACTH was significantly greater in the pregnant

ewes in this study. I theorize this may be due to the relatively high basal levels on Day 0 in the

vehicle-treated nonpregnant ewes. Plasma cortisol in the vehicle-infused nonpregnant ewes was

higher on Day 0 than all other days, based on two-way RM ANOVA followed by Duncan’s

Multiple Range test within vehicle-treated ewes (p<0.05). Although not significant, there is an

apparent trend for ACTH to exhibit the same pattern in these ewes. Meanwhile, this pattern does

not occur in the vehicle-treated pregnant ewes. This data supports unpublished observations in

our lab that the excitable nature of this species is somewhat subdued during pregnancy in

response to human interaction and experimental procedures compared to nonpregnant ewes.

At the same time, due to unexpected fluctuations in peripheral hemodynamic endpoints in

this study, the HPA axis responses to either vehicle or fluoxetine infusion should be interpreted

with caution. Additionally, in some cases there were detectable differences in these variables

even on Day 0, when fluoxetine from the osmotic pump should not have yet begun to reach the

tip of the catheter. As expected hematocrit and plasma sodium concentrations were not altered

by treatment with fluoxetine. However, in nonpregnant ewes, hematocrit decreased over time in

either treatment group. Again, the elevated hematocrit levels at the beginning of the study are

likely the result of stress from surgery or human interaction. Like the cortisol and ACTH

concentrations, these levels appear to normalize over time. Based on three-way RM ANOVA,

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plasma protein and plasma potassium were both significantly affected by fluoxetine. However,

two-way RM ANOVA followed by Duncan’s Multiple Range test performed within each group

suggested that the plasma protein effect of fluoxetine is only significant in the pregnant ewes.

As for plasma potassium, a similar analysis was unable to detect significant effects in either

group when analyzed separately, and suggested that fluoxetine only tended to have an effect over

time in the nonpregnant ewes (p=0.059 for interaction of treatment and time). In any case, the

differential ACTH and cortisol responses to subchronic fluoxetine cannot be attributed to

secondary effects from changes in blood pressure, hematocrit, plasma protein, or electrolyte

concentrations.

Throughout the literature, reductions in meal size and eating rate are often observed

following peripheral or central injections of serotonergic agents and these changes are consistent

with alterations in the mechanisms of satiety (Blundell 1986). Interestingly, despite the lack of

significance by three-way RM ANOVA, the food intake data are suggestive of time effects and

differences in response to icv fluoxetine between nonpregnant and pregnant ewes. Based on the

literature and apparent trends of the food intake data in this study, separate two-way RM

ANOVA’s were performed. Post-hoc Duncan’s tests revealed significant increases in food

intake in both groups of vehicle-treated ewes on Days 3 and 5 as compared to Day 0 (two-way

RM ANOVA followed by Duncan’s Multiple Range test performed on vehicle-treated ewes;

p<0.05). This is consistent with unpublished observations in our lab that include a typical

pattern of reduced feeding during the first few post-operative days, with a gradual recovery over

time. Typically, we allow animals to recover from surgery for a period of 5 days which includes

daily intramuscular prophylactic antibiotic injections and rectal temperature assessment before

beginning any experimentation, and by this time, the feeding effects of surgery have diminished.

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In this study, however, the experimentation began on the second post-operative day, before full

recovery of feeding has occurred. We were therefore able to observe any effects that icv

infusion of fluoxetine might have on this feeding recovery period and whether or not these

effects differ between pregnant and nonpregnant ewes. Additionally, post-hoc Duncan’s

Multiple Range tests suggested that in the fluoxetine-treated nonpregnant ewes, food intake was

lower on Day 2 and Day 4 relative to Day 0 of the infusion (p<0.05), whereas there were no

significant differences in food intake in the pregnant ewes during fluoxetine infusion. The data

suggest that this food intake recovery period for the pregnant ewes may not have been affected

by fluoxetine administration, while significant reductions in food intake in the nonpregnant ewes

are evident by 4 days of fluoxetine infusion. These results suggest an alteration in central

serotonin-mediated satiety pathways during pregnancy. As discussed in Chapter 2, serotonin is

thought to play a role in regulation of food intake mediated, at least in part, through its effects at

the POMC neurons of the arcuate nucleus of the hypothalamus. Future investigations to

characterize the subtle differential responses to fluoxetine between nonpregnant and pregnant

ewes observed in this study should therefore include this pathway.

Summary

The exact mechanism of serotonergic stimulation of the HPA axis is not entirely

understood. These studies may also demonstrate variability in responses as they relate to

duration and dose of fluoxetine administration. The differential responses between nonpregnant

and pregnant ewes may be explained by differences at the gene level, the protein level, or post-

translational differences between pregnant and nonpregnant ewes in terms of serotonin-mediated

HPA axis activation. Additionally, these differences could be occurring at the hypothalamus,

hippocampus or the brain stem, although our lateral ventricular infusion of fluoxetine would be

expected to primarily target forebrain, hypothalamic or hippocampal, sites of serotonin action.

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Ovarian hormones might be influencing the responses to fluoxetine in these ewes. Our data

suggest that progesterone may play a role in attenuating the CRH or ACTH response to serotonin

stimulation as it was shown to be inversely and significantly related to ACTH responses to

fluoxetine in Study I. Additionally, the presence of serotonergic receptors and reuptake

transporters in the hippocampus as well as its innervation by serotonergic neurons would suggest

that it is a likely site for future investigations to explain potential differences among the

reproductive states. Meanwhile, serotonergic neurons themselves are known to express

corticosteroid receptors and therefore posses the potential to be regulated by circulating

glucocorticoid, directly (Harfstrand et al. 1986, Fuxe et al. 1987, Morimoto et al. 1996).

Despite the fact that the acute findings disprove our hypothesis that pregnancy is a state of

elevated serotonergic tone which in turn would play a role in elevating basal HPA axis activity

during pregnancy, these studies have, however, uncovered potential differences in responsivity to

serotonergic agents between the pregnant and postpartum states in terms of HPA axis activation.

The findings of the subchronic study, on the other hand, were suggestive of increased

serotonergic responsivity in pregnant ewes as an increase in plasma cortisol concentration was

observed sooner in these animals compared to the nonpregnant ewes in response to icv fluoxetine

infusion. Aside from the vast difference in dose and duration of administration, the seemingly

contradictory findings of Study I and Study II might also be related to differences between

postpartum and nonpregnant ewes in terms of either basal HPA axis regulation or serotonin

system activity. However, previously, our lab has reported comparable findings in studies on

pregnant compared to nonpregnant and pregnant compared to postpartum ewes in terms of

alterations in HPA axis feedback. Additionally, the observation that subchronic fluoxetine

infusion had less of a hindering effect on post-operative feeding recovery in the pregnant ewes,

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may provide evidence that central serotonin-mediated feeding regulation may be altered in

pregnancy. The major implications of these studies are that if differences similar to those

measured in this study are also evident in humans, these findings could suggest a need for

adjustment of treatment regimens for depression, anxiety, and/or HPA axis dysregulation as a

woman transitions between these reproductive states.

This research was supported by an R01 grant from the NIH (DK38114) to Maureen Keller-

Wood.

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Figure 4-1. Study I: Plasma ACTH following acute icv fluoxetine (FLX) or vehicle (VEH).

Grey boxes indicate time points in which the increase in plasma ACTH following acute icv fluoxetine (3.3 mg) was significantly greater for the postpartum (PP) ewes than it was for the same ewes during pregnancy (P) based on two-way RM ANOVA when given fluoxetine, followed by Duncan’s Multiple Range test. ** Indicates significantly different from ACTH concentration during saline infusion at the corresponding time point for both pregnant and postpartum states. * Indicates significantly different from ACTH concentration during saline infusion at the corresponding time point for ewes in postpartum state only. Values are means ± SEM and considered significant at p<0.05.

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Figure 4-2. Study I: Plasma cortisol following acute icv fluoxetine (FLX) or vehicle (VEH).

Plasma cortisol was significantly higher during pregnancy; and there was a significant increase in plasma cortisol following acute icv fluoxetine (3.3 mg) injection, both during pregnancy (P) and in the postpartum (PP) period (main effects of group, treatment, and time as well as a time and treatment interaction by three-way RM ANOVA, p<0.05). ** Indicates significant difference from vehicle-infusion for both groups at the corresponding time point for the corresponding group.

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Figure 4-3. Study II: Plasma ACTH response to subchronic icv infusion of fluoxetine (FLX) or

vehicle (VEH). Three-way RM ANOVA indicated a significant interaction of treatment and time (p<0.05), and the time course of the plasma ACTH response to icv infusion of fluoxetine tended to vary between nonpregnant (NP) and pregnant (P) ewes (interaction of time, group, and treatment, p= 0.057). Two-way RM ANOVA followed by Duncan’s Multiple Range test revealed increased plasma ACTH on Day 2 of the infusion in fluoxetine-treated ewes compared to those given vehicle, whereas nonpregnant ewes remained unchanged until Day 6 of fluoxetine infusion compared to both vehicle-infused ewes and to all previous days of vehicle infusion. * Indicates significantly greater than vehicle-infused ewes of the same group on the corresponding day of infusion.

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Figure 4-4. Study II: Plasma cortisol response to subchronic icv infusion of fluoxetine (FLX) or

vehicle (VEH). The time course of the plasma cortisol response to subchronic icv fluoxetine varies between nonpregnant (NP) and pregnant (P) ewes (three-way RM ANOVA: interaction of time, group, and treatment, p<0.05). In response to fluoxetine infusion, significant elevation of cortisol is observed on Day 6 for the nonpregnant ewes. * Indicates significantly greater than levels observed in vehicle-treated ewes at the same time point (two-way RM AVOVA followed by Duncan’s Multiple Range test within nonpregnant ewes, interaction of treatment and time, p<0.05).

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Figure 4-5. Study II: Mean arterial pressure (MAP) during subchronic icv infusion of fluoxetine

(FLX) or vehicle (VEH) in (A) nonpregnant and (B) pregnant ewes. Mean arterial pressure did not significantly change in response to fluoxetine infusion, based on three-way RM ANOVA. Data are expressed as group means ± SEM and considered significant at p<0.05.

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Figure 4-6. Study II: Hematocrit and plasma solute concentrations during subchronic icv

fluoxetine (FLX) or vehicle (VEH) infusion. Hematocrit (A) decreased over time in nonpregnant ewes and was not altered by treatment with fluoxetine in either group (main effect of time, interaction of time and group, p<0.05). Plasma protein (B) concentrations were significantly altered by fluoxetine over time and were lower in pregnant ewes (main effects of treatment and group, interaction of time and treatment). Plasma sodium (C) concentration was not different between groups or in response to fluoxetine infusion. Plasma potassium (D) concentrations were significantly altered by fluoxetine over time, but this effect varied between groups (main effect of treatment, interaction of time, treatment, and group). Values are group means ± SEM and considered significant at p<0.05 when analyzed by three-way RM ANOVA.

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Figure 4-7. Study II: Daily food intake during subchronic icv fluoxetine or vehicle infusion.

There was a significant increase in food intake in both groups of vehicle-treated ewes on Days 3 and 5 as compared to Day 0 (* indicates significantly different from same group at Day 0 at p<0.05, based on two-way RM ANOVA followed by Duncan’s Multiple Range test within vehicle-treated ewes). Overall, there was no main effect of time on daily food intake in the fluoxetine treated ewes. However in the fluoxetine-treated nonpregnant ewes there was a significant decrease in food intake at Day 2 and Day 4 relative to that on Day 0, whereas there were no significant differences in food intake in the pregnant ewes during fluoxetine infusion (** indicates significantly different from Day 0 in same group at p<0.05, based on two-way RM ANOVA followed by Duncan’s Multiple Range test within nonpregnant ewes).

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CHAPTER 5 HYPOTHALAMIC EXPRESSION OF GENES RELATED TO HPA AXIS REGULATION

AND THE SEROTONERGIC SYSTEM IN EWES

Introduction

In our lab, we use pregnant, nonpregnant, and postpartum ewes as our experimental model;

and one of the benefits of an in vivo model is the ability to assess relative gene expression in

intact physiological systems. Because the hypothalamus is the point of integration for all

upstream inputs driving or inhibiting hypothalamic-pituitary-adrenal (HPA) axis activity,

investigating relevant gene expression patterns in this region is a logical starting point.

Therefore, the objective of this study was to characterize relative mRNA expression levels of

HPA axis- and serotonergic system-relevant genes in the hypothalamus between nonpregnant,

pregnant, and post-partum ewes. Any differences found here may at least partially explain

differences in basal HPA axis activity during pregnancy. Additionally, we hope to at shed some

light on the possible mechanisms for the differential HPA axis responses activation between

pregnant and nonpregnant or postpartum ewes in response to MR antagonism or selective

serotonin reuptake transporter inhibition discussed in the preceding chapters of this dissertation.

It is known that HPA axis negative feedback is mediated by corticosteroid binding to the

low affinity, glucocorticoid receptor (GR) and the higher affinity, mineralocorticoid receptor

(MR) (Keller-Wood & Dallman 1984, Reul & de Kloet 1985, Reul et al. 1987, Bradbury et al.

1991, de Kloet et al. 1993). Due in part to the affinity profiles, MR are thought to be the major

steroid receptor involved in regulation of basal HPA axis activity. At the same time, axons of

the serotonergic system have been shown to project to several components of the HPA axis,

while several animal studies and human trials have demonstrated increases in plasma ACTH and

cortisol concentrations in response to administration of serotonin, its precursors, serotonin

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receptor agonists, and selective serotonin reuptake inhibitors (for review, see: Carrasco & Van de

Kar 2003).

Our lab is interested in understanding the mechanisms for elevated basal plasma ACTH

and cortisol concentrations during pregnancy in the human. The overall hypothesis of the lab is

that ovarian steroid hormones which are elevated in pregnancy, such as progesterone and

estrogen, may play a role in modifying regulatory mechanisms of HPA axis activity. Classically,

steroid hormones such as cortisol, progesterone and estrogen are known to modulate gene

expression as their respective receptors are members of the nuclear receptor family.

We hypothesize that relative mRNA expression might be increased for either or both CRH

and AVP thereby driving basal HPA axis activity at a higher level. On the other hand, a

reduction in negative feedback by basal levels of cortisol in pregnancy might be occurring in a

system with reduced MR expression. With less MR being transcribed, ultimately fewer

receptors might be present which could mean reduced HPA axis inhibition by basal levels of

cortisol as demonstrated previously in our lab (Keller-Wood 1998). The results in Chapter 3

suggest a differential early response to 4 hours of intravenous MR antagonism by canrenoate

between pregnant and nonpregnant ewes, in which the early (<2 hours) response of the pregnant

ewes is blunted. This would also suggest that MR expression might be reduced during

pregnancy. Changes in GR expression patterns are not likely as pregnant ewes have normal

inhibition of ACTH in response to high levels of cortisol (Keller-Wood 1998).

Serotonergic responsivity was originally hypothesized to be elevated during pregnancy as a

possible mechanism for increased basal HPA axis activity; and we expected elevations in ovarian

hormones might decrease hypothalamic 5-HT1A receptor mRNA and increase 5-HT2A. This

hypothesis is based on studies in rats which describe such changes in response to ovarian steroids

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in other brain regions (Sumner & Fink 1995, Sumner & Fink 1997, Osterlund & Hurd 1998).

Several groups have also suggest that glucocorticoids might tonically inhibit the expression of 5-

HT1A (Mendelson & McEwen 1992, Chalmers et al. 1993, Chalmers et al. 1994, Briones-Aranda

et al. 2008). On the other hand, the results of the acute fluoxetine study (Study I) discussed

earlier suggest that serotonergic responsivity is in fact depressed compared to the postpartum

state. We would therefore expect hypothalamic 5-HT1A might be elevated and/or 5-HT2A

expression levels might be reduced in pregnancy relative to the postpartum state. Based on

results of the subchronic study (Study II), the pregnant ewes might have increased 5-HT2A

relative to nonpregnant ewes. A reduction in SERT expression relative to postpartum ewes

might also explain the reduced response to the selective serotonin reuptake inhibitor in the

pregnant ewes in the acute study. The differential food intake effects of subchronic fluoxetine

administration between pregnant and nonpregnant ewes suggest that hypothalamic POMC

expression might be lower during pregnancy allowing for reduced inhibition by serotonergic

agents.

Materials and Methods

Euthanasia and Tissue Recovery

All animals used in these studies were euthanized with an intravenous injection of a

pentobarbital/ phenytoin solution (15-20 ml; Euthasol®, Virbac AH, Fort Worth, Texas) using an

indwelling venous catheter. Ewes were sacrificed in their home pens to minimize stress during

this procedure. Immediately after the ewes were moved to the necropsy room, the carotid

arteries were catheterized for perfusion with ice-cold 10% DMSO/0.9% saline solution. This

procedure cools the forebrain and pituitary and slows the enzymatic degradation of mRNA and

protein that might have otherwise occurred during removal and dissection of the brain. Relevant

brain structures (hippocampus and hypothalamus) and other tissues including pituitary were

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removed quickly using baked, RNase-free instruments, placed into RNase-free containers,

immediately snap-frozen in liquid nitrogen and stored at −80oC for future protein and mRNA

quantification.

At the time of tissue collection, the hypothalamus was divided at the midline into right and

left halves and each piece was stored separately in the −80oC freezer. The relevant nuclei to HPA

axis activation (PVN) are present in each half as they are bilateral and found adjacent to the third

ventricle, which forms part of midline division of the hypothalamus. The following data

describe mRNA expression levels from one half of the hypothalamus, while the other half has

been saved for extraction and quantification of proteins.

RNA Extraction and Quantification

RNA was extracted from the hypothalami of nonpregnant, pregnant (between 130 and 146

days of gestation, term is approximately 147 days), and postpartum (4 ± 1 days postpartum)

ewes. Tissues were first homogenized in Trizol® (GIBCO/BRL, Grand Island, New York)

according to the manufacturer’s directions. Genomic DNA was removed using RNeasy Plus

Mini Kits (Qiagen Inc., Valencia, California). The absorbance of a diluted aliquot of each

sample was then measured at 260 and 280 nm for determination of RNA concentration and

purity using a spectrophotometer.

Reverse Transcription and Real-Time Quantitative PCR

Reverse transcription in a thermocycler for 10 minutes at 25 °C followed by 120 minutes at

37 °C was performed using a high capacity cDNA archive kit (Applied Biosystems; Foster City,

California), followed by storage at −20oC. Quantitative polymerase chain reaction (qPCR) was

then performed and analyzed using the Taqman® Universal PCR Master Mix and the ABI Prism

7000 Sequence Detection System according to the manufacturers’ instructions (Applied

Biosystems, Foster City, California). Each PCR reaction contains a total volume of 25 ul which

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includes 2.5 μl of cDNA product from the reverse transcription step as the template and 12.5 ul

Taqman® Master Mix. Reactions were performed under the following conditions: initial

incubation at 95°C for 10 min, followed by 40 cycles of 95°C denaturation for 15 sec and 60°C

annealing for 1 min. The relative mRNA expression levels in the hypothalamus of the following

genes were investigated in this study: mineralocorticoid receptor (MR), glucocorticoid receptor

(GR), corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), serotonin receptor

1A (5-HT1A), serotonin receptor 2A (5-HT2A), serotonin reuptake transporter (SERT), and

proopiomelanocortin (POMC). The template amount for each gene was 20 ng of cDNA, except

for MR which utilized 100 ng of cDNA in each reaction. The probe and primer sequences along

with their concentrations are listed in Table 5-1. All probe/primer sequences and concentrations

have been previously optimized and validated for use in quantitative PCR via prior experiments

in this lab. Each sample was analyzed in triplicate and all samples for each gene were analyzed

on the same 96-well plate. β-actin was also run for each sample as the housekeeping gene.

Data Analysis

One-way analysis of variance (ANOVA) was performed using the relative cycle threshold

(ΔCt) method (Livak & Schmittgen 2001). Briefly, the threshold concentration (Ct) was

determined from a log–linear plot of the PCR product signal versus the cycle number. Then ΔCt

was calculated as the mean Ct for a set of triplicates in a given sample for the housekeeping gene

(β-actin) minus the mean Ct for a set of triplicates in the same animal for the gene of interest.

One-way ANOVA was then performed using these values with p<0.05 as the criterion for

significance. Post-hoc analysis was performed using Duncan’s multiple range test. On data sets

that were not normally distributed and/or had unequal variance, Kruskal-Wallis one-way

ANOVA on ranks was performed. Data in figures are expressed as group means of fold change

relative to expression in the nonpregnant group (NP) ± standard error of the mean. Relative gene

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expression or fold change for each gene of interest was calculated as 2(− ΔΔCt); where ΔΔCt was

calculated as ΔCt for a given animal minus the mean ΔCt for the nonpregnant group.

For most of the genes including β-actin, there were 7 nonpregnant, 10 pregnant, and 12

post-partum ewes included in the analysis. For 5-HT2A, an initial run using fewer animals (NP,

n=4; P, n=10; PP, n=10) indicated that differences among these groups were unlikely and was

therefore not run on the additional five samples. For both POMC and AVP, two of the

nonpregnant ewes demonstrated late amplification relative to the rest of this group (shifts of 4

and 7 cycles to the right for POMC and approximately 9 cycles for AVP) and were therefore left

out of the analysis. We hypothesize that due to the location of the nuclei expressing POMC and

AVP, that these areas may have been inadvertently excluded during dissection.

Results

Glucocorticoid Receptor (GR)

There were no significant differences in ΔCt between nonpregnant, pregnant, and

postpartum ewes for expression levels of glucocorticoid receptor mRNA (p=0.825). The average

ΔCts for each group were 11.6 ± 0.20, 11.50 ± 0.28, and 11.39 ± 0.11, respectively. Relative

expression compared to nonpregnant ewes is depicted in Figure 5-1.

Mineralocorticoid Receptor (MR)

There were no significant differences in ΔCt between nonpregnant, pregnant, and

postpartum ewes for expression levels of mineralocorticoid receptor mRNA (p=0.722). The

average ΔCts for each group were 5.29 ± 0.35, 5.77 ± 0.13, and 5.67 ± 0.12, respectively.

Relative expression compared to nonpregnant ewes is depicted in Figure 5-2.

Corticotropin-Releasing Hormone (CRH)

There were no significant differences in ΔCt between nonpregnant, pregnant, and

postpartum ewes for expression levels of corticotropin-releasing hormone mRNA (p=0.782).

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The average ΔCts for each group were 10.73 ± 0.54, 10.34 ± 0.47, and 10.38 ± 0.22,

respectively. Relative expression compared to nonpregnant ewes is depicted in Figure 5-3.

Arginine Vasopressin (AVP)

There were no significant differences in ΔCt between nonpregnant, pregnant, and

postpartum ewes for expression levels of arginine vasopressin mRNA (p=0.209). The average

ΔCts for each group were -1.18 ± 0.53, -0.36 ± 0.34, and 0.27 ± 0.54, respectively. Relative

expression compared to nonpregnant ewes is depicted in Figure 5-4.

5-HT1A Receptor

There were no significant differences in ΔCt between nonpregnant, pregnant, and

postpartum ewes for expression levels of 5-HT1A receptor mRNA (p=0.449). The average ΔCts

for each group were 7.41 ± 0.18, 7.70 ± 0.16, and 7.76 ± 0.20, respectively. Relative expression

compared to nonpregnant ewes is depicted in Figure 5-5.

5-HT2A Receptor

There were no significant differences in ΔCt between nonpregnant, pregnant, and

postpartum ewes for expression levels of 5-HT2A receptor mRNA (p=0.471). The average ΔCts

for each group were 9.06 ± 0.30, 8.61 ± 0.20, and 8.77 ± 0.18, respectively. Relative expression

compared to nonpregnant ewes is depicted in Figure 5-6.

Serotonin Reuptake Transporter (SERT)

There were no significant differences in ΔCt between nonpregnant, pregnant, and

postpartum ewes for expression levels of serotonin reuptake transporter mRNA (p=0.832). The

average ΔCts for each group were 11.83 ± 0.30, 11.74 ± 0.16, and 11.71 ± 0.09, respectively.

Relative expression compared to nonpregnant ewes is depicted in Figure 5-7.

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Proopiomelanocortin (POMC)

Expression of POMC mRNA was significantly lower in pregnant and postpartum ewes

compared to nonpregnant ewes as indicated by comparison of ΔCt across groups using one-way

ANOVA followed by Duncan’s Multiple Range test (p=0.036). The average ΔCts were 8.01 ±

0.41 for nonpregnant, 9.84 ± 0.32 for pregnant, and 9.77 ± 0.47 for postpartum ewes. Relative

expression (fold change) compared to nonpregnant ewes is depicted in Figure 5-8 (0.3 ± 0.1 for

pregnant and 0.5 ± 0.1 for postpartum ewes).

Discussion

Because the studies discussed previously in this dissertation have suggested differences in

HPA axis activation between these reproductive states, relative gene expression at the level of

the hypothalamus was investigated in order to elucidate a mechanism for these potential

differences. The specific objective of this study was to characterize any differences in relative

mRNA expression levels of HPA axis- and serotonergic system-relevant genes in the

hypothalamus of nonpregnant, pregnant, and post-partum ewes. We hypothesized that changes

at the transcriptional level could at least partially explain regulatory differences in basal HPA

axis activity between the pregnant and nonpregnant states.

More specifically, one possible mechanism for the increased basal ACTH and cortisol of

pregnancy and the blunted early HPA axis response to intravenous MR blockade in pregnant

ewes would be a decrease in hypothalamic MR expression. Contrary to our hypothesis,

however, there were no differences in nonpregnant, pregnant, and postpartum expression of

hypothalamic MR expression. There were also no detectable differences in hypothalamic GR,

CRH, or AVP gene expression among the reproductive groups studied here. On the other hand

these data are consistent with previous findings in our lab that have also shown no differences in

hippocampal MR or GR between pregnant and nonpregnant ewes (unpublished data by Yi Hua),

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nor were there differences between adrenal intact and adrenalectomized ewes (Richards et al.

2003), indicating a lack of effect by ovarian or adrenal hormones on these genes in the

hippocampus. However, several studies in rats have suggested that hippocampal GR and MR are

negatively regulated by adrenal hormones (Tornello et al. 1982, Sapolsky et al. 1984, Sapolsky

& McEwen 1985, Herman et al. 1989, Reul et al. 1989, Kalman & Spencer 2002) and possibly

estrogen (Burgess & Handa 1993, Carey et al. 1995, Castren et al. 1995).

Extensive evidence indicates a stimulatory role for serotonin on HPA axis activity. We

originally hypothesized that increased serotonergic responsivity might at least in part explain the

increase in basal ACTH and cortisol associated with pregnancy. However, based on the reduced

responses to acute serotonin reuptake transporter blockade during pregnancy, we expected

reduced hypothalamic expression of 5-HT2A or SERT and/or increases in hypothalamic 5-HT1A

in pregnant ewes. Whereas, based on the influence of ovarian hormones on this system as

discussed in Chapter 2, we might have predicted increased expression of 5-HT2A and decreased

expression of 5-HT1A, but likely still a decrease in SERT expression. The prediction for 5-HT1A

expression would be similar if the elevated cortisol of pregnancy is considered; however, the

data is limited regarding glucocorticoid modulation of 5-HT2A or SERT expression. Our

hypotheses were disproved as none of the serotonergic genes investigated in this study were

significantly different between nonpregnant, pregnant or postpartum states.

As a result of the interesting effects of subchronic icv infusion of fluoxetine on food intake

discussed in Chapter 4, we investigated the possibility of differential hypothalamic expression of

proopiomelanocortin (POMC) between nonpregnant and pregnant ewes. The data suggested a

trend for fluoxetine to reduce feeding over time in the nonpregnant ewes, whereas there was no

effect of fluoxetine on daily food intake in the pregnant ewes. Serotonergic agents such as

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fluoxetine are known to be anorexogenic in both animal models and in humans; and serotonin is

thought to play a role in POMC-mediated satiety mechanisms discussed in Chapter 2.

Specifically, POMC neurons in the arcuate nucleus of the hypothalamus, which are known to be

responsive to peripheral energy balance signals such as ghrelin and leptin, are thought to be

stimulated by serotonergic agents (Heisler et al. 2002). Upon activation, these neurons release

melanocortins (cleavage products of the precursor POMC) which then act on melanocortin

receptors to ultimately inhibit feeding (Cone 2006). Therefore, the reduced expression of POMC

mRNA in pregnant compared to nonpregnant ewes, might suggest a mechanism for increased

feeding during pregnancy and might at least partially explain the lack of an anorexogenic effect

of fluoxetine in these ewes.

Although relative expression of these genes was not shown to be altered in the

hypothalamus across these reproductive states for the majority of the genes analyzed in this

study, alterations at the gene level may still be occurring in other brain regions that are known to

influence HPA axis activity. For the purposes of this study, the hypothalamus was chosen as a

starting point for investigating possible differential patterns of expression across the reproductive

states as it is the site of convergence for all upstream brain regions which are thought to regulate

cortisol. The hypothalamus receives signals from both inhibitory and stimulatory sources; and

the resultant cortisol release is from an integration of these inputs. It is also known that MR, GR,

serotonin receptors and serotonin reuptake transporters are expressed in many other brain regions

besides the hypothalamus. It is therefore entirely possible that expression patterns of the genes

of interest to these studies may be altered during pregnancy in other brain regions such as the

hippocampus or at the level of the brain stem which houses the serotonergic cell bodies. I

propose that there may be increased expression of the 5-HT1A autoreceptor at the level the brain

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stem as a possible mechanism for reduced serotonergic tone in the pregnant ewes which could

explain the differential response to acute icv fluoxetine.

Specifically, we know from previous reports that both MR and GR are expressed in the

hippocampus; a region thought to inhibit HPA axis activity and that this area also receives inputs

from serotonergic neurons. The 5-HT1A receptor, for example, is known to be one of the most

highly expressed serotonin receptors in the hippocampus (Hoyer et al. 1986, Pazos et al. 1987,

Chalmers & Watson 1991, Chalmers et al. 1993, Pucadyil et al. 2005). This region of the brain

is therefore a prime target for future gene expression studies.

Additionally, it is possible that changes in hypothalamic expression of these genes are

simply being diluted by not specifically isolating and analyzing the paraventricular nucleus from

these ewes. It is also entirely possible, however, that any changes in basal HPA axis regulation

involving these systems that may occur during pregnancy are taking place after gene

transcription. Such changes might include alterations in the regulation of translation or post-

translational differences. Studies have shown, for example that hormone-dependent

phosphorylation of steroid receptors imparts stability and therefore influences receptor levels

available for binding within the cytosol and subsequent transactivation (for review see, Weigel

1996). It is also important to note that the current study has, by no means, represented an

exhaustive investigation of all possible genes within the hypothalamus that are known be

involved in HPA axis regulation, nor can we disregard the possibility of yet undiscovered

systems that influence cortisol regulation that may be altered during pregnancy.

This research was supported by an R01 grant from the NIH (DK38114) to Maureen Keller-

Wood.

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Table 5-1. Sequences of probe and primer sets for all genes analyzed in ovine hypothalamus

along with the respective concentrations added to each reaction.

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Figure 5-1. Relative hypothalamic glucocorticoid receptor (GR) mRNA expression. No

significant differences between nonpregnant (NP), pregnant (P), and postpartum (PP) ewes. Data are expressed as fold change from nonpregnant ewes ± SEM.

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Figure 5-2. Relative hypothalamic mineralocorticoid receptor (MR) mRNA expression. No

significant differences between nonpregnant (NP), pregnant (P), and postpartum (PP) ewes. Data are expressed as fold change from nonpregnant ewes ± SEM.

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Figure 5-3. Relative hypothalamic corticotropin-releasing hormone (CRH) mRNA expression.

No significant differences between nonpregnant (NP), pregnant (P), and postpartum (PP) ewes. Data are expressed as fold change from nonpregnant ewes ± SEM.

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Figure 5-4. Relative hypothalamic arginine vasopressin (AVP) mRNA expression. No

significant differences between nonpregnant (NP), pregnant (P), and postpartum (PP) ewes. Data are expressed as fold change from nonpregnant ewes ± SEM.

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Figure 5-5. Relative hypothalamic 5-HT1A receptor mRNA expression. No significant

differences between nonpregnant (NP), pregnant (P), and postpartum (PP) ewes. Data are expressed as fold change from nonpregnant ewes ± SEM.

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Figure 5-6. Relative hypothalamic 5-HT2A receptor mRNA expression. No significant

differences between nonpregnant (NP), pregnant (P), and postpartum (PP) ewes. Data are expressed as fold change from nonpregnant ewes ± SEM.

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Figure 5-7. Relative hypothalamic serotonin reuptake transporter (SERT) mRNA expression.

No significant differences between nonpregnant (NP), pregnant (P), and postpartum (PP) ewes. Data are expressed as fold change from nonpregnant ewes ± SEM.

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Figure 5-8. Relative hypothalamic proopiomelanocortin (POMC) mRNA expression. Based on

one-way ANOVA, followed by Duncan’s Multiple Range test, delta Ct was significantly greater in both pregnant (P) and postpartum (PP) ewes compared to nonpregnant (NP) ewes, suggesting reduced hypothalamic POMC mRNA expression during pregnancy and early postpartum. Data are expressed as fold change from nonpregnant ewes ± SEM. * Indicates significantly different from nonpregnant group (p<0.05).

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CHAPTER 6 SUMMARY

The overall goal of the research being done in our lab is to investigate the mechanisms of

maternal adaptation to pregnancy. More specifically, we are interested in the regulation of the

hypothalamic-pituitary-adrenal (HPA) axis during pregnancy. In the literature, several systems

have been found be associated with HPA axis regulation in normal physiology, either in an

inhibitory or stimulatory manner. The studies contained in this manuscript focus on two of those

systems and are aimed at comparing: (1) the relative role of the mineralocorticoid receptor (MR)

and (2) relative serotonergic responsivity between pregnant and non-pregnant sheep as they

relate to negative feedback control of basal HPA axis activity. In order to investigate the

relationship of these systems to HPA axis regulation during pregnancy, we studied pregnant,

nonpregnant and postpartum ewes.

For the first study, a mineralocorticoid antagonist, canrenoate was given intravenously to

both pregnant and nonpregnant ewes continuously over a period of 4 hours. Blockade of this

receptor systemically, allowed us to determine the relative role of both central and peripheral

MR in the regulation of both basal HPA axis activity and hemodynamic changes that occur

during pregnancy. Peripheral MR are known to be important in normal physiology for

electrolyte and fluid balance via binding by the mineralocorticoid, aldosterone at the renal

tubule, while central MR are thought to be the major steroid receptor involved in regulation of

basal HPA axis activity. The hypothesis for this part of my dissertation work was that systemic

blockade of MR would stimulate ACTH and cortisol and this effect may be blunted in the

pregnant ewe, reflecting an inherent reduction in the ability of MR to participate in negative

feedback during pregnancy as an underlying hypothesis in our lab is that progesterone

antagonizes cortisol negative feedback at MR. If this inherent reduction is in fact present, it

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could provide one mechanism for increased basal HPA axis activity during pregnancy. These

results did in fact support my hypothesis as the response pattern of the plasma cortisol and

ACTH measured hourly during infusion with the MR antagonist, canrenoate, differed between

pregnant and nonpregnant ewes. The cortisol response was blunted in pregnant ewes during the

first half of canrenoate infusion and was reflected in the pattern of ACTH release which is

therefore suggestive of central or pituitary MR-mediated effects. The responses during the

second half of the infusion support the ‘underfill’ hypothesis of pregnancy as homeostatic factors

are upregulated to a greater extent in pregnant ewes in response to the subtle hemodynamic

effects of canrenoate.

For the second study, a selective serotonin reuptake inhibitor (SSRI), fluoxetine was given

intracerebroventricularly either acutely or continuously over a period of 6 days to pregnant and

nonpregnant ewes. Blockade of the serotonin reuptake transporter, allows the neurotransmitter

serotonin to remain in the synaptic cleft for a longer period, thereby enhancing its effects.

Administration through this route allows us to avoid any peripheral effects of fluoxetine that

might secondarily influence HPA axis activity. The serotonergic system has been shown, in

numerous animal models including sheep, to increase plasma ACTH and cortisol concentrations.

The hypothesis for this part of my dissertation work was that if increased serotonergic tone is

partially responsible for elevation in HPA axis activity during pregnancy, than administration of

an SSRI would demonstrate this via enhanced SSRI-stimulated ACTH and cortisol release in

pregnant compared to nonpregnant or postpartum ewes. The results of the acute study disproved

our hypothesis, in directionality but may still have uncovered potential differences in

responsivity to serotonergic agents between the pregnant and postpartum states in terms of HPA

axis activation, whereas the results of our subchronic infusion study may support our hypothesis.

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Additionally, the observation that fluoxetine infusion had less of an inhibitory effect on post-

operative feeding recovery in the pregnant ewes, may provide evidence that central serotonin-

mediated feeding regulation may also be altered in pregnancy. As we know from the literature,

most if not all of the regions involved in the serotonin-mediated stimulation of ACTH and

cortisol are highly abundant in both corticosteroid and ovarian hormone receptors allowing for

the possibility of modulation by these steroids. Previous reports have demonstrated highly

variable effects across brain region and across species which are further compounded by variable

effects based on the duration of exposure to the steroids.

Because the studies discussed previously in this manuscript have suggested differences in

HPA axis activation between these reproductive states, I then sought to investigate whether such

differences are occurring as a result of changes at the gene expression level. The objective of the

final study was to characterize any differences in relative mRNA expression levels of HPA axis-

and serotonergic system-relevant genes in the hypothalamus of nonpregnant, pregnant, and post-

partum ewes. We hypothesized that changes at the transcriptional level could help to explain

basal HPA axis regulation differences between the pregnant and nonpregnant states.

Specifically, we expected that we might see a decrease in hypothalamic MR expression.

Contrary to our hypothesis, however, there were no differences in nonpregnant, pregnant, and

postpartum expression of hypothalamic MR expression. This suggests that the differential

response to canrenoate as described in Chapter 3 is not due a decrease in hypothalamic MR

expression during pregnancy, but rather might support our lab’s alternative hypothesis that

progesterone is antagonizing cortisol negative feedback at MR. There were also no detectable

differences in hypothalamic GR, CRH, or AVP gene expression among the reproductive groups

studied here. Meanwhile, extensive evidence indicates a stimulatory role for serotonin on HPA

113

axis activity. We formulated multiple hypotheses based on evidence for glucocorticoid and

ovarian hormone modulation of serotonergic system-related genes from the literature.

Additionally, we hoped to explain the differential responses to fluoxetine that were described in

Chapter 4 through alterations in hypothalamic gene expression patterns. Our hypotheses were

disproved as none of the serotonergic genes investigated in this study were significantly different

between nonpregnant, pregnant or postpartum states. On the other hand, the differential daily

food intake response to subchronic fluoxetine between nonpregnant and pregnant ewes suggested

that POMC expression during pregnancy might be reduced, thereby causing a reduction in

downstream signally to satiety centers. In fact, hypothalamic POMC expression was

significantly reduced in pregnant ewes compared to nonpregnant ewes.

The overall conclusions of this work are that (1) the role of MR in mediating the negative

feedback effects of cortisol does, in fact, appear to be altered in pregnancy, (2) an increase in

serotonergic responsivity does not explain the increase in basal HPA axis activity during

pregnancy, and (3) relative MR, GR, CRH, AVP, 5-HT1A, 5-HT2A, and SERT expression were

not altered across the reproductive states investigated here, while POMC expression was reduced

during pregnancy.

Figure 6-1 is a representative model of HPA axis regulation as it relates to these studies,

based on the consensus from the literature. As we well know, the hypothalamus is responsible

for stimulating secretion of ACTH release by the anterior pituitary. These studies have

uncovered potential modulation of MR-mediated negative feedback and serotonergic system-

mediated stimulation of the HPA axis, which could be at the level of the hypothalamus, but may

also be occurring in upstream brain regions such as the hippocampus or brain stem.

114

Our lab has previously shown that corticosteroids are important for many changes that

must occur during pregnancy such as maternal volume expansion, uterine blood flow, and fetal

homeostasis which impact maternal and fetal health. It is also important to note that the lack of

global changes in expression of key HPA axis regulatory genes, which are typically known to be

altered in response to chronic stress, further indicates that pregnancy is not perceived as a

stressor. As an understudied, basic adaptive process of successful pregnancy, it is important that

we continue to characterize the regulation of the HPA axis. Due to the importance of

corticosteroids in regulating such processes, several systems are in place to ensure its tight

regulation. The elevations in basal plasma ACTH and cortisol that occur during pregnancy are,

therefore, undoubtedly the result of coordinated alterations in many of these systems and

therefore, alterations in regulation of the HPA axis by other mechanisms still remain to be

investigated.

Our understanding of HPA axis regulation both in normal physiology and in pregnancy is

still far from complete. Interestingly, however, if differences similar to those observed in these

studies are also evident in humans, the findings could suggest a need for adjustment of treatment

regimens in women for HPA axis dysregulation as well as depression and/or anxiety as a she

transitions between these reproductive states in order to protect the health of both the mother and

her developing fetus.

115

Figure 6-1. Proposed roles for central corticosteroid receptors and the serotonergic system in

regulating basal hypothalamic and pituitary release during pregnancy. It is known that 5-HT1A autoreceptors are found at the level of the serotonergic cell bodies in the dorsal raphe nuclei of the brain stem. The triangle represents an inhibitory interneuron that presumably expresses 5-HT1A receptors and may be located within the hypothalamus or the hippocampus, therefore activation of these receptors will ultimately result in stimulation of the HPA axis. 5-TH2A is excitatory and found in high abundance in the hypothalamus. Meanwhile MR and GR are found throughout the circuit as well, with MR being highly expressed in the hippocampus. Activation of either of these receptors by glucocorticoids serves to inhibit the HPA axis. Meanwhile, activation of POMC at the arcuate nucleus inhibits feeding and this effect is thought to be enhanced by serotonin. The represents excitatory effects, while the ┬ represents inhibitory effects.

116

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BIOGRAPHICAL SKETCH

Melissa Dawn Lingis was born Melissa Dawn Landen in 1976 in Fort Lauderdale, Florida

to David and Pamela Landen. Melissa developed a strong interest in science and critical thinking

as a member of the gifted program at Nova, a magnet school for Broward County, which she

attended from kindergarten through the twelfth grade. Immediately upon graduating high school,

Melissa began her undergraduate studies with the University of Florida during the summer of

1994. Melissa completed a Bachelor of Science degree in animal sciences in 1998 and began

applying to veterinary college to pursue her childhood dream of becoming a small animal

veterinarian.

As the end of her undergraduate studies was drawing near, Melissa began working as a

veterinary technician in several small animal practices both in Gainesville and in Fort

Lauderdale. Over time, Melissa slowly began to realize that she was not as enthusiastic about a

career in veterinary medicine as she had been as a child. After much consideration, she

eventually returned to the University of Florida in 2003 to attend graduate school as a means to

pursue a career in scientific research. In August 2004, Melissa completed a non-thesis Master of

Agriculture degree in animal sciences with a focus on reproductive biology, as this was one of

her major interests during her undergraduate studies.

While completing her master’s degree, she began working in the lab of Dr. Maureen

Keller-Wood as a laboratory technician. Melissa found the research exciting and enjoyed the

mentorship so much that she chose to extend her graduate school experience by adding to the

work being done in the lab. She was accepted into the doctorate program in the Department of

Pharmacodynamics and chose Dr. Keller-Wood as her major professor. Melissa’s dissertation

work focused on the relative roles of the mineralocorticoid receptor and the serotonergic system

in the regulation of basal maternal hypothalamic-pituitary-adrenal axis activity during pregnancy

in the ewe. Her work was funded by an R01 grant from the National Institutes of Health

(DK38114) awarded to Maureen Keller-Wood.

Melissa married Robert Lingis in 2006 and gave birth to their son, Matthew, in the summer

of 2008. Throughout the last year of her doctorate studies, she juggled motherhood, research,

and writing to the best of her abilities; ultimately receiving her PhD in August 2009. Melissa

looks forward to beginning post-doctoral work in the lab of Dr. Kirk Conrad in the College of

Medicine, also at the University of Florida. Here she will study the role of relaxin and its

receptor in cardiovascular and renal physiology. Following her post-doctoral work, Melissa

hopes to pursue a career in pre-clinical research at the industrial level and later return to

academia as a professor and mentor to students interested in a career in scientific research.