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2014

Cellular Mechanisms and Neural Control of Sodium Appetite in

Male Rats
Laura Ann Grafe
University of Pennsylvania, lauraagrafe@gmail.com

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Grafe, Laura Ann, "Cellular Mechanisms and Neural Control of Sodium Appetite in Male Rats" (2014).
Publicly Accessible Penn Dissertations. 1290.
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Cellular Mechanisms and Neural Control of Sodium Appetite in Male Rats

Abstract
Sodium appetite is a reliable and robust behavior displayed by a broad range of species. Despite its key
role in survival, the biological basis of this behavior remains undefined. The goal of this thesis is to
elucidate the cellular signaling and neural circuitry underlying sodium appetite. More specifically, I focus
on the central actions of the hormones aldosterone and Angiotensin II (AngII) in eliciting this crucial
ingestive behavior. First, I tested the hypothesis that a signaling protein downstream of the AngII receptor,
Mitogen Activated Protein Kinase (MAPK), underlies sodium appetite induced by endogenous AngII. I
demonstrated through both behavioral pharmacology and protein expression that sodium appetite
requires MAPK activation but thirst does not. Next, I tested the hypothesis that aldosterone and AngII
potentiate sodium appetite through inactivation of an inhibitory signal. Using functional neuroanatomy
and reversible lesions, I revealed that behavioral cooperativity between aldosterone and AngII involves the
alleviation of an inhibitory oxytocin signal relayed from the paraventricular nucleus of the hypothalamus
to the organum vasculosum lateral terminalis. Finally, I tested the hypothesis that an increase in
motivation for sodium is associated with an increase in mesolimbic dopamine activity. Using a
progressive ratio schedule of reinforcement and functional neuroanatomy, I discovered that the
combination of AngII and aldosterone induces a selective drive for sodium, which is associated with an
increase in neural activity and markers of dopamine synthesis in the ventral tegmental area and nucleus
accumbens. Together, these findings demonstrate that MAPK signaling is important for AngII-induced
sodium appetite, and potentiation of sodium appetite by aldosterone plus AngII requires both inhibition of
oxytocin secretion and activation of downstream mesolimbic circuitry.

Degree Type
Dissertation

Degree Name
Doctor of Philosophy (PhD)

Graduate Group
Neuroscience

First Advisor
Loretta M. Flanagan-Cato

Keywords
Appetite, Brain, Motivation, Rat, Signals, Sodium

Subject Categories
Behavior and Ethology | Neuroscience and Neurobiology | Social and Behavioral Sciences

This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1290

CELLULAR MECHANISMS AND NEURAL CONTROL OF SODIUM APPETITE IN MALE RATS

Laura Ann Grafe

A DISSERTATION

in

Neuroscience

Presented to the Faculties of the University of Pennsylvania

in

Partial Fulfillment of the Requirements for the

Degree of Doctor of Philosophy

2014

Supervisor of Dissertation

__________________________
Dr. Loretta M. Flanagan-Cato
Associate Professor of Psychology, University of Pennsylvania

Graduate Group Chairperson

__________________________
Dr. Joshua I. Gold, Associate Professor of Neuroscience, Perelman School of Medicine
at the University of Pennsylvania

Dissertation Committee

Dr. Kendra K. Bence, Associate Professor of Animal Biology, University of Pennsylvania

School of Veterinary Medicine (Chair)

Dr. Irwin Lucki, Professor of Pharmacology and Psychiatry, University of Pennsylvania

Dr. Marc Schmidt, Associate Professor of Biology, University of Pennsylvania

Dr. Lawrence P. Reagan, Associate Professor of Pharmacology, Physiology, and

Neuroscience, University of South Carolina School of Medicine

 
 
CELLULAR MECHANISMS AND NEURAL CONTROL OF SODIUM APPETITE IN MALE RATS

COPYRIGHT

2014

Laura Ann Grafe

This work is licensed under the

Creative Commons Attribution-
NonCommercial-ShareAlike 3.0
License

To view a copy of this license, visit

http://creativecommons.org/licenses/by-ny-sa/2.0
 ACKNOWLEDGEMENT

First and foremost, I would like to thank my advisor, Lori Flanagan-Cato. I met

Lori by taking her neuroendocrinology class in the spring semester of my first year of

graduate school. She struck me as a very intelligent, passionate, and warm person; I

decided to do a rotation in her lab, and the rest is history! Throughout the years she has

been extremely approachable and supportive, and has given me advice on anything

from writing a manuscript to childcare options in your faculty years. She reminds me

how important it is to stay on top of the literature and also to extend your knowledge

beyond the scope of your thesis in order to be a well-rounded and successful scientist.

She helped me prepare for my career with journal clubs on how to improve your CV and

encouraged me to attend job talks so I would know what to expect in the years to come,

for which I am extremely grateful. I also enjoy the fact that she made a lab facebook

page to promote our work and posts scientific articles that she finds interesting. I

appreciated our happy hours, where we could relax a bit and talk about non-science

things, which allowed me to realize that we have a lot in common! I am going to miss

working in this lady lab a lot, and Lori is one of the main reasons for that!

I am also incredibly lucky to have such a supportive husband who has helped me

get through some of the most stressful times in graduate school. He is my own personal

cheerleader, filling me with confidence when I have had a failed experiment or when I

feel inferior to other scientists in the field. Besides listening to me talk about science

constantly, Corey also comes into the lab occasionally to help me with experiments that

require some manual labor (e.g., cutting plastic cannulae so they can fit next to each

other on the rat skull). He also makes life easier by driving me to lab at odd hours and

one time even coming to have dinner with me while I waited for my rats to stop pressing
iii  
 
their levers in a progressive ratio experiment. He is my rock, and I love him very much

for believing in me.

I would not be where I am today if I didn't have the love and support of my

immediate family, including my mom, dad, and two sisters. I am so grateful to have

parents that always valued education, instilled in me the drive to succeed, and supported

me through college. They each helped me grow, especially in writing and science, two

things that are integral to a PhD in neuroscience! My sisters have also been there for me

along the way, listening to me talk about graduate school, and also sharing important life

milestones like marriage throughout this very busy time!

In addition to Lori being an awesome advisor, the lab environment has been

phenomenal thanks to Sarah Ferri, the graduate student who I have been working next

to for my entire PhD. Though we do not work on the same projects, Sarah has showed

me how to perform various lab techniques and is always helpful in finding reagents,

troubleshooting experiments, or pitching in when I need a hand. As we went through

graduate school, we bonded over each hoop we had to jump through and became very

close. Not only do I respect her as a scientist, but I value her as a friend. I enjoyed girl

talk as breaks from lab work, and I could not have asked for a better lab mate!

I would like to thank my committee for all of their helpful feedback and for

challenging me throughout my graduate career. Kendra Bence, the chair of my

committee, has been extremely supportive and has given me sound advice, especially

on signaling, during my studies. She also gave me information about an assay

measuring p-MAPK, which was integral to me acquiring my first publication. She has

been an extremely fair and reliable chair, and is always a friendly face to see at

committee meetings. Irwin Lucki, who I met when first interviewing at Penn and also had

the pleasure of doing a rotation with, is a very creative and wise scientist. I value his
 
iv  
 
opinions at committee meetings, and especially appreciate his knack for data

presentation, which has helped me significantly in talks and manuscripts. Marc Schmidt,

a systems neuroscientist at heart, has helped me consider the big picture from time to

time during my committee meetings, and really makes me think about the circuitry for the

behavior I am studying. I appreciate his non-rodent perspective, which often makes me

think of things I hadn't thought of before. Lastly, I would like to thank Larry Reagan of

South Carolina School of Medicine, for graciously agreeing to be on my committee and

making the trip to Philadelphia for my defense! So far, I have enjoyed his friendly emails

and sincere interest in the work I have sent him.

I have also had the pleasure of working with our collaborator, Daniel Yee of the

University of Pennsylvania Vet School. I would like to thank him for his encouragement,

advice, trouble shooting and technical assistance with Western blots, and for listening to

numerous practice talks and lab meetings of mine.

I would additionally like to acknowledge two of my favorite undergraduate

assistants, Klil Babin and Annie Takacs. Klil was with me for the first half of my graduate

career Annie my second half. I was lucky enough to have them as two competent,

dependable, funny, and bright little helpers that made my experiments go more

smoothly. I will forever be grateful for the many mornings and weekends they came in to

help me with my studies.

Finally, I want to thank my extended family, neighbors, and my amazing friends

for their never-ending support and encouragement, advice, and good humor throughout

my graduate career. They all are unique and have special places in my heart.

I am so lucky to have worked with so many brilliant and caring faculty and staff in

the Neuroscience Graduate Group. Specifically, I would like to thank the Chairs of our

program during my PhD, Mikey Nusbaum, Rita Balice-Gordon, and Josh Gold, and also
v    
 
the neuroscience coordinator, Jane Hoshi. Every day I was grateful to be part of this

institution, and part of the Neuroscience Program, which is filled with amazing faculty

and students. It is no surprise that we won best graduate group at Society for

Neuroscience this year!

 
vi  
 
 ABSTRACT
 

CELLULAR MECHANISMS AND NEURAL CONTROL OF SODIUM APPETITE IN MALE RATS

Laura Ann Grafe

Dr. Loretta M. Flanagan-Cato

Sodium appetite is a reliable and robust behavior displayed by a broad range of

species. Despite its key role in survival, the biological basis of this behavior remains

undefined. The goal of this thesis is to elucidate the cellular signaling and neural

circuitry underlying sodium appetite. More specifically, I focus on the central actions of

the hormones aldosterone and Angiotensin II (AngII) in eliciting this crucial ingestive

behavior. First, I tested the hypothesis that a signaling protein downstream of the AngII

receptor, Mitogen Activated Protein Kinase (MAPK), underlies sodium appetite induced

by endogenous AngII. I demonstrated through both behavioral pharmacology and

protein expression that sodium appetite requires MAPK activation but thirst does not.

Next, I tested the hypothesis that aldosterone and AngII potentiate sodium appetite

through inactivation of an inhibitory signal. Using functional neuroanatomy and

reversible lesions, I revealed that behavioral cooperativity between aldosterone and

AngII involves the alleviation of an inhibitory oxytocin signal relayed from the

paraventricular nucleus of the hypothalamus to the organum vasculosum lateral

terminalis. Finally, I tested the hypothesis that an increase in motivation for sodium is

associated with an increase in mesolimbic dopamine activity. Using a progressive ratio

schedule of reinforcement and functional neuroanatomy, I discovered that the

combination of AngII and aldosterone induces a selective drive for sodium, which is

associated with an increase in neural activity and markers of dopamine synthesis in the

 
vii  
 
ventral tegmental area and nucleus accumbens. Together, these findings demonstrate

that MAPK signaling is important for AngII-induced sodium appetite, and potentiation of

sodium appetite by aldosterone plus AngII requires both inhibition of oxytocin secretion

and activation of downstream mesolimbic circuitry.

 
viii  
 
 TABLE OF CONTENTS

ACKNOWLEDGEMENT ...................................................................................................... III  

ABSTRACT ............................................................................................................................ VII  

LIST OF FIGURES ............................................................................................................... XII  

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

Fluid Balance ......................................................................................................................................2  

The Link Between Sodium Intake and Hypertension ......................................................................... 4  

The Renin-Angiotensin-Aldosterone System (RAAS) ..................................................................5  

Peripheral RAAS...................................................................................................................................... 5  
Central RAAS........................................................................................................................................... 6  

Neural Circuitry Underlying Sodium Appetite ...............................................................................8  

Brain Sites of AngII Action in Sodium Appetite ................................................................................... 8  
Brain Sites of Aldosterone Action in Sodium Appetite ..................................................................... 10  
Intersection of AngII and Aldosterone Circuits in Sodium Appetite................................................ 11  

Cellular Signaling Underlying Sodium Appetite.......................................................................... 13  

Angiotensin II Receptors ...................................................................................................................... 13  
Aldosterone Receptors ......................................................................................................................... 14  
Crosstalk ................................................................................................................................................. 15  

Novel Questions Addressed by this thesis .................................................................................. 16  

What signal is required for endogenous AngII-induced sodium appetite? Is this signal unique to
sodium appetite? ................................................................................................................................... 16  
What signaling and circuitry underlies Aldosterone and AngII potentiation of sodium appetite?
................................................................................................................................................................. 17  
Does the Aldosterone and AngII potentiation of sodium appetite involve an increase in
mesolimbic dopamine activity?............................................................................................................ 18  

CHAPTER 2: ENDOGENOUS ANGIOTENSIN II-INDUCED P44/42 MITOGEN-

ACTIVATED PROTEIN KINSAE ACTIVATION MEDIATES SODIUM APPETITE
BUT NOT THIRST OR NEUROHYPOPHYSEAL SECRETION IN MALE RATS
.....................................................................................................................................................20  

SUMMARY.......................................................................................................................................... 21  

INTRODUCTION ................................................................................................................................ 22  

 
ix  
 
MATERIALS AND METHODS........................................................................................................... 24  

RESULTS............................................................................................................................................ 30  

DISCUSSION...................................................................................................................................... 35  

PERSPECTIVES ................................................................................................................................ 40  

ACKNOWLEDGEMENTS .................................................................................................................. 41  

CHAPTER 3: THE ROLE OF THE HYPOTHALAMIC PARAVENTRICULAR

NUCLEUS AND THE ORGANUM VASCULOSUM LATERAL TERMINALIS IN
THE CONTROL OF SODIUM APPETITE IN MALE RATS ......................................47  

SUMMARY.......................................................................................................................................... 48  

INTRODUCTION ................................................................................................................................ 49  

MATERIALS AND METHODS........................................................................................................... 51  

RESULTS............................................................................................................................................ 58  

DISCUSSION...................................................................................................................................... 64  

CONCLUSIONS ................................................................................................................................. 69  

ACKNOWLEDGEMENTS .................................................................................................................. 69  

CHAPTER 4: ALDOSTERONE AND ANGII-INDUCED SODIUM APPETITE IS

ASSOCIATED WITH MESOLIMBIC ACTIVATION ....................................................78  

SUMMARY.......................................................................................................................................... 79  

INTRODUCTION ................................................................................................................................ 80  

MATERIALS AND METHODS........................................................................................................... 82  

RESULTS............................................................................................................................................ 88  

DISCUSSION...................................................................................................................................... 94  

ACKNOWLEDGEMENTS ................................................................................................................ 101  

CHAPTER 5: GENERAL CONCLUSIONS AND FUTURE DIRECTIONS ........ 106  

APPENDIX ............................................................................................................................ 119  

x    
 
REFERENCES..................................................................................................................... 122  

 
xi  
 
 LIST OF FIGURES

Fig 1.1. Locations of nuclei in the rat brain implicated in sodium appetite ........................ 19  

Fig 1.2. Cellular signaling implicated in sodium appetite ..................................................... 19  

Figure 2.1. Examining Furo/Cap-MAPK activation in the brain........................................... 42  

Figure 2.2. Furo/Cap activates MAPK in key brain areas; this is reduced by an AT1R and
MEK inhibitor....................................................................................................................... 43  

Figure 2.3. Furo/Cap increases urine output as well as water and sodium intake........... 44  

Figure 2.4. Furo/Cap sodium intake but not water intake is mediated by MAPK. ........... 45  

Figure 2.5. Furo/Cap-induced Vasopressin and Oxytocin release is not mediated by

MAPK................................................................................................................................... 46  

Fig 2.6. AngII-induced Vasopressin and Oxytocin Release is not mediated by MAPK. .. 46  

Figure 3.1. Erk1/2 is not necessary for the DOC/AngII potentiation of sodium appetite. 70  

Figure 3.2. DOC pretreatment enhanced AngII-induced OVLT activation and reduced
PVN activity and OT release. ........................................................................................... 72  

Figure 3.3. The IP3 receptor is required for DOC/AngII potentiation of sodium appetite.74  

Figure 3.4. PVN inactivation enhances AngII-induced sodium appetite. ........................... 76  

Figure 4.1. DOC/AngII treatment increases motivation for sodium. ................................. 102  

Figure 4.2. DOC/AngII treatment increases cFos expression in the ventral tegmental
area and nucleus accumbens. ....................................................................................... 103  

Figure 4.3. PVN inactivation enhances AngII-induced cFos expression in the ventral
tegmental area and nucleus accumbens...................................................................... 104  

Figure 4.4. DOC/AngII treatment increases tyrosine hydroxylase activation in the ventral
tegmental area and nucleus accumbens...................................................................... 105  

Figure 5.1. Model Circuit Diagram for Aldosterone- and AngII-induced Sodium Appetite
............................................................................................................................................ 118  

Figure A.1. AngII-induced oxytocin staining in the OVLT is reduced by DOC

pretreatment...................................................................................................................... 119  

Figure A.2. DOC increases cFos activation in the BNST................................................... 120  

 
xii  
 
Figure A.3. Tyrosine Hydroxylase in the Ventral Tegmental Area correlates with cFos in
the OVLT, but not sodium intake. .................................................................................. 121  

 
xiii  
 
 CHAPTER 1: INTRODUCTION

The goal of this dissertation is to better understand the cellular signaling and

neural circuitry underlying sodium appetite. First described by Curt Richter in 1936, this

motivated behavioral state drives animals to consume sodium in response to a sodium

deficiency. Moreover, Richter discovered that removal of the adrenal gland caused an

increase in sodium intake, and when sodium solutions were removed, rats died within

five days (Richter, 1936). The importance of this behavior was further demonstrated in

a subsequent case study of a child with an insatiable sodium appetite, which kept him

alive until a low sodium diet at the hospital resulted in his death (Wilkins & Richter,

1940). Though sodium appetite is critical for survival, key aspects of the biological basis

of this behavior remain unclear.

Scientific laboratories have designed several ways to induce sodium appetite so

that we may study its etiology. These preparations include a sodium deficient diet,

subcutaneous injection of mineralocorticoids, furosemide diuresis, and central

administration of AngII (Wolf, McGovern, & Dicara, 1974). Each of these preparations

differ in terms of time course for the development of sodium appetite and type of fluid

balance disturbance. For example, a sodium deficient diet must be administered to rats

for over a week in order to induce an observable sodium intake (Fregly, Harper, &

Radford, 1965). A low dose treatment with a mineralocorticoid, such as

deoxycorticosterone acetate (DOC), is slightly faster, inducing sodium appetite in a

three-day period. Furosemide diuresis, which causes excretion of sodium through the

kidney, only takes about 24 hours to induce sodium intake (Jalowiec, 1974). However,

1  
 
central AngII has the most rapid effects, stimulating sodium intake within minutes

(Buggy & Jonklaas, 1984).

In this dissertation, I use two different preparations of sodium appetite, one

causing a rapid increase in central AngII, and the other using both mineralocorticoid

pretreatment and central AngII, to study the cellular mechanisms and neural control of

sodium appetite. I first focus on cellular signals, namely mitogen-activated protein

kinase and inositol trisphosphate, in contributing to this behavior. I then use functional

neuroanatomy and reversible lesions to identify important brain areas involved in the

control of sodium appetite, focusing on initial targets like circumventricular organs and

moving towards downstream areas such as the mesolimbic dopamine system.

This introductory chapter describes our current understanding of the biological

basis of sodium appetite and some key aspects that still remain unclear. This includes a

general discussion of fluid balance, the hormone systems that control sodium appetite,

and both the neural circuitry and cellular players known to underlie this behavior.

Finally, I will present novel questions that form the basis of my dissertation research.

Fluid Balance

The human body consists of sixty percent water (Abbott, 1946). One third of this

water is contained in the extracellular fluid. Organisms precisely maintain the ratio of

water compared to electrolytes such as sodium to maintain sufficient extracellular fluids,

a process called fluid homeostasis (Jones & Bellamy, 1964). Many systems in the body

coordinate body fluid status, including the kidney, cardiovascular system, central

nervous system, and adrenal glands (Denton, McKinley, & Weisinger, 1996). Disruption
2    
 
of sodium balance can have severe consequences such as high blood pressure, heart

failure, and even death (McKelvie, 2011).

Fluid disturbances are detected by both the kidney and stretch sensitive

receptors in the atria, aorta, and carotid (Folkow, 1971). The kidney regulates the

composition of extracellular fluid by selectively filtering water and sodium for excretion

(Bradbury, 1973). Substrates to be conserved are reabsorbed into the plasma, while

any remaining fluid is excreted into urine (Berne & Levy, 1988). Fluid perturbations

affect vascular stretch receptors by altering neural firing via the glossopharyngeal or

vagus nerve to the nucleus of the solitary tract and area postrema in the hindbrain

(Johnson & Thunhorst, 1997). These brain areas then release hormones that correct

fluid balance. For example, the hormones aldosterone and vasopressin are released in

fluid deficiency to cause renal reabsorption of sodium and water, respectively. Neural

control of renal blood flow can be achieved by release of noripenephrine on sympathetic

nerve terminals, as juxtaglomerular cells in the kidney have adrenergic receptors (Gill,

1979). Stimulation of renal nerves induces sodium and water retention; denervation

results in natriuresis and diuresis (Berne & Levy, 1988).

Though physiological responses aim to restore fluid homeostasis, minimizing

fluid loss is often not sufficient to reestablish balance. In this case, ingestive behavior is

required to replace the fluid lost (Johnson & Thunhorst, 1997). Cellular dehydration

occurs when sodium concentration extracellularly increases (drawing water from the

cell), or when extracellular fluid volume decreases; these situations induce osmotic thirst

and hypovolemic thirst, respectively (Almli, 1970). Osmotic thirst only requires water

intake, while hypovolemic thirst requires both water and sodium intake (Daniels, Yee, &

Fluharty, 2007). Osmotic control takes precedence over volumetric control in the body;
3    
 
there are inhibitory signals to prevent sodium appetite until osmosensors detect dilute

fluid. This may explain why sodium appetite is delayed compared to thirst (Avrith &

Fitzsimons, 1980). Without these ingestive behaviors to replace fluid lost, death would

ensue. Despite the criticality of this behavior, the biological basis of sodium appetite

remains unclear.

The Link Between Sodium Intake and Hypertension

Humans are genetically programmed to ingest approximately 0.25 grams of

sodium a day, but the average salt intake in most countries is roughly 10 grams per day

(He & MacGregor, 2010). As previously mentioned, a major function of the kidney is to

excrete excess sodium consumed. However, excreting such a large amount of sodium

is sometimes difficult, especially as one ages (Alderman, 2000). Epidemiological studies

have shown that societies with access to an abundance of sodium have higher blood

pressure than populations that do not (He & MacGregor, 2010). Not only this, but

several additional studies show that on average, high sodium intake is linked to an

increased blood pressure, which is then a risk factor for stroke and heart attack (Mann,

2013). Clinical trials have also shown that reducing sodium intake decreases blood

pressure (Vollmer et al., 2001).

Skepticism remains about whether lowering sodium intake over time in

normotensive humans will decrease the risk for hypertension, as there is some

conflicting data from previous studies (Midgley, Matthew, Greenwood, & Logan, 1996).

However, this is mostly due to poorly controlled studies that examine people with high

and low sodium intake, rather than having long-term trial where subjects restrict their

4    
 
sodium intake (Mann, 2013). In the end, it cannot be denied that there is a link between

high sodium intake and hypertension, and it should not be neglected. Better

understanding the biological underpinnings of sodium appetite may help in

characterizing the link between this critical behavior and hypertension.

The Renin-Angiotensin-Aldosterone System (RAAS)

It took decades for scientists to make progress in understanding how Richter’s

adrenalectomy experiments induced such robust sodium appetite. These studies

revealed that adrenal steroids, such as aldosterone, were responsible for this behavior

(Wolf, 1965). The contributions of another hormone, Angiotensin II, were much later

determined to be important for sodium appetite as well (Buggy & Jonklaas, 1984).

These hormones are part of the Renin-Angiotensin-Aldosterone System (RAAS), which,

as one might guess, is essential for sodium balance (Reid, 1985). Detection of low

blood volume or pressure stimulates RAAS both peripherally and centrally (Denton et al.,

1996). The actions produced by RAAS reestablish homeostasis and are thereby

necessary for survival (Fitzsimons, 1998).

Peripheral RAAS

When blood pressure is low, an enzyme called renin is released from stretch-

sensitive juxtaglomerular cells in the kidney (Reid, 1985). Renin converts a precursor

hormone called angiotensinogen, concentrated in the liver, to Angiotensin I (Peach,

1977). Angiotensin I is converted by Angiotensin Converting Enzyme (ACE), secreted

by the lung, to Angiotensin II (AngII).

5    
 
 Upon activation, AngII increases blood pressure to normal levels through several

avenues. Peripherally, AngII causes vasoconstriction, including contraction of the renal

smooth muscle, altering renal blood flow and glomerular flitration to reabsorb more fluids

(Berne & Levy, 1988). Additionally, AngII induces the release of aldosterone from the

adrenal gland, which then reabsorbs sodium in the kidney, thereby retaining more fluid

(Andersson, 1977). Aldosterone achieves this feat by increasing proteins on the

membrane that enhance the permeability of sodium so it is more readily absorbed

(Berne & Levy, 1988). In both cases, more fluid is retained, exerting more force on the

vasculature and thereby supporting blood pressure.

Central RAAS

In addition to its existence in the periphery, there is also a central RAAS (Wright

et al., 1984). However, a single brain cell that expresses all of the components of RAAS

has not yet been found; formation of active AngII in the brain may require multiple cell

interactions (Bader, 2010). Previous studies have revealed that manipulation of

peripheral RAAS with dexamethasone does not affect central RAAS, underscoring their

independence (Ganong, 1984). Nevertheless, brain RAAS is not completely

independent from the periphery, as AngII generated in the body can gain access to the

brain in areas with an incomplete blood brain barrier (Fitzsimons & Stricker, 1971).

Centrally, AngII stimulates vasopressin and oxytocin release, sympathetic

activation, and both thirst and sodium appetite (Reid, 1984). Vasopressin stimulates

water reabsorption in the kidney during hypovolemia, while oxytocin maintains osmotic

balance by inducing natriuresis and inhibiting sodium appetite (Cross & Wakerley,

6    
 
1977). AngII facilitates sympathetic activation in the autonomic nervous system by

inducing norepinephrine release (Ganong, 1984). While the onset of AngII-induced thirst

is immediate, sodium appetite it delayed; however, sodium intake is not secondary to

increased water intake (Avrith & Fitzsimons, 1980). Studies of transgenic mice

complement those of central administration of the neuropeptide: while mice with

increased AngII expression become hypertensive, those with reduced AngII synthesis

have decreased blood pressure, vasopressin secretion, and sympathetic activity (Bader,

2010). Together, these physiological and behavioral responses induced by central AngII

restore body fluid balance.

In addition to AngII as a critical effector of central RAAS, aldosterone plays an

important role in both blood pressure regulation and sodium appetite (Wolf, 1964).

Similar to AngII, there is some evidence of aldosterone synthesis in the brain, but the

majority of this hormone is made peripherally (Gomez-Sanchez et al., 1997). Indeed,

the absence of peripheral aldosterone induces a robust sodium appetite, as revealed by

studies in adrenalectomized rats with the inability to retain sodium (Richter, 1936).

However, a high dose of mineralocorticoids, allowing central action, also stimulates

ingestion of large volumes of sodium (Rice & Richter, 1943). Much after scientists

discovered AngII and aldosterone were separately important for sodium appetite, a study

revealed that the combination of both hormones potentiates sodium appetite but not

thirst (Fluharty & Epstein, 1983). This phenomenon has been observed in rats, pigeons

and baboons and is not secondary to natriuresis or diuresis (Fluharty & Epstein, 1983;

Massi & Epstein, 1990; Shade et al., 2002). However, it is thirty years later, and the

mechanism by which this potentiation occurs still remains unknown.

7    
 
Neural Circuitry Underlying Sodium Appetite

Neuroanatomical tract tracing and functional mapping methods have implicated

numerous brain structures involved in sodium appetite. This neural network allows

visceral information reflecting fluid balance to be distributed into several loci that then act

as neural and endocrine ports of access into the brain (Johnson & Thunhorst, 1997).

There is a long list of brain structures, and consequently, many neurotransmitters that

are thought to be involved in the circuitry underlying this behavior including

norepinephrine, GABA, glutamate, and acetylcholine (Barnes, DeWeese, & Andresen,

2003; Dendorfer, Raasch, Tempel, & Dominiak, 1998; Oz, Yang, O'donovan, & Renaud,

2005; Ozaki, Soya, Nakamura, Matsumoto, & Ueta, 2004). Below, I will discuss brain

regions thought to be important for AngII action, aldosterone action, and where these

two hormones may interact in the brain to potentiate sodium appetite.

Brain Sites of AngII Action in Sodium Appetite

Peripheral AngII gains access to the brain by way of circumventricular organs,

namely the subfornical organ (SFO) and organum vasculosum lateral terminalis (OVLT),

which are brain regions with an incomplete blood brain barrier (Ganong, 1984). In these

circumventricular organs, two major activities contributing to fluid balance take place: 1)

plasma sodium levels are sensed through a specific sodium channel and 2) AngII binds

to its receptor; information concerning the current fluid status is then passed to other key

brain areas (Weisinger et al., 1996). Not only do these circumventricular organs contain

osmosensors themselves, but they likely receive input from other osmosensors in the

body (Johnson & Thunhorst, 1997). The OVLT also receives afferent inputs from the

8    
 
SFO and hypothalamus and projects to several hypothalamic and extrahypothalamic

areas (Camacho & Phillips, 1981).

In addition to acting as a hormone, AngII may be released from axon terminals

(Ferguson, Washburn, & Latchford, 2001). Studies that lesioned axon terminals

projecting from the SFO to the OVLT abolished drinking, which may support a

neurotransmitter-like role for AngII (Eng & Miselis, 1981). Moreover, early studies

demonstrated that ablation of the SFO itself decreases water intake (Simpson &

Routtenberg, 1973), while lesions in the OVLT abrogate sodium intake (Fitzsimons,

1980). Contrarily, infusion of AngII into the SFO induces water intake, while AngII into

the OVLT induces both water and sodium intake (Fitts & Masson, 1990). However,

more recent studies indicate that both circumventricular organs are important for sodium

appetite (Fitts, Freece, Van Bebber, Zierath, & Bassett, 2004). In order for these

circumventricular organs to contribute to drinking behavior, they must project to higher

order brain areas.

Besides its efferent projections to the OVLT, the SFO is known to have fibers

terminating in the paraventricular (PVN) and supraoptic nuclei (SON) of the

hypothalamus (McKinley, Badoer, & Oldfield, 1992; Miselis, 1981). The magnocellular

neurons of these hypothalamic nuclei mediate AngII-induced oxytocin and vasopressin

release (Bisset, Clark, & Errington, 1971). The parvocellular neurons in the PVN,

however, are responsible for sending projections to autonomic centers in the brainstem

and spinal cord (Sofroniew, Weindl, Schrell, & Wetzstein, 1981). Studies that

electrolytically lesioned the hypothalamus eliminated sodium appetite, demonstrating the

importance of this part of the forebrain in exhibiting this behavior (Wolf, 1967).

9    
 
 Tracing and lesion experiments have highlighted the lateral hypothalamus as an

important part of AngII-induced sodium appetite circuitry. As mentioned previously, the

OVLT projects to many hypothalamic areas, including the lateral hypothalamus

(Camacho & Phillips, 1981). Experiments that lesion the lateral hypothalamus report

complete abrogation of sodium appetite, demonstrating its vital role in this behavior

(Wolf, 1964; Wolf, 1967). Recent tracing studies have revealed that particular

orexinergic neurons in the lateral hypothalamus project to the ventral tegmental area,

known to be involved in motivation (Fadel & Deutch, 2002; Narita et al., 2006). It is well

established that dopaminergic neurons in the ventral tegmental area project to the

nucleus accumbens, which then projects to the ventral pallidum to generate goal

directed movement (Carelli, 2002). These possible connections to the mesolimbic

dopamine system suggest that sodium appetite is a motivational-affective state rather

than just a reflexive activation of motor behavior.

Brain Sites of Aldosterone Action in Sodium Appetite

The major site of aldosterone action in the brain is the nucleus of the solitary tract

(NTS) in the hindbrain (Geerling & Loewy, 2009). This brain area contains both the

mineralocorticoid receptor (MR), a steroid receptor to which aldosterone binds, and the

enzyme hydroxysteroid dehydrogenase (HSD2), which inactivates glucocorticoids and

prevents them from binding to MRs (de Kloet et al., 2000; Geerling, Kawata, & Loewy,

2006; Geerling & Loewy, 2009; Riftina, Angulo, Pompei, & McEwen, 1995). As

glucocorticoids are much higher in concentration than aldosterone in the brain, and have

high affinity for MR, HSD2 is necessary to allow aldosterone action (Anderson &

 
10  
 
Fanestil, 1976). However, HSD2 neurons are more than just aldosterone sensors, as

they are activated by prolonged sodium deficiency even after adrenalectomy (Geerling

et al., 2006; Geerling, Chimenti, & Loewy, 2008).

When an animal is sodium-deficient, the HSD2 neurons in the NTS fire,

indicating a need for sodium (J. Lu, Sherman, Devor, & Saper, 2006; Shekhtman,

Geerling, & Loewy, 2007). Moreover, the rostral NTS receives gustatory input via the

chorda tympani, allowing for sodium detection and taste (Geerling et al., 2006). The

sodium need and detection signals are integrated in the forebrain, namely, in the central

amygdala and bed nucleus of the stria terminalis (BNST). Lesions in either brain region

decrease sodium intake but not thirst (Zardetto-Smith, Beltz, & Johnson, 1994). These

integration sites then project to motor pattern generators that lead to sodium appetite.

After some consumption, post ingestive signals from the gut are received by non-HSD2

neurons in the NTS to signal the brain to stop ingesting salt (Schwartz, Woods, Porte,

Seeley, & Baskin, 2000). The direct connections in these circuits still remain unclear.

Intersection of AngII and Aldosterone Circuits in Sodium Appetite

After examining the brain areas for AngII and aldosterone action, it appears that

these hormones have separate initial targets in the brain. It has been proposed that

behavioral cooperativity of AngII and aldosterone on sodium ingestion may be based on

disinhibition (Stricker & Verbalis, 1996). In particular, AngII acts centrally to excite

oxytocin neurons, promoting hormone release from the neurohypophysis. Concomitant

central release of oxytocin inhibits sodium appetite (McKinley, Allen, Burns, Colvill, &

Oldfield, 1998). Conversely, physiological conditions that diminish oxytocin activity

 
11  
 
increase sodium appetite (Stricker & Verbalis, 1987). Of particular relevance,

deoxycorticosterone acetate (DOC), a precursor of aldosterone, reduces AngII-induced

activation of the oxytocin neurons in the PVN and SON (Roesch, Blackburn-Munro, &

Verbalis, 2001; Stricker & Verbalis, 1996; Stricker & Verbalis, 2004). The BNST is a

candidate for integration of this information, as its GABAergic neurons that receive input

from the NTS project to the PVN, allowing for inhibition of oxytocin (Cullinan, Herman, &

Watson, 1993; Dong, Petrovich, Watts, & Swanson, 2001). However, it remains

unknown where in the brain sodium appetite is inhibited by oxytocin.

Other brain regions, like the central nucleus of the amygdala and lateral

hypothalamus, are also thought to be important areas for integration of information

between aldosterone and AngII. Both brain regions are heavily interconnected with the

BNST, and as mentioned before, also have an established role in sodium intake

(Johnson, de Olmos, Pastuskovas, Zardetto-Smith, & Vivas, 1999; Wolf, 1967). The

central amygdala is heavily innervated by inputs from the NTS, and may influence

ingestive behaviors by its output to forebrain sites that regulate motor activity

(Bourgeais, Gauriau, & Bernard, 2001). Oppositely, the lateral hypothalamus may

receive information directly from the OVLT, which then may project to the NTS,

influencing sodium appetite (Kelly & Watts, 1996; Kelly & Watts, 1998).

The ultimate goal is to delineate the complete circuit responsible for sodium

appetite, beginning with primary input sites, and ending with motor output channels in

the brainstem and spinal cord. While many pieces of the puzzle still remain, many areas

of importance have been highlighted. But, within each brain area lie specific receptors to

which each hormone must bind, and even more complicated downstream signals at

work underlying this fascinating behavior.

 
12  
 
Cellular Signaling Underlying Sodium Appetite

The initial brain targets discussed above are important for AngII and aldosterone

action because they contain receptors to which these hormones can bind. It is through

actions on their respective receptors that AngII and aldosterone induce signaling

cascades, which can affect neural excitability and plasticity, thereby altering

neurotransmission in these complex circuits. Below, I will discuss signaling pathways

thought to be important for AngII action, aldosterone action, and how these two

hormones may use cellular signals to potentiate sodium appetite.

Angiotensin II Receptors

AngII acts on two different highly specific receptors, termed Angiotensin Type 1

(AT1) and Angiotensin Type 2 (AT2) receptors (Swanson, Marshall, Needleman, &

Sharpe, 1973). AT1 receptors are expressed on all of the brain areas previously

discussed for AngII action, while AT2 receptors decline in expression after development

and are restricted to regions involved in motor and sensory control (Reagan et al., 1994;

Gallinat, Busche, Raizada, & Sumners, 2000). While AT1 receptors mediate the actions

of AngII on blood pressure, the secretion of oxytocin and vasopressin, and water and

sodium intake, AT2 receptors are implicated in apoptosis and antagonistic roles to AT1

receptors (McKinley et al., 1996; Sugaya et al., 1995). Expectedly, the signaling

underlying these receptors oppose one another (Huang, Richards, & Sumners, 1996).

As the AT1 receptor is involved in our behavior of interest, namely sodium appetite, we

focused on the downstream signaling of this specific receptor subtype.

 
13  
 
 The AT1 receptor belongs to the super family of G protein coupled receptors and

associates with Gq to stimulate inositol 1,4,5-triphosphate (IP3) production and Ca2+

mobilization (Beresford MJ, 1992; Enjalbert et al., 1986). Consequently, AT1 receptor

activation increases calcium currents, which increases the firing frequency of that neuron

(Sumners, Zhu, Gelband, & Posner, 1996). In vitro research uncovered a parallel signal

transduction pathway that can occur without Gq coupling. This pathway induces

phosphorylation of MAPK Kinase (MEK), which activates the p42 and p44 isoforms of

mitogen-activated protein kinase (MAPK) (Sadoshima, Qiu, Morgan, & Izumo, 1995),

also referred to as extracellular signal-regulated kinase 1 and 2. Subsequently, p44/42

MAPK generates downstream changes in cellular activity, including changes in gene

expression (Clark, Balla, Jones, & Catt, 1992; Lee, El-Shewy, Luttrell, & Luttrell, 2008;

LeHoux & Lefebvre, 2006). While many studies examining AT1R-induced p44/42 MAPK

activation were performed in vitro, p44/42 MAPK activation also occurs in the SFO and

PVN after a systemic AngII injection (Wei, Yu, Zhang, & Felder, 2009). As these AT1R

signaling molecules were detected in the brain, researchers have begun to investigate

their role in the neural actions of AngII, including sodium appetite.

Aldosterone Receptors

Aldosterone binds to the MR, a steroid receptor located primarily in the NTS, as

well as the SFO, OVLT, and amygdala (Geerling & Loewy, 2009). The MR is important

for sodium appetite, as demonstrated in studies using MR antisense oligonucleotides to

inhibit these actions (Sakai, Ma, Zhang, McEwen, & Fluharty, 1996; Xue et al., 2011).

When aldosterone binds to the MR, chaperone proteins dissociate, and the receptor

 
14  
 
translocates from the cytoplasm to the nucleus (Binart, Lombes, Rafestin-Oblin, &

Baulieu, 1991; Lombes, Kenouch, Souque, Farman, & Rafestin-Oblin, 1994; Rafestin-

Oblin, Farman, Cassingena, Ronco, & Vandewalle, 1993). Once in the nucleus, the MR

associates with transcription factors like AP-1 or NFκB to change the expression of

certain genes (Viengchareun et al., 2007). Conversely, the MR can have non-genomic

actions in the cytoplasm, where both ERK and IP3 signaling are induced (Dooley,

Harvey, & Thomas, 2012). Interestingly, non-genomic signaling often enhances the

genomic MR signaling, either through facilitation of nuclear translocation or synergistic

effects on the same target molecules (Grossmann & Gekle, 2009).

Crosstalk

Previous literature has demonstrated that AngII and aldosterone can mutually

enhance each other’s actions in the same cell. For example, AngII stimulates

aldosterone production and stimulates nuclear localization of MR, whereas aldosterone

increases AngII binding, AT1R levels, ACE activity, and AngII signaling in peripheral

tissues (Harada et al., 2001; Hirono et al., 2007; Jaffe & Mendelsohn, 2005; Mazak et

al., 2004; Min et al., 2005; Ullian, Schelling, & Linas, 1992; Xiao, Puddefoot, Barker, &

Vinson, 2004; Xue et al., 2011). Centrally, DOC, a precursor of aldosterone, was found

to increase AngII sensitive neurons and receptors per neuron in the medial septum

(Thornton & Nicolaidis, 1994). Interaction of both aldosterone and AngII in the brain is

required for a robust sodium appetite; this has been confirmed by using central receptor

antagonists of the two hormones (Sakai, Nicolaidis, & Epstein, 1986). Thus, it may be

 
15  
 
through augmentation of central signaling that these two hormones potentiate sodium

appetite.

Novel Questions Addressed by this thesis

Numerous brain areas and signaling pathways that may be important for sodium

appetite have been examined, but the exact circuits and cascades that trigger this

behavior are still unknown. For my thesis, I have used two preparations of sodium

appetite: 1) furosemide with a low dose of captopril (an ACE inhibitor) to stimulate

endogenous AngII production and 2) low doses of DOC and AngII. Through use of

these preparations, I have studied novel questions concerning the cellular signals and

circuitry underlying sodium appetite as described below.

What signal is required for endogenous AngII-induced sodium appetite? Is this signal
unique to sodium appetite?

Administration of AngII intracerebroventricularly causes both sodium and water

intake (Epstein, Fitzsimons, & Simons, 1969). Recent studies have suggested that

divergent cellular signals downstream of the AT1 receptor can give rise to these

separable behavioral phenomena (Daniels, Yee, Faulconbridge, & Fluharty, 2005).

More specifically, IP3 signaling underlies water intake, while MAPK signaling underlies

sodium appetite (Daniels D, Mietlicki EG, Nowak EL, Fluharty SJ, 2009). This finding

was novel and intriguing; however, it remained unclear whether the result reflected a

normal physiological process or a pharmacological phenomenon based on exogenously

administered AngII. In Chapter 2 of this thesis, I use a preparation of endogenous AngII

 
16  
 
production to establish the physiological significance of AngII-induced p44/42 MAPK on

sodium appetite. In addition, I examine the contribution of AT1 receptor signaling in

another action of central AngII, namely, neurohypophysial secretion.

What signaling and circuitry underlies Aldosterone and AngII potentiation of sodium
appetite?

Aldosterone and AngII cooperate centrally to potentiate sodium appetite

(Fluharty & Epstein, 1983). However, the mechanism by which this occurs is unknown.

Studies in peripheral tissue reveal reciprocal enhancement of cellular signaling, but not

all signaling proteins thought to be involved have been thoroughly examined (King,

Harding, & Moe, 1988). In vivo studies suggest convergence of separate neural

systems. Specifically, there are some data that support the hypothesis that aldosterone

inhibits AngII-induced oxytocin secretion, thereby disinhibiting sodium appetite (Stricker

& Verbalis, 1987). However, the site of oxytocin action to inhibit sodium appetite and the

pathway by which aldosterone disinhibits oxytocin remain unknown. In Chapter 3 of this

thesis, I first investigate the role of MAPK and IP3 in aldosterone and AngII potentiation

of sodium appetite through use of pharmacological inhibitors and measures of signal

transduction. I then examine the oxytocin disinhibition hypothesis concerning

aldosterone and AngII potentiation of sodium appetite by way of reversible lesions and

functional neuroanatomy.

 
17  
 
Does the Aldosterone and AngII potentiation of sodium appetite involve an increase in
mesolimbic dopamine activity?

Rats run faster down a runway to receive a sodium reward if both aldosterone

and AngII are present compared to either hormone alone, suggesting an increase in

motivation for this reinforcement (D. M. Zhang, Stellar, & Epstein, 1984). Additionally,

increases in dopamine activity have been observed in the nucleus accumbens in rats

depleted of sodium by a diuretic, a treatment that increases both aldosterone and AngII

(Roitman, Patterson, Sakai, Bernstein, & Figlewicz, 1999). While aldosterone and AngII

induce robust sodium ingestion, only the initial brain targets, and not downstream

circuitry of this behavior, have been well studied. It is unclear if an increase in

mesolimbic activity underlies this increase in motivation for sodium induced by central

cooperation of aldosterone and AngII. In Chapter 4 of this thesis, I first examine

motivation for sodium induced by cooperation of aldosterone and AngII using a

progressive ratio schedule of reinforcement. I then measure both neural and dopamine

activity in the ventral tegmental area and nucleus accumbens after both hormone

treatments to determine if an increase in mesolimbic activity is correlated with an

increased motivation for sodium.

The following studies aim to elucidate the cellular signaling and neuronal circuitry

involved in central aldosterone- and AngII-induced sodium appetite. This will advance

basic science knowledge by expanding our understanding of appetitive behavior that is

critical for survival. This research is also translational, given than a derangement of the

RAAS is responsible for 30 percent of hypertension cases (Mizuno et al., 1991); Patients

may live longer and healthier lives if inhibitors of sodium appetite can assist them in

reducing their salt intake.

 
18  
 
  

Fig 1.1. Locations of nuclei

in the rat brain implicated in
sodium appetite

Saggital plane of the rat brain

highlighting brain areas that
are important for AngII- and
aldosterone- induction of
sodium appetite.
Abbreviations: NUACC =
Nucleus Accumbens, BNST =
bed nucleus stria terminalis,
SFO = subfornical organ,
OVLT = organum vasculosum
lateral terminalis, CEA =
central amygdala, PVN =
paraventricular nucleus of the
hypothalamus, LH = lateral
hypothalamus, VTA = ventral
tegmental area, NTS =
nucleus of the solitary tract

Fig 1.2. Cellular signaling

implicated in sodium
appetite

A simplified illustration showing

the cellular signaling underlying
both AngII- and aldosterone-
induced sodium appetite.
AngII actions on the AT1R
induces parallel IP3 and MAPK
signaling, while aldosterone
may have both non-genomic
and genomic actions on the
MR. Non-genomic MR actions
allow for possible crosstalk with
AngII signaling proteins.
Though it is pictured in the
same cell, it may not be.
Abbreviations: AT1R = AngII
Type 1 Receptor, MR =
Mineralocorticoid receptor.

 
19  
 
 CHAPTER 2: ENDOGENOUS ANGIOTENSIN II-INDUCED P44/42 MITOGEN-
ACTIVATED PROTEIN KINSAE ACTIVATION MEDIATES SODIUM APPETITE BUT
NOT THIRST OR NEUROHYPOPHYSEAL SECRETION IN MALE RATS

Laura A. Felgendreger1, Steven J. Fluharty1,2,4, Daniel K. Yee2, and Loretta M. Flanagan-

Cato1,3,4

Neuroscience Graduate Group1, Departments of Animal Biology2 and Psychology3, and

the Mahoney Institute of Neurological Sciences4, University of Pennsylvania,

Philadelphia Pennsylvania, USA

Abbreviated title: AngII-induced MAPK

Key words: Fluid Homeostasis; Diuresis; Circumventricular Organs; Vasopressin;

Oxytocin

This work originally appeared in Journal of Neuroendocrinology, 25(2):97-106.

20  
 
SUMMARY

The renin-angiotensin-aldosterone system makes a critical contribution to body

fluid homeostasis, and abnormalities in this endocrine system have been implicated in

certain forms of hypertension. The peptide hormone angiotensin II (AngII) regulates

hydromineral homeostasis and blood pressure by acting on both peripheral and brain

targets. In the brain, AngII binds to the angiotensin type 1 receptor (AT1R) to stimulate

thirst, sodium appetite and both arginine vasopressin (AVP) and oxytocin (OT) secretion.

The present work used an experimental model of endogenous AngII to examine the role

of p44/42 mitogen-activated protein kinase (MAPK) as a signaling mechanism to

mediate these responses. Animals were given a combined treatment of furosemide and

a low dose of captopril (furo/cap), a diuretic and an angiotensin converting enzyme

inhibitor, respectively, to elevate endogenous AngII levels in the brain. Furo/cap induced

p44/42 MAPK activation in key brain areas that express AT1R, and this effect was

reduced with either a centrally administered AT1R antagonist (irbesartan) or a p44/42

MAPK inhibitor (U0126). Additionally, furo/cap treatment elicited water and sodium

intake, and irbesartan markedly reduced both of these behaviors. Central injection of

U0126 markedly attenuated furo/cap-induced sodium intake but not water intake.

Furthermore, p44/42 MAPK signaling was not necessary for either furo/cap- or

exogenous AngII-induced AVP or OT release. Taken together, these results indicate

that p44/42 MAPK is required for AngII-induced sodium appetite, but not thirst or

neurohypophysial secretion. This result may allow for discovery of more specific

downstream targets of p44/42 MAPK to curb sodium appetite, known to exacerbate

hypertension, while leaving thirst and neurohypophysial hormone secretion undisturbed.

 
21  
 
INTRODUCTION

The renin-angiotensin-aldosterone system promotes body fluid homeostasis by

coordinating physiological and behavioral responses to correct fluid perturbations

(Bakris, 2010; Peach, 1977). Hypotensive and hypovolemic events prompt the release

of the enzyme renin from the kidneys, which initiates a catalytic cascade that ultimately

elevates blood levels of the hormone angiotensin II (AngII) (Ferguson et al., 2001).

Peripherally, AngII constricts blood vessels to support blood pressure. Circulating AngII

binds to brain receptors in the subfornical organ (SFO) and organum vasculosum of the

lateral terminalis (OVLT), brain regions with an incomplete blood-brain barrier. In

addition, AngII produced locally by the brain renin-angiotensin system acts on relays in

the median pre-optic nucleus (MNPO), paraventricular nucleus of the hypothalamus

(PVN) and supraoptic nucleus (SON) (Geerling & Loewy, 2008). Acting upon these

central nuclei, AngII prompts thirst, sodium appetite (Epstein, Fitzsimons, & Rolls, 1970;

Findlay & Epstein, 1980), and both arginine vasopressin (AVP) and oxytocin (OT)

secretion (Marc & Llorens-Cortes, 2011). While the physiological actions of AngII are

well known, our understanding of its cellular actions is incomplete.

The central actions of AngII are mediated by the angiotensin type 1 receptor

(AT1R), which belongs to the super family of G protein coupled receptors and associates

with Gq to trigger inositol 1,4,5-triphosphate (IP3) production and Ca2+ mobilization

(Beresford MJ, 1992; Enjalbert et al., 1986). In vitro research uncovered a parallel

signal transduction pathway that phosphorylates MAPK Kinase (MEK), which activates

mitogen-activated protein kinase (MAPK) (Sadoshima et al., 1995) including the p42 and

p44 isoforms, also referred to as extracellular signal-regulated kinase 1 and 2. In turn,

p44/42 MAPK provokes a variety of downstream changes in cellular activity, such as

 
22  
 
changes in gene expression (Clark et al., 1992; Lee et al., 2008; LeHoux & Lefebvre,

2006). Although the majority of studies examining AT1R-induced p44/42 MAPK

activation were performed in vitro, p44/42 MAPK activation also occurs in the SFO and

PVN after a systemic AngII injection (Wei et al., 2009). As these AT1R signaling

molecules were detected in the brain, researchers have begun to investigate their role in

the neural actions of AngII.

A previous study employed signaling selective AT1R ligands and inhibitors to

reveal that the inositol triphosphate (IP3) branch of AT1R signal transduction mediates

thirst, whereas the p44/42 MAPK signaling pathway mediates sodium appetite (Daniels

D, Mietlicki EG, Nowak EL, Fluharty SJ, 2009; Daniels D, Yee DK, Faulconbridge LF,

Fluharty SJ, 2005). This finding was novel and intriguing; however, it remained unclear

whether the result reflected a normal physiological process or a pharmacological

phenomenon based on exogenously administered AngII. Therefore, to establish the

physiological significance of AngII-induced p44/42 MAPK on sodium appetite, we

exploited a preparation for endogenous AngII-induced sodium appetite (Thunhorst &

Johnson, 1994). More specifically, a combined treatment of furosemide and a low dose

of captopril (furo/cap), a loop diuretic and an inhibitor of angiotensin converting enzyme

(ACE) inhibitor, respectively, elevates brain levels of AngII. The low dose of captopril

blocks the conversion of diuresis-provoked angiotensin I to AngII in the periphery, but

still allows conversion in circumventricular organs, which contain very high levels of ACE

in comparison to the periphery (Chai, Allen, Adam, & Mendelsohn, 1986). The high

level of endogenous AngII production in the brain elicits rapid and robust thirst and

sodium ingestion (Thunhorst, Morris, & Johnson, 1994). We used this model to test the

hypothesis that p44/42 MAPK phosphorylation mediates sodium, but not water, ingestion

 
23  
 
induced by endogenous AngII production.

Given the myriad actions of AngII in the brain, it is important to consider the

signaling pathways involved in other AngII-induced effects, such as neuroendocrine

responses. In addition to its effects on water and sodium ingestion, central AngII is a

potent stimulus for AVP and OT secretion (Andersson, Eriksson, Fernandez, Kolmodin,

& Oltner, 1972). While AVP promotes renal water reabsorption, OT increases renal

sodium excretion and centrally inhibits sodium appetite (Conrad, Gellai, North, & Valtin,

1993; Ganong, 1977; Stricker & Verbalis, 1996). AT1R-expressing neurones in the SFO

send axonal projections to the PVN and SON, where AVP and OT neuronal activity is

increased to promote hormone release from the neurohypophysis (McKinley et al.,

1998). We addressed the role of p44/42 MAPK signaling in AngII-induced AVP and OT

by measuring plasma levels of the neurohypophysial hormones after either furo/cap

treatment or icv AngII in the presence of an AT1R antagonist or MEK inhibitor. Overall,

our experiments better define the participation of p44/42 MAPK signaling in the

regulation of fluid balance.

MATERIALS AND METHODS

Animals

Adult male Sprague-Dawley rats (n = 83) that weighed between 225-250 g were

obtained from Charles River Laboratories (Wilmington, MA, USA). Rats were pair-

housed in plastic tubs with standard bedding and with food and water available ad

libitum, except during experimental procedures. The temperature in the colony was

maintained at 22 °C with a 12:12 h reversed light/dark cycle. Animals were allowed at

 
24  
 
least one week to acclimate to the colony before any procedures were performed. The

Institutional Animal Care and Use Committee of the University of Pennsylvania approved

all procedures with animals.

Drugs

Furosemide (Abbott Laboratories, N. Chicago, IL) and captopril (SQ-14225,

Bristol-Meyers-Squibb Pharmaceutical Research Institute, Princeton, NJ) were

administered subcutaneously (sc) at doses of 10 mg/kg and 5 mg/kg body weight,

respectively, in 0.9% sterile saline. Equal volume 0.9% sterile saline was administered in

control animals. The specific doses for each drug given intracerebroventricularly (icv)

were as follows: 20 ng AngII (Bachem, King of Prussia, PA), 0.4 ug irbesartan (a

generous gift from Dr. Sal Lucania, Bristol Myers Squibb), 2 nmol U0126 dissolved in

10% DMSO (Promega, Madison, WI). Artificial cerebrospinal fluid (aCSF; R&D Systems,

Minneapolis, MN) plus 10% DMSO was administered icv as a vehicle control. All

injections were administered icv in a volume of 2 ul.

Surgeries

Surgeries were performed under aseptic conditions. Animals were anaesthetised

with a combination of ketamine and xylazine (70 and 5 mg/kg, respectively,

intraperitoneally [ip]) before being fixed in a stereotaxic frame and implanted with 26-

gauge guide cannulae (Plastics One, Roanoke, VA, USA) aimed at the lateral ventricle.

The coordinates (0.48 mm caudal to bregma, 1.6 mm from mid-line and 4.2 mm ventral

to dura mater) were chosen to allow an internal injection cannula to extend beyond the
 
25  
 
guide cannula into the ventricular space. The cannulae were fixed in place with dental

cement and bone screws, and the animals were allowed at least five days to recover

before verification procedures were performed. After surgery, animals were injected with

yohimbine (0.11 mg/kg, Ben Venue Laboratories Bedford, OH) and orally administered

Children’s Tylenol (30 mg/ml, McNeil PPC Inc, Fort Washington, PA). Upon awakening,

animals were singly housed.

Five days after surgery and prior to undergoing experimental treatments, animals

were tested for correct cannula placement and patency. They were given an injection of

20 ng of AngII diluted in aCSF icv via a Hamilton syringe connected with PE-10 tubing to

an injector that terminated 1 mm beyond the guide cannula. Animals were excluded

from the experiment if they failed to demonstrate a drinking response in less than 30

seconds, consuming at least 3 ml of water, in two separate AngII challenges. Animals

began testing three days after the icv test injections.

Enzyme Linked Immunosorbent Assays

Experiment 1. Animals were pretreated with either vehicle, irbesartan, or

U0126 icv. Fifteen minutes after the icv injection, they were given furosemide, sc,

followed 10 minutes later by captopril, sc. Thirty minutes after the captopril injection,

animals were rapidly decapitated without anaesthesia and the brains were flash frozen in

hexane over dry ice. Rats were not administered anaesthesia because it is known to

increase phosphorylated p44/42 MAPK levels, which would compromise our results

(Khan & Watts, 2004). Micropunches (1 mm diameter) of the brain regions of interest

(OVLT, SFO, PVN, SON, and primary somatosensory cortex) were obtained from 300-

 
26  
 
µm sections cut on a cryostat. OVLT/SFO and PVN/SON punches were combined from

each rat to ensure detectable levels for analysis. The micropunched tissue samples

were immersed in the lysis buffer provided in the Pathscan p44/42 MAPK Sandwich

ELISA kit (Cell Signaling Technology, Danvers, MA) plus 1 mM

phenylmethanesulfonylfluoride (Sigma, St Louis, MO). The brain tissue samples were

sonicated three times for one second (incubated on ice between bursts) followed by

centrifugation at 14,000 rpm at 4°C for 10 minutes. Supernatant was collected and a

protein assay was performed on five microliters of each sample. Based on the protein

levels detected by Bicinchoninic Acid Assay, appropriate amounts of lysis buffer,

sample, and sample diluent were mixed for each sample. This mixture (100 µl) was

loaded into corresponding wells in both the Total and Phospho p44/42 MAPK Sandwich

ELISA kits. The ELISA was performed according to the protocol provided by each kit.

Behavioral Study

For behavioral experiments, rats were removed from their home cages

approximately one hour before the onset of the dark phase. The rats were weighed and

then placed in individual wire mesh-bottomed cage equipped with a funnel and

calibrated tube for the collection of urine, and with two 25-ml bottles available for

drinking tap water and 1.5% saline, each marked with 0.2 ml graduations. The rats were

allowed to acclimate for one hour before the experiment began. After the last drug

treatment was given, water, saline, and urine levels were measured at 15 min intervals

for the first hour and then at 30 min intervals for the remaining time.

Experiment 2A. The goal of the first behavioral experiment was to examine the

 
27  
 
role of p44/42 MAPK activation in mediating the effects of endogenous AngII on water

and sodium intake. The animals were assigned to one of four treatments in a crossover

design, which allowed each rat to receive all treatment conditions over the course of the

study without all the animals receiving the drug combinations in the same order. The

control condition was two vehicle injections (0.9% saline, sc) with a 10-min interval

between them. All other treatments included injections of furosemide and, 10 min later,

captopril. Icv injections were given 15 minutes before the furosemide injection. Fluid

ingestion was measured for three hours when animals were given furo/cap or saline.

Based on this initial study, it was noted that the induced ingestion mainly occurred within

the first 90 minutes; therefore, subsequent studies used this time course. To assess the

role of AT1R, animals were treated with irbesartan icv, and to assess the role of p44/42

MAPK, they were injected with U0126 icv. Water and 1.5% saline intakes as well as

urine output, were measured for 90 minutes.

Experiment 2B. The goal of the second behavioral experiment was to examine

the role of p44/42 MAPK activation on water intake in the absence of sodium ingestion.

The animals were treated with either vehicle or U0126 icv followed 15 minutes later by

furosemide, and 10 minutes later by captopril in a crossover design. Rather than being

presented with two bottles, as in the first experiment, animals were presented with a

single bottle containing tap water. Water intake was monitored for the next 90 minutes.

AVP and OT Secretion

Experiment 3A. Rats were pretreated centrally with either vehicle, irbesartan, or

U0126, followed by either vehicle or furo/cap, sc as described above. Thirty minutes

 
28  
 
after the captopril injection rats were rapidly decapitated and trunk blood was collected

into 10 ml BD Vacutainer blood collection heparinised glass tubes on ice (Franklin

Lakes, NJ). The thirty-minute time point was chosen based on the emergence of

diuresis and drinking behavior in our initial furo/cap studies. Additionally, the ELISA

study showed that U0126 effectively inhibited p44/42 MAPK activation at this time point.

Previous research examined a ninety-minute time point after furo/cap treatment, but did

not observe increases in oxytocin (Thunhorst et al., 1994). The tubes then were

centrifuged at 4oC at 1300 relative centrifugal force (rcf) for 10 min. The plasma

supernatant was extracted with acetone and petroleum ether. Plasma levels of AVP and

OT were measured with a specific radioimmunoassay performed by the laboratory of Dr.

Joseph G. Verbalis, as described previously (Verbalis, McHale, Gardiner, & Stricker,

1986). The standard curve for each peptide was linear between 0.5 and 10.0 pg per

tube with the use of a synthetic AVP and between 0.25 and 5 µU per tube with the OT

standard (Bachem Americas, Inc, Torrance, California). The minimum detectable

concentration of AVP or OT in extracted plasma was 0.5 pg/ml and 0.25 µU/ml,

respectively. The AVP antiserum (R-4) displayed < 1% cross-reactivity with OT, and the

OT antiserum exhibited < 1% cross-reactivity with AVP.

Experiment 3B. Rats were assigned to one of the following pretreatment

groups: vehicle or U0126 (1mM) followed 15 minutes later by AngII (20 ng) icv. Two or

fifteen minutes after the last icv injection, rats were rapidly decapitated and their trunk

blood was collected. The two- and fifteen-minute time points were based on the

emergence of drinking behavior in previous exogenous AngII studies and selected to

establish the range of neurohypophysial hormone levels in the plasma after the

treatments. Previous studies used a 90-second time point and observed increases in

 
29  
 
plasma vasopressin (Gohlke et al., 2002). The protocol described in Experiment 3A was

then followed to measure AVP and OT levels.

Statistical Analysis

Data are presented as the mean ± the standard error of the mean. For the

behavioral studies, comparisons were made between the treatment conditions and over

time using repeated measure analysis of variance (two-way ANOVA; time point x

treatment). For the p44/42 MAPK ELISA and AVP/OT radioimmunoassay results,

comparisons were made between the treatment groups (one-way ANOVA). When

warranted, planned comparison post-hoc t-tests were performed. All hypothesis tests

used a=0.05 as the criterion level of significance. Statistical analyses were conducted

using Prism 2.0 software (La Jolla, CA).

RESULTS

Furo/cap treatment activates p44/42 MAPK in the brain

In Experiment 1, rat brain punches were collected from coronal slices for several

brain regions, namely, OVLT, SFO, PVN, SON and primary somatosensory cortex as a

control brain region (depicted in Figure 2.1). As shown in Figure 2.2A, total p44/42

MAPK levels between all of the treatment groups in the OVLT/SFO were not different

(ANOVA F (5,19) = 0.5, p>0.05). However, p44/42 MAPK phosphorylation in the

OVLT/SFO differed between treatment groups (ANOVA (F (5,19) = 5.9, p<0.01). More

specifically, furo/cap treatment increased phospho p44/42 absorbance levels compared

 
30  
 
to control (1.6 ± 0.0 vs 1.1 ± 0.1, p<0.001). Furo/cap treatment paired with icv AT1R

antagonist irbesartan or MEK inhibitor U0126 significantly reduced absorbance levels

compared to furo/cap with vehicle pretreatment (1.1 ± 0.1 vs 1.1 ± 0.0 vs 1.6 ± 0.0,

respectively; p<0.01).

Likewise, Figure 2.2B illustrates that total p44/42 MAPK levels were equivalent

for all treatment groups in the PVN/SON (ANOVA F (5,19) = 0.2, p>0.05). However,

p44/42 MAPK phosphorylation in the PVN/SON of the hypothalamus differed between

treatment groups (ANOVA F (5,19) = 10.4, p<0.001). In particular, furo/cap treatment

augmented phospho p44/42 absorbance levels compared to control (1.7 ± 0.1 vs 1.1 ±

0.0, p<0.001). Pretreatment with either icv irbesartan or U0126 reduced absorbance

levels of phospho p44/42 MAPK compared to furo/cap with vehicle pretreatment (1.2 ±

0.1 vs 1.0 ± 0.1 and 1.7 ± 0.1, respectively; p<0.001).

As shown in Figure 2.2C, the control brain region S1 had similar levels of p44/42

total MAPK regardless of treatment group (ANOVA (F (5,19) = 0.4, P>0.05). Unlike

OVLT/SFO and PVN/SON, S1 did not show an increase in p44/42 MAPK

phosphorylation in response to furo/cap treatment (ANOVA F (5,19) = 0.1, P>0.05).

The role of p44/42 MAPK in furo/cap-induced water and sodium ingestion

Consistent with previous work (Thunhorst et al., 1994), the furo/cap protocol

produced a rapid diuresis and prompt water and sodium ingestion (as shown in Figure

2.3). A two-way ANOVA established a strong main effect of furo/cap treatment on urine

output (F (1, 112) = 72.4, p<0.0001). Animals that received furo/cap treatment excreted

 
31  
 
significantly more urine during the three hours after treatment than controls (13.6 ± 1.1

versus 2.9 ± 0.5 ml, p<0.001). This increase in urine output was first significant within 15

minutes of treatment (p<0.01). These animals also drank water robustly in response to

the furo/cap treatment compared with controls (F (1,112) = 23.5, p<0.001; 7.8 ± 1.4

versus 0.8 ± 0.2 ml, p<0.001). In addition, furo/cap-treated rats consumed substantial

amounts of 1.5% NaCl solution compared with controls during this three hour time period

(F (1,112) = 9.0, p<0.01; 5.7 ± 1.5 versus 0.7 ± 0.2 ml, p<0.001). The time point at

which the furo/cap group intake became significantly elevated compared with the control

group was 45 min for water intake (p<0.01) and 60 min for sodium intake (p<0.05) after

the captopril injection.

Experiment 2A verified that furo/cap-induced water and sodium ingestion could

be attributed, at least in part, to AT1Rs. In particular, rats were pretreated centrally with

the AT1R antagonist irbesartan (200 ng/ul) 15 min before furo/cap treatment (Hines,

Fluharty, & Yee, 2003) A two-way ANOVA established a main effect of irbesartan on

furo/cap induced water intake compared to furo/cap controls (F (1,275) = 11.2, p<0.01).

A similar main effect occurred with irbesartan treatment and furo/cap-induced sodium

intake (F (1,270) = 9.2, p<0.01). As shown in Figure 2.4A, irbesartan pretreatment

decreased furo/cap-induced water intake by 50% over 90 minutes (5.8 ± 0.6 versus 2.9

± 0.5 ml, p<0.001), first becoming significant at 45 minutes (p<0.001). Sodium intake

was reduced by 56% at 90 minutes (4.6 ± 0.6 versus 2.0 ± 0.5 ml, p<0.001), first

becoming significantly attenuated at 45 minutes (p<0.01). This level of suppression of

furo/cap-induced water and sodium intake by an AT1R antagonist is consistent with

previous findings (Thunhorst et al., 1994).

Next, we investigated whether furo/cap-induced water and sodium ingestion

 
32  
 
could be attributed, at least in part, to p44/42 MAPK signaling. In particular, rats were

pretreated centrally with the MEK inhibitor U0126 15 min before the furo/cap treatment.

The dose of U0126 was chosen based on previous efficacy (Daniels D, Mietlicki EG,

Nowak EL, Fluharty SJ, 2009), and was validated in our Experiment 1. A two-way

ANOVA did not reveal a main effect of U0126 on furo/cap-induced water intake

compared to controls (F (1,275) = 2.5, p = 0.1). However, there was a significant

interaction between treatment and time (p<0.01). U0126 decreased furo/cap-induced

water intake by 30% at 90 minutes (5.8 ± 0.6 versus 4.1 ± 0.5 ml, p< 0.01), as shown in

Figure 2.4A. A two-way ANOVA revealed a main effect of U0126 on furo/cap-induced

sodium intake (F (1,280) = 13.0, p<0.001). U0126 reduced sodium intake by 60% (4.5

± 0.6 versus 1.8 ± 0.5 ml, p < 0.001), first becoming significant at 30 min (p<0.05). Urine

volume measurement confirmed that neither irbesartan nor U0126 pretreatment the

interfered with the diuretic effectiveness of the furo/cap treatment (12.3 ± 0.9 versus 12.4

± 1.4 versus 14.0 ml, respectively; data not shown).

Previous work that suggested that p44/42 MAPK signaling mediates AngII-

induced sodium appetite but not thirst; however, we observed a reduction in water intake

in the U0126-treated animals, albeit not until the 90-min time point. We reasoned that

this delayed suppression of water intake was an artifact of the two-bottle test, given that

consumption of hypertonic saline would provide an osmotic stimulus for water intake.

Thus, as U0126 suppresses sodium ingestion, it may indirectly effect to reduce water

intake. This interpretation of the data is supported by the fact that U0126 suppressed

sodium intake at 30 min, and subsequently reduced water intake at 90 min. To test this

hypothesis, the effect of U0126 was examined in a single-bottle test. In this case, a two-

way ANOVA revealed that water intake was not significantly decreased with U0126

 
33  
 
treatment (F (1,105) = 1.2, p = 0.3), compared with animals only treated with furo/cap.

Furo/cap versus furo/cap + U0126 groups drank 4.3 ± 0.8 versus 5.0 ± 0.9 ml,

respectively, at the 90-minute time point (Figure 2.4B).

The role of p44/42 MAPK in furo/cap- and AngII-induced AVP and OT release

Experiment 3A investigated whether or not p44/42 MAPK signaling mediated

furo/cap-induced AVP and OT secretion. Rats were pretreated with vehicle, irbesartan,

or U1026 icv paired with either vehicle or furo/cap sc. Thirty minutes after captopril

treatment, plasma AVP levels differed between treatment groups (Figure 2.5; ANOVA (F

(5,19) = 6.2, P<0.01). Post hoc analysis revealed that furo/cap stimulated AVP

compared to vehicle treatment (1.3 ± 0.2 vs 0.6 ± 0.1 pg/ml, p<0.05). However, when

animals were pretreated centrally with irbesartan or U0126, furo/cap-induced AVP

secretion was not significantly reduced compared with vehicle pretreatment (1.4 ± 0.1 vs

1.7 ± 0.3 vs 1.3 ± 0.2 pg/ml, p>0.05). Similarly, OT levels differed between groups thirty

minutes after captopril treatment (ANOVA (F (5,19) = 3.3, P<0.05). Post hoc evaluation

indicated that furo/cap stimulated OT compared to control (5.6 ± 1.0 vs 1.8 ± 0.5 pg/ml,

p<0.05). However, furo/cap treatment paired with irbesartan or U0126 centrally did not

reduce OT levels compared to furo/cap with vehicle pretreatment (6.0 ± 1.6 vs 4.7 ± 0.9

vs 5.6 ± 1.0 pg/ml, p>0.05).

In Experiment 3B, rats were pretreated centrally with either vehicle or U0126

followed by AngII (20 ng). Two or fifteen minutes after treatment, plasma AVP differed

between the groups (Figure 2.6; ANOVA (F (2,38) = 5.6, p<0.01). Given that there were

no significant differences between the 2 and 15 min time points within each treatment
 
34  
 
group, these data were combined. Post-hoc analysis indicated that AngII treatment

stimulated AVP secretion compared with vehicle treatment (2.5 ± 0.3 versus 1.5 ± 0.2

pg/ml, p<0.01). This level of AngII-induced AVP secretion is consistent with previous

findings (Reis, Saad, Camargo, Elias, & Antunes-Rodrigues, 2010). However, when

animals were pretreated with U0126, the AngII-induced AVP secretion was not

significantly different than when AngII was given alone (2.3 ± 0.2 versus 2.5 ± 0.3 pg/ml,

p = 0.7). Likewise, plasma OT levels was increased 2 and 15 min after AngII treatment

compared with vehicle (Figure 2.6; ANOVA F (2,35) = 5.3, p<0.01). Post hoc analyses

revealed that AngII treatment stimulated OT secretion compared with vehicle treatment

at levels congruous with previous findings (14.2 ± 2.8 versus 6.0 ± 1.3 pg/ml, p<0.01)

(Reis et al., 2010). However, AngII-induced OT secretion was not diminished by U0126

pretreatment (14.2 ± 2.8 versus 13.5 ± 2.4 pg/ml, p = 0.9).

DISCUSSION

The goal of our experiments was to uncover the role of p44/42 MAPK in both

behavioral and physiological actions of central AngII. Experiment 1 demonstrated

p44/42 MAPK activation in certain brain regions after endogenous AngII production,

which was inhibited by an AT1R antagonist. Experiment 2 established that endogenous

AngII production promotes sodium appetite, but not thirst, via p44/42 MAPK activation.

Experiment 3 found that p44/42 MAPK activation does not mediate furo/cap- or AngII-

induced AVP and OT secretion, underscoring the selective role of p44/42 MAPK in

AngII-induced sodium appetite.

 
35  
 
 While previous findings have demonstrated the part played by p44/42 MAPK in

AngII-induced sodium appetite, our studies confirm and extend these results (Daniels D,

Yee DK, Faulconbridge LF, Fluharty SJ, 2005). For example, although studies using

exogenous AngII-induced sodium appetite demonstrated the stimulation of p44/42

MAPK activation by AngII, they did not show its reduction by MEK inhibitor U0126 in

relevant brain regions (Daniels D, Mietlicki EG, Nowak EL, Fluharty SJ, 2009; Daniels D,

Yee DK, Faulconbridge LF, Fluharty SJ, 2005). The present studies demonstrate that

furo/cap induces p44/42 MAPK activation in relevant forebrain areas, and that our doses

of central irbesartan and U0126 restore phospho p44/42 MAPK to control levels. The

furo/cap treatment produces high levels of circulating renin and angiotensin I, and

central AT1R appears to mediate a large proportion of the observed water and sodium

ingestion (Fitzsimons & Stricker, 1971; Thunhorst et al., 1994). The present results

further support the notion that AT1R-p44/42 MAPK activation is responsible for the

increase in sodium appetite observed in our experiments, and that the reduction of the

behavior by U0126 is due to decreased p44/42 MAPK activation in relevant AT1R

containing brain regions. Moreover, our studies provided an additional control to

examine the unique role of p44/42 MAPK in sodium appetite through use of a single

water bottle test.

Nevertheless, our studies present their own limitations. For example, AngII

levels in the brain may be unusually elevated by the peripheral blockade of angiotensin I

conversion using the furo/cap model. However, in this protocol AngII production is

constrained by physiological availability of the prohormone (Thunhorst et al., 1994).

Another consideration is that furo/cap treatment may activate other interoceptive

systems apart from the renin-angiotensin-aldosterone system. For example, low- and

 
36  
 
high-pressure baroreceptors would detect the diuresis-induced loss of blood volume and

pressure, respectively, and relay this information to brainstem structures that may

promote thirst, sodium appetite, or both (Thunhorst & Johnson, 1994). Such

baroreceptor-mediated signals may explain the partial effect of irbesartan pretreatment

on water and sodium intake, and its ineffectiveness on AVP and OT secretion. Another

limitation of our studies is the combining of brain regions for the p44/42 MAPK ELISA.

An immunohistochemical approach would allow for better localization of activated p44/42

MAPK. However, the objectivity, reproducibility, and semi-quantitative nature of ELISAs

offer a clear advantage over the challenges of quantifying immunohistochemistry.

Lastly, the icv injections used in our studies are not as spatially discrete as parenchymal

injections. Future studies using local microinjections will be informative to pinpoint the

contribution of parallel signaling pathways in specific brain regions.

While demonstrating that AT1R-MAPK mediates sodium appetite, our studies

indicate that water intake and the secretion of AVP and OT do not require p44/42 MAPK

activation. Circulating AngII accesses AT1R located on neurones in the

circumventricular organs, such as the SFO and the OVLT (Geerling & Loewy, 2008)

whose neurones project to the magnocellular neurones in the PVN and SON, which also

express AT1R (McKinley et al., 1998; Wei et al., 2009). Centrally administered AngII

induces c-Fos expression in the PVN and SON and increases the firing rate of AVP and

OT neurones (Renaud & Bourque, 1991; Roesch et al., 2001) resulting in plasma levels

of these hormones being elevated within minutes after AngII injection (Keil, Rosella-

Dampman, Emmert, Chee, & Summy-Long, 1984). These hormones contribute to fluid

balance, with AVP prompting water retention and OT stimulating natriuresis (Conrad et

al., 1993; Ganong, 1977). In furo/cap-treated animals, the AT1R antagonist irbesartan

 
37  
 
does not reduce AVP and OT secretion, which suggests that other mechanisms, such as

baroreceptors, trigger neurohypophysial release in this circumstance. Experiment 3B

demonstrates that even when AngII is centrally administered to prompt

neurohypophysial release, p44/42 MAPK activation is not required. However, it is

possible that a role for AT1R and p44/42 MAPK in furo/cap-induced AVP and OT

release could be found at different time points. At present, it seems as though acute

AVP and OT secretion must rely on other signaling pathways, such as IP3-mediated

ionotropic events, or genotropic or structural changes mediated by other MAPK family

members.

The present study focused on a subset of the MAPK family, namely p44/42. The

role of other MAP kinases in the central effects of AngII remains unclear. It is possible

that p38 MAPK and/or JNK play a role in mediating the central actions of AngII not

blocked by the p44/42 MAPK inhibitor. Additionally, as shown in previous studies, it is

possible for some MAP kinases, but not others, to be involved in mediating central AngII

effects, such as AT1R upregulation (Wei et al., 2009). It would be important for future

studies to investigate the role of other MAP kinases in drinking behavior and

neurohypophysial release.

Studying the signaling molecules involved in the central actions of AngII is the

first step to understanding how central AngII exerts its physiological effects, but much

remains to be uncovered about the cellular mechanisms by which they mediate these

actions. For example, there are several plausible mechanisms by which p44/42 MAPK

may mediate sodium appetite in the brain. One possibility is that AngII-MAPK signaling

elicits sodium appetite by changing neuronal excitability in circumventricular neurones or

forebrain integration sites that project to motor generators involved in salt ingestive
 
38  
 
behavior (Geerling & Loewy, 2008; Schrader et al., 2006). This rapid effect on synaptic

transmission could explain the relatively fast time course of furo/cap-induced sodium

appetite (Numakawa et al., 2002). Alternatively, p44/42 MAPK may be involved in long-

term gene transcription and protein synthesis for sodium and potassium channel

subunits or AT1R. For example, MAPK up-regulates AT1R in the SFO and PVN, and

increased levels of AngII binding to its receptor may explain the robust sodium appetite,

although not the lack of p44/42 MAPK-mediated water ingestion (Wei et al., 2009).

Other downstream consequences of p44/42 MAPK activation include rearrangements of

the cytoskeleton, which could include structural neural plasticity (Olsen, Reszka, &

Abraham, 1998; Schenk et al., 2005). This mechanism is highlighted in a study by

Swank and Sweatt, which found that p44/42 MAPK activation is important for plasticity in

taste learning (Swank & Sweatt, 2001). Other research has demonstrated that sodium

appetite is associated with dendritic growth and the development of dendritic spines in

the nucleus accumbens (Roitman, Na, Anderson, Jones, & Bernstein, 2002). p44/42

MAPK activation may underlie strengthened connections within this brain region

associated with incentive sensitization, which would explain the sudden avidity for

sodium. It will be imperative for future studies to discern what downstream

consequences of p44/42 MAPK signaling contribute to AngII-induced sodium appetite,

keeping in mind there may be several parallel mechanisms.

Extending beyond drinking behaviors and neurohypophysial secretion, central

AngII has a multitude of actions in the brain. For example, AngII causes sympathetic

stimulation, hypothalamic-pituitary-adrenal activation, increased sympathetic tone, and

ultimately an increase in mean arterial pressure (Peach, 1977). Other investigators have

begun to uncover the AT1R signaling mediating these actions. Specifically, Wei et al

 
39  
 
have shown that p44/42 MAPK mediates the effect of central AngII to increase

sympathetic nerve activity in heart failure, another syndrome adversely affected by salt

intake (Wei, Yu, Zhang, Weiss, & Felder, 2008). An important avenue for future

investigation is to determine the locus within the network of AngII-modulated neural

circuitry wherein sodium appetite and sympathetic activity share a common dependence

on p44/42 MAPK activation. Another key question to address is the effect of elevated

p44/42 MAPK activity in specific brain regions on the many central actions of AngII.

In summary, these studies provide new evidence that the diverse effects of

central AngII rely on divergent signaling mechanisms. More specifically, we found that

sodium appetite requires p44/42 MAPK activation, whereas thirst and neurohypophysial

hormone secretion do not. It will be important for future studies to assess the role of

both p44/42 MAPK and other MAPK kinases in other central AT1R actions that remain

undefined, such as hypothalamic-pituitary-adrenal activation. Moreover, it will be critical

to find the downstream targets of MAPK and determine the cellular mechanism of AngII

in exerting these central effects.

PERSPECTIVES

Elements of the renin-angiotensin-aldosterone system are frequently implicated

in the pathogenesis of hypertension, although the cause of hypertension is often

unknown (Marc & Llorens-Cortes, 2011). Hypertension is a common and serious health

concern (Karppanen & Mervaala, 2006). One in three adults have chronically elevated

blood pressure, which shortens a person’s life span by three to five years (Del Giudice,

Pompa, & Aucella, 2010). The identification of signaling-selective subsets of AngII

 
40  
 
action in the brain presents an opportunity to more precisely target central pathways that

regulate blood pressure (Felder, Yu, Zhang, & Wei, 2009; Margolius & Bodenheimer,

2010)

ACKNOWLEDGEMENTS
 

This research was supported by R01HL091314. Preliminary results were reported at the

Society for Neuroscience meeting (San Diego, 2010) and the World Congress for

Neurohypophysial Hormones (Boston, 2011). We gratefully acknowledge the dedicated

assistance of Klil Babin. We also greatly appreciate the expertise of Drs. Joseph

Verbalis and Qin Xu at Georgetown University in conducting radioimmunoassays for

arginine vasopressin and oxytocin.

 
41  
 
 Figure 2.1. Examining Furo/Cap-MAPK
activation in the brain.

Drawing of a coronal hemi section of rat

brain depicting the brain regions of interest
for p44/42 MAPK activation assays. Circles
within each brain region represent 1 mm
micropunches collected for the p44/42
MAPK ELISAs. The brain regions examined
include circumventricular organs OVLT and
SFO, as well as the PVN and SON of the
hypothalamus. Additionally, the S1 was
collected as a control brain region to assay
P44/42 MAPK activation. Abbreviations: S1
= primary somatosensory cortex, CPu =
caudate putamen, MS = medial septal
nucleus, OVLT = organum vasculosum of
the lateral terminalis, SFO = subfornical
organ, fi = fimbria of the hippocampus, ic =
internal capsule, PVN = paraventricular
nucleus of the hypothalamus, SON =
supraoptic nucleus of the hypothalamus.

 
42  
 
Figure 2.2. Furo/Cap activates MAPK in key brain areas; this is reduced by an AT1R and
MEK inhibitor.

Bar graphs illustrating the levels of total p44/42 MAPK and phospho p44/42 MAPK in the
OVLT/SFO (panel A), PVN/SON (panel B), and S1 (panel C) after either vehicle or furo/cap
treatment sc and either vehicle, irbesartan, or U0126 administered icv (n = 3-5/group). Total
p44/42 MAPK levels do not change, whereas furo/cap treatment increases phosphorylated
p44/42 MAPK in the OVLT/SFO and PVN/SON, but not in S1. Both irbesartan and U0126 prevent
this furo/cap induced increase in p44/42 MAPK phosphorylation. Abbreviations: OVLT = organum
vasculosum of the lateral terminalis, SFO = subfornical organ, PVN = paraventricular nucleus of
the hypothalamus, SON = supraoptic nucleus of the hypothalamus, S1 = primary somatosensory
cortex.

 
43  
 
Figure 2.3. Furo/Cap increases urine output as well as water and sodium intake.

Line graphs illustrating the effect of furo/cap treatment on urine volume (panel A) and water and
sodium intake during a two-bottle test (panel B). On the x axis, zero minutes represents the time
of captopril injection. Compared with vehicle treatment, rats given furo/cap treatment excreted
significantly more urine within 15 minutes (n=16). Furo/Cap also induced a robust intake of water
and 1.5% sodium within 45 minutes and 60 minutes of treatment, respectively. Asterisks indicate
that p<0.01 when comparing Furo/Cap to vehicle treatment.

 
44  
 
Figure 2.4. Furo/Cap sodium intake but not water intake is mediated by MAPK.

Line graphs illustrating the effect of blocking AT1R receptors and MAPK activation after furo/cap
treatment in a two bottle test (panel A) and in a one bottle test (panel B). On the x axis, zero
minutes represents the time of captopril injection. In the two bottle test, compared with Furo/Cap
+ Vehicle treatment, rats given the Furo/Cap + AT1R antagonist irbesartan treatment consumed
significantly less water within 45 minutes after treatment (n=28). Irbesartan also reduced the
intake of 1.5% saline within 45 minutes of treatment. Compared with Furo/Cap + vehicle
treatment, rats given Furo/Cap + the MEK inhibitor U0126 treatment consumed significantly less
water within 90 minutes after treatment and significantly less saline within 30 minutes of treatment
(n=28). In the one bottle test, compared with vehicle treated, rats given the MEK inhibitor U0126
treatment consumed similar amounts of water (n=12). Asterisks indicate that p<0.05 when
comparing to Furo/Cap + Veh treatment. Stats for Furo/Cap + Veh compared to Veh are shown in
Figure 2.3.

 
45  
 
 Figure 2.5. Furo/Cap-induced
Vasopressin and Oxytocin release is
not mediated by MAPK.

Bar graphs illustrating the role of p44/42

MAPK activation on Furo/Cap-induced
neurohypophysial secretion. Plasma AVP
(Panel A) and OT (Panel B) after vehicle
or Furo/Cap treatment combined with
either vehicle, irbesartan, or U0126
administered icv (n= 3-5/group).
Furo/Cap-induced AVP and OT secretion
was not diminished with either the AT1R
antagonist irbesartan or the MEK inhibitor
U0126. Abbreviations: AVP = arginine
vasopressin, OT = oxytocin.

Fig 2.6. AngII-induced Vasopressin and Oxytocin

Release is not mediated by MAPK.

Bar graphs illustrating the effect of inhibiting p44/42

MAPK activation on AngII-induced neurohypophysial
hormone secretion. Plasma AVP (Panel A) and OT
(Panel B) levels two and fifteen minutes after icv Veh,
AngII, or U0126 plus AngII (n = 10-16/group). AngII-
induced AVP and OT levels were not inhibited with
the MEK inhibitor U0126. Abbreviations: AVP =
arginine vasopressin, OT = oxytocin.

 
46  
 
CHAPTER 3: THE ROLE OF THE HYPOTHALAMIC PARAVENTRICULAR NUCLEUS
AND THE ORGANUM VASCULOSUM LATERAL TERMINALIS IN THE CONTROL OF
SODIUM APPETITE IN MALE RATS

Laura A. Grafe1, Annie Takacs2, Daniel K. Yee3, and Loretta M. Flanagan-Cato1,2,4

Neuroscience Graduate Group1, Departments of Psychology2 and Animal Biology3

and the Mahoney Institute of Neurological Sciences4, University of Pennsylvania,

Philadelphia Pennsylvania, USA 19104

Abbreviated title: Hypothalamic control of sodium appetite

Key words: Aldosterone; Angiotensin; Oxytocin; Receptor Signaling; Sodium Appetite

This work is in revision for resubmission to the Journal of Neuroscience.

 
47  
 
SUMMARY

Angiotensin II (AngII) and aldosterone cooperate centrally to produce a robust

sodium appetite. The intracellular signaling and circuitry that underlie this interaction

remain unspecified. Male rats pretreated with deoxycorticosterone (DOC; a synthetic

precursor of aldosterone) followed by central injections of AngII exhibited a marked

sodium intake, as classically described. Disruption of inositol trisphosphate (IP3)

signaling, but not extracellular-regulated receptor kinase 1 and 2 (ERK1/2) signaling,

prevented the cooperativity of DOC and AngII on sodium intake. The pattern of

expression of the immediate early gene product cFos was used to identify key brain

regions that may underlie this behavior. DOC pretreatment augmented AngII-induced

cFos expression in the organum vasculosum lateral terminalis (OVLT) and conversely

dampened cFos induction in the paraventricular nuclei (PVN) of the hypothalamus.

Finally, to explore the possible link between activity in the PVN and the OVLT, action

potentials in the PVN were inhibited with intraparenchymal lidocaine injections. As

predicted, this treatment blocked AngII-induced cFos expression in the PVN and

exaggerated AngII-induced sodium ingestion. Intriguingly, this treatment also increased

the number of neurons in the OVLT expressing cFos. Collectively, these results suggest

that the behavioral cooperativity between DOC and AngII involves the alleviation of an

inhibitory signal relayed from the PVN to the OVLT.

 
48  
 
INTRODUCTION
 

The hormones angiotensin (AngII) and aldosterone are key defenders of

electrolyte balance, blood volume, and blood pressure (Peach, 1977). In the event of

sodium depletion, hypovolemia, and/or hypotension, AngII and aldosterone are

produced, which engage homeostatic responses, including a prodigious drive for sodium

consumption (Ganong, 1977). Steady progress has been made in revealing the

independent natriorexic effects of AngII and aldosterone; however, the mechanism for

the cooperative actions of AngII and aldosterone to promote a greater than additive

effect on sodium ingestion remains unclear.

Humoral AngII and aldosterone appear to have separate initial targets in the

brain. Circulating AngII binds to Angiotensin Type 1 Receptors (AT1R) in the subfornical

organ (SFO) and organum vasculosum lateral terminalis (OVLT), brain regions with an

incomplete blood-brain barrier (Ganong, 1984). The SFO and OVLT relay information to

the paraventricular nucleus (PVN) and supraoptic nucleus (SON) (McKinley et al., 1992;

Miselis, 1981) to prompt secretion of arginine vasopressin (AVP) and oxytocin (Bisset et

al., 1971). The downstream network involved in ingestive behavior remains unspecified.

The AT1R, as a G protein coupled receptor, associates with Gq to trigger inositol 1,4,5-

triphosphate (IP3) production and Ca2+ mobilization (Beresford MJ, 1992; Enjalbert et

al., 1986; Smith et al., 1984; Taylor, Smith, & Exton, 1990). In parallel, mitogen-

activated protein kinase kinase (MEK) becomes phosphorylated, which activates the p42

and p44 isoforms of mitogen-activated protein kinase, also referred to as extracellular

signal-regulated kinase 1 and 2 (ERK1/2) (Sadoshima et al., 1995). ERK1/2 is activated

in the SFO and PVN after a central AngII injection (Wei et al., 2009). Furthermore,

whereas the IP3 signaling mediates AngII-induced thirst, the ERK1/2 signaling pathway
 
49  
 
mediates sodium appetite (Daniels et al., 2005; Daniels, Mietlicki, Nowak, & Fluharty,

2009; Felgendreger, Fluharty, Yee, & Flanagan-Cato, 2013). Aldosterone-specific

actions rely on the enzyme hydroxysteroid dehydrogenase 2 (HSD2), which oxidizes

glucocorticoids into an inactive metabolite (Funder, Pearce, Smith, & Smith, 1988). MR

and HSD2 are co-localized in the nucleus of the solitary tract (NTS), which receives

gustatory projections, possibly modulating the taste of sodium (Geerling et al., 2006).

Given that the actions of AngII and aldosterone have different primary sites of action in

the brain, determining the site of convergence may help to explain their behavioral

cooperativity.

It has been proposed that the natriorexic effect of Aldo and AngII on sodium

ingestion may be based on disinhibition (Blackburn, Demko, Hoffman, Stricker, &

Verbalis, 1992; Stricker & Verbalis, 1987). Of particular relevance, mineralocorticoid

treatment reduces AngII-induced activation of the OT neurons in the PVN and SON

(Roesch et al., 2001; Stricker & Verbalis, 1996; Stricker & Verbalis, 2004), although, the

exact role of oxytocin in the natriorexic effect of AngII is not clear (Blackburn et al.,

1992; Fitts et al., 2003; Rigatto, Puryear, Bernatova, & Morris, 2003). To investigate the

neural integration that underlies the Aldo and AngII potentiation of sodium appetite, we

took a multidisciplinary approach using behavioral pharmacology, measures of signal

transduction, reversible lesions, and functional neuroanatomy. The results implicate a

novel inhibitory link from the PVN to the OVLT, inhibited by mineralocorticoids,

comprising a disinhibitory control of sodium ingestion.

 
50  
 
MATERIALS AND METHODS

Animals

Adult male Sprague-Dawley rats (n = 68) that weighed between 225-250 g were

obtained from Charles River Laboratories (Wilmington, MA, USA). Rats were housed in

groups of two in plastic tubs with standard bedding and with food and water available ad

libitum, except during experimental procedures. The temperature in the colony was

maintained at 22 °C with a 12:12 h reversed light/dark cycle. Animals were allowed at

least one week to acclimate to the colony before any procedures were performed. The

Institutional Animal Care and Use Committee of the University of Pennsylvania approved

all procedures with animals.

Drugs

Deoxycorticosterone Acetate (DOC; Sigma, St Louis, MO) was administered

subcutaneously (sc) at a dose of 0.25 mg in 0.2 mL of sesame oil (Sigma, St Louis, MO).

DOC is typically used instead of aldosterone because it penetrates the blood brain

barrier more easily than Aldo due to its low capacity for hydrogen bond formation

(Geerling & Loewy, 2009). Sesame oil was administered at .2mL sc as a control. The

specific doses for each drug given icv were as follows: 2.0 or 20.0 ng AngII in a volume

of 2.0 ul (Bachem, King of Prussia, PA), 2.0 nmol U0126 dissolved in 10% DMSO in a

volume of 2.0 ul (Promega, Madison, WI), 20.0 ng Xestospongin-C (XC) dissolved in 2%

DMSO in a volume of 2.0 ul (Tocris Bioscience, Minneapolis, MN), 8.5 nmol/side of 2%

lidocaine in a volume of .4 ul (Phoenix Pharmaceuticals, Burlingame, CA), 10.0 µg

oxytocin in a volume of 1 µL (Bachem, King of Prussia, PA). Artificial cerebrospinal fluid

 
51  
 
(aCSF; R&D Systems, Minneapolis, MN) plus 10% DMSO was administered icv as a

vehicle control in a volume of 2.0 ul.

Surgeries

Surgeries were performed in aseptic conditions. Animals were anesthetized with

isofluorane gas before being fixed in a stereotaxic frame and implanted with a 26-gauge

guide cannulae (Plastics One, Roanoke, VA, USA) aimed at the lateral ventricle. For the

lateral ventricle surgeries, coordinates were: 0.48 mm caudal to bregma, 1.6 mm from

mid-line and 4.2 mm ventral to dura mater. For the lidocaine experiments, an additional

26-gauge double guide cannula (0.8 mm apart) was implanted into the PVN. For PVN

double cannulae, coordinates were: 1.8mm caudal to bregma, 0.4 mm from the mid-line

and 7.7 mm ventral to dura mater. These coordinates were chosen to allow an internal

injection cannula to extend beyond the guide cannula into either the ventricular space or

the PVN itself. The cannulae were fixed in place with dental cement and bone screws,

and the animals were allowed at least five days to recover before verification procedures

were performed. After surgery, animals were injected with yohimbine (0.11 mg/kg, Ben

Venue Laboratories Bedford, OH). Upon awakening, animals were singly housed.

Five days after surgery and prior to undergoing experimental treatments, animals

were tested for correct lateral ventricle cannula placement and patency. They were

given an icv injection of 20.0 ng of AngII diluted in aCSF via a Hamilton syringe

connected with PE-10 tubing to an injector that terminated 1 mm beyond the guide

cannula. Animals were excluded from the experiment if they failed to demonstrate a

drinking response in less than 30 seconds, consuming at least 3 ml of water, in two

separate AngII challenges. Animals began behavioral testing three days after the icv
 
52  
 
test injections.

Behavioral Studies

Rats were placed in individual wire mesh-bottomed cages with water and food

available ad libitum. One day prior to injections, rats acclimated for one hour to two 25-

ml bottles containing tap water and 1.5% saline, each marked with 0.2 ml graduations.

Vehicle or DOC (0.25 mg) was administered to each rat in 0.2 mL of sesame oil twice

daily (ten hours apart) for three days.

To investigate the role of ERK1/2 in this model of water and sodium

consumption, rats were assigned to specific icv conditions on the fourth day: Vehicle,

U0126, AngII, or U0126 plus AngII. U0126 was administered 15 min before AngII in the

last condition (Daniels et al., 2009; Felgendreger et al., 2013). To examine the role of

the OVLT activation in the potentiation of sodium appetite, rats were assigned to specific

icv conditions on the fourth day: Vehicle, XC, AngII, or XC plus AngII. XC was

administered 20 min before AngII in the last condition (Brennan et al., 2008; Li, Ji, &

Neugebauer, 2011). To evaluate the role of the PVN in DOC and AngII-induced sodium

appetite, rats were administered either veh or lidocaine directly into the PVN over one

min, followed 5 min later by injection of vehicle or AngII icv (Fernandes, Tavares, Pelosi,

& Correa, 2007; Flanagan, Dohanics, Verbalis, & Stricker, 1992). The bilateral lidocaine

injection was performed using a dual infusion pump (Harvard Apparatus, Holliston, MA).

In an additional study examining the role of oxytocin in sodium appetite, oxytocin or

vehicle was administered icv 15 min prior to injections of lidocaine and AngII

(Blackburn, Stricker, & Verbalis, 1992).

 
53  
 
 For all behavioral experiments, after the last drug treatment was given, rats were

returned to their cages, and water and saline levels were measured at 15 min intervals

for the first hour and then at 30 min intervals for the next hour. Food was not available

during behavioral testing.

Western Blot

Animals were pre-treated twice daily for three days (ten hours apart) with

vehicle or DOC, as described above. On day four, rats were administered either vehicle,

2 ng AngII, or 20 ng AngII icv. Two minutes after the last injections, animals were rapidly

decapitated and brains were flash frozen in hexane over dry ice. Using a cryostat, 1 mm

punches of brain regions of interest (OVLT, SFO, PVN, SON) were collected from 300

µm slices. The OVLT and SFO were combined and the PVN and SON punches were

combined from each rat to ensure sufficient tissue. Punches were immersed in lysis

buffer containing 25 mM Tris-HCl (pH 8), protease inhibitors (pepstatin, leupeptin,

aprotinin), and phosphatase inhibitors (sodium pyrophosphate, sodium fluoride, sodium

molybdate, phenylarsin oxide, and sodium orthovanadate). The OVLT/SFO and

PVN/SON punches were immersed in 50 and 100 uL of lysis buffer, respectively. Brain

punches were sonicated for 3 seconds followed by centrifugation at 14,000 rpm in 4°C

for 15 minutes. Supernatant was collected and a Bicinchoninic acid (BCA) protein assay

was performed on five microliters of each sample. Based on the protein levels detected

by BCA, appropriate amounts of sample and sample buffer were loaded into wells of a

sodium dodecyl sulfate polyacrylamide gel. Western blot for phospho- and total ERK1/2

was performed using the LiCor Odyssey System. The following antibodies were used:

 
54  
 
phospho-ERK1/2 (Thr202/Tyr204) (E10) Mouse mAb #9106 (Cell Signaling, Danvers,

MA); Erk 1 Antibody (C-16): sc-93 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA);

IRDye 800CW Goat anti-Mouse IgG (H + L) (Li-Cor Biosciences, Lincoln, NE); IRDye

680LT Goat anti-Rabbit IgG (H + L) (Li-Cor Biosciences, Lincoln, NE).

cFos immunohistochemistry

To observe brain activation after DOC and AngII treatments, rats were pre-

treated twice daily for three days (ten hours apart) with vehicle or DOC. On day 4, rats

were administered either vehicle or 20 ng AngII icv. To determine the efficacy of XC in

blocking calcium release, rats were injected icv with either vehicle, XC, AngII, or XC plus

AngII. To examine the effect PVN inactivation on cFos expression in other brain areas,

rats were injected with either: lidocaine or vehicle in the PVN, followed 5 min later by

either: vehicle or 20 ng AngII icv. Sixty minutes after the last icv injection in each study,

each rat was anesthetized with 50 mg/kg ketamine and 20 mg/kg xylazine,

intraperitoneally (ip). They were perfused transcardially with 100 mL of heparinized

saline followed by 200 mL 4% paraformaldehyde (Electron Microscopy Sciences, Fort

Washington, PA). The brains were isolated, post-fixed in paraformaldehyde overnight at

4°C, then submerged in 20% sucrose in .1M phosphate buffer for three days. Coronal

sections were cut on a freezing microtome into three serial sets of 40-um-thick sections.

These slices encompassed the organum vasculosum of the lateral terminalis (OVLT),

subfornical organ (SFO), paraventricular nucleus of the hypothalamus (PVN), and the

supraoptic nucleus of the hypothalamus (SON). One set from each animal underwent

immunohistochemical staining and analysis.

 
55  
 
 Sections were washed in Tris-buffered saline (TBS; pH 7.4) then incubated with

a cFos antibody (1:500, sc-52, rabbit; Santa Cruz Biotechnology, Santa Cruz, CA) in

TBS with .2% TritonX-100 and 3% normal donkey serum (Jackson Immunoresearch;

West Grove, PA) overnight at 4°C. After several washes, sections were incubated with a

Biotin-SP-conjugated AffiniPure Donkey Anti-rabbit IgG (1:100, Jackson

Immunoresearch) in TBS with .2% TritonX-100 and 3% normal donkey serum for 2

hours at room temperature. After several washes, sections were incubated with the

Vectastain ABC kit (Vector Laboratories, Burlingame, CA) for one hour. This was

followed by another set of washes before staining with 3’3’-diaminobenzidine (Sigma-

Aldrich, St Louis, MO) for 10 minutes. After a final set of washes, sections were

mounted on slides, air-dried, and cover slipped with DPX mounting media (Electron

Microscopy Sciences: Fort Washington, PA).

Images were acquired with a digital camera (Diagnostic Instruments, Sterling

Heights, MI, model RTKE), maintaining the same microscope and camera settings to

ensure the same level of light and exposure for all images. Background was subtracted

from images using Photoshop, and images were thresholded to the same level (200).

Images were further analyzed in NIH Image J using standardized boxes for each brain

region (calculated using Paxinos & Watson Rat Brain Atlas), using the analyze particles

function. Pixel size minimum was 15 and circularity was set to 0.35.

Oxytocin Secretion

Animals were pre-treated twice daily for three days (ten hours apart) with vehicle

or DOC, as described previously. On day four, rats were administered either vehicle or

 
56  
 
20 ng AngII icv. Two minutes after the last injections, animals were rapidly decapitated

and trunk blood was collected into 10 ml BD Vacutainer blood collection heparinized

glass tubes on ice (Franklin Lakes, NJ). The tubes then were centrifuged at 4oC at

1300 relative centrifugal force for 10 min. The plasma supernatant was extracted with

acetone and petroleum ether. Plasma levels of oxytocin and AVP were measured with a

specific radioimmunoassay performed by the laboratory of Dr. Joseph G. Verbalis, as

described previously (Verbalis et al., 1986). The standard curve for each peptide was

linear between 0.25 and 5 µU per tube with the oxytocin standard and between 0.5 and

10.0 pg per tube with the use of a synthetic AVP (Bachem Americas, Inc, Torrance,

California). The minimum detectable concentration of oxytocin or AVP in extracted

plasma was 0.25 µU/ml and 0.5 pg/ml, respectively. The oxytocin antiserum exhibited <

1% cross-reactivity with AVP, and the AVP antiserum (R-4) displayed <1% cross-

reactivity with oxytocin.

Statistical Analysis

Data are presented as the mean ± the standard error of the mean. For all

experiments, comparisons were made between treatment groups (with either a one-way,

two-way, or three-way ANOVA). When warranted, planned comparison post-hoc t-tests

were performed. All hypothesis tests used α=0.05 as the criterion level of significance.

Statistical analyses were conducted using Prism 2.0 software (La Jolla, CA) for one-way

or two-way ANOVAs and Statistica 12 Ultimate Academic Bundle (Tulsa, OK) for three-

way ANOVAs.

 
57  
 
RESULTS

The Role of ERK1/2 in DOC and AngII-induced Sodium Appetite

Consistent with previous research (Fluharty & Epstein, 1983), DOC pretreatment

potentiated AngII-induced sodium intake, but not water intake (Figure 3.1A). A two-way

ANOVA established a main effect for both AngII (F(2,54) = 10.7, p<0.001) and DOC

(F(1,54) = 29.6, p<0.001) on sodium intake, as well as an interaction between the two

treatments (F(2,54) = 3.2, p = 0.046; n = 12 rats/treatment group). Bonferonni post-hoc t

tests revealed a significant difference between each dose of AngII and the respective

DOC pre-treated condition (p<0.001 for the 2 ng AngII dose; p = 0.020 for the 20 ng

AngII dose). Regarding water intake, a two-way ANOVA supported a main effect for

AngII (F(2, 60) = 31.1, p<0.001), but not DOC (F(1,60 = 0.7, p = 0.400), to increase

water intake.

The role of ERK1/2 in the interaction between AngII and DOC was investigated

by using the MEK inhibitor U0126 (Figure 3.1B). U0126 significantly reduced sodium

intake induced by 20 ng AngII alone (6.6±1.8 versus 2.2±1.0 ml, p = 0.003; planned

comparison t tests; n = 18 rats/treatment group), as shown previously (Daniels et al.,

2009; Felgendreger et al., 2013). However, the MEK inhibitor U0126 did not reduce

sodium intake elicited by combined DOC and AngII treatments; in the 2ng AngII

condition, U0126 enhanced DOC/AngII induced sodium appetite (5.1±0.9 versus 8.5±0.8

ml, p = 0.010, for the 2.0 ng dose of AngII; 11.3±1.4 versus 12.6±2.3 ml, p = 0.270, for

the 20 ng dose of AngII). U0126 did not diminish the effect of AngII on water

consumption.

 
58  
 
 The effect of these hormonal treatments on the activation of ERK1/2 in OVLT,

SFO, PVN, and SON then was measured by Western blot analysis, as shown in Figure

3.1C. A two-way ANOVA supported a main effect of AngII alone, but not DOC alone, on

ERK1/2 phosphorylation (F(2,20)=4.1, p = 0.030; n = 6 rats/treatment group) for both

OVLT/SFO and PVN/SON. Post-hoc tests revealed that both doses of AngII significantly

increased phosopho-ERK1/2 compared with vehicle treatment (OVLT/SFO: 0.5±0.0 and

0.5±0.0 versus 0.3±0.0, p = 0.049 and 0.043); PVN/SON: 0.5±0.0 and 0.4±0.0 versus

0.3±0.0, p = 0.025 and 0.048). Additionally, there was a significant interaction between

DOC and AngII treatments for these brain regions (OVLT/SFO: (F(2,20) = 4.1, p =

0.031); PVN/SON: (F(2,20) = 3.9. p = 0.035)), such that in the presence of DOC, AngII

treatment no longer activated ERK1/2. In short, these results did not support the

hypothesis that DOC potentiated the effects of central AngII on sodium appetite by

further increasing phospho-ERK1/2 levels in relevant brain regions. Nevertheless, the

negative interaction between DOC and AngII on ERK1/2 activation in circumventricular

and neuroendocrine regions suggested that further investigation of these areas was

warranted.

The interaction of DOC and AngII on cFos activation in the brain

To further explore the neurological mechanisms that underlie the behavioral

cooperativity of DOC and AngII, cFos immunohistochemistry was used to detect a

unique pattern of brain activity that might correspond to the behavioral effects of these

hormones. As illustrated in Figure 3.2A, brain sections from OVLT, SFO, PVN, and

SON were analyzed for cFos immunolabeling. In the OVLT, cFos levels were

 
59  
 
significantly enhanced compared to vehicle for all treatment groups (Vehicle: 8.7±1.7,

DOC: 34.5±6.5, AngII: 29.0, DOC and AngII: 50.4; p = 0.010, p = 0.004, p = 0.020; n = 6

rats/treatment group, 4-6 sections per brain area). In the SFO, cFos levels were similar

for vehicle and DOC, but both AngII and DOC/AngII treatments significantly enhanced

staining (Vehicle: 11.5±2.6, DOC: 22.1±6.0, AngII: 56.8±5.9, DOC and AngII: 47.4±7.6; p

= 0.200, p = 0.001, p = 0.004). AngII significantly increased cFos staining compared to

vehicle in the PVN (126.3±5.3 versus 59.4±6.9, p<0.001), and DOC reduced AngII-

induced cFos staining to vehicle levels (126.3±5.3 versus 72.5±14.4, p = 0.006). In the

SON, AngII induced a significant amount of cFos staining compared to vehicle (40.4±6.6

versus 14.6±3.7, p = 0.020). There was a trend for DOC to reduce AngII-induced Fos

staining compared to AngII alone (25.4±5.0 versus 40.4±6.6, p = 0.100). In short, the

most striking interactions between DOC and AngII on cFos labeling were the enhanced

cFos staining in the OVLT and the suppressed cFos levels in the PVN.

The observation that DOC diminished the AngII-induced Fos activation in the

PVN is reminiscent of previous studies that demonstrated the DOC inhibits oxytocin

neuron activity in the PVN, thereby disinhibiting sodium intake (Roesch et al., 2001).

We tested whether the DOC treatment was associated with alterations in AngII-induced

neurohypophysial secretion. Plasma levels of oxytocin and AVP were measured by

radioimmunoassay after DOC, AngII, or both hormone treatments (Figure 3.2B). A two-

way ANOVA revealed a main effect for both AngII and DOC, as well as an interaction

between the two hormones on oxytocin secretion (F(1,27) = 4.2, p = 0.049; F(1,27) =

4.4, p = 0.045; F(1,27) = 5.7, p = 0.020, n = 8 rats/treatment group). AngII increased

oxytocin levels significantly compared to vehicle (12.8±2.9 versus 6.4±0.8 pg/ml, p =

0.048). DOC reduced these AngII-induced oxytocin levels back down to control levels
 
60  
 
(12.8±2.9 versus 5.1±1.2 pg/ml, p = 0.035). In contrast, there was no effect of DOC on

AngII-induced AVP secretion.

Taken together, these results demonstrate a correlation between cFos

expression in the OVLT and a suppression of PVN oxytocin activity with the natriorexic

effect of combined DOC and AngII. We next sought to determine whether disruption of

cFos induction in the OVLT with an IP3 receptor antagonist would impair the behavioral

cooperativity of DOC and AngII.

The Role of IP3 in DOC and AngII-induced Sodium Appetite

IP3 signaling was blocked with an antagonist to the IP3 receptor, namely

xestospongin-C (XC). First the bioactivity of centrally administered XC was verified by

assaying AngII-induced cFos immunohistochemistry in Figure 3.3A. Given that cFos is

an immediate early gene activated by IP3-induced intracellular calcium signaling, an

effective IP3 receptor antagonist should prevent calcium signaling, and thereby cFos

expression (Clark, Balla, Jones, & Catt, 1992; Daniels et al., 2005; Jomphe, Levesque,

& Trudeau, 2003; Templeton, Wang, & Miralem, 1998; W. Zhang et al., 2012). A two-

way ANOVA indicated main effects of AngII and XC, and an interaction on cFos

expression (F(1,16) = 4.7, p = 0.045; F(1,16) = 6.3, p = 0.020, F(1,16) = 6.8, p = 0.018,

respectively) in the OVLT (n= 6 rats/treatment group, 4-6 sections per brain area). In

particular, Bonferroni post-hoc tests suggest that XC pretreatment significantly reduced

the number of AngII-induced cFos-labeled neurons (9.4±2.5 versus 29.6±4.1, p = 0.002).

Labeling in the SFO exhibited a main effect for AngII, but not XC (F(1,15) = 7.2, p =

0.020; F(1,15) = 0.9, p = 0.360). However, there was a significant interaction between
 
61  
 
AngII and XC (F(1,15) = 5.3, p = 0.040). Specifically, Bonferonni post-hoc tests

designated a significant reduction in cFos expression between AngII and XC plus AngII

(30.0± 4.3 versus 15.1±4.3, p = 0.035).

Having demonstrated that the XC treatment effectively blocked IP3-induced cFos

in relevant brain regions, the role of IP3 in AngII and DOC induced sodium appetite was

investigated (shown in Figure 3.3B). A three-way ANOVA indicated that sodium intake

differed between treatment groups (F(1,68) = 6.5, p = 0.010; n = 10 rats/treatment

group). Consistent with Figure 3.1, DOC enhanced AngII-induced sodium intake (p =

0.006). XC did not affect sodium ingestion in animals treated with AngII alone (8.3±1.8

versus 8.3±1.7 ml, p = 0.990). However, XC reduced the DOC and AngII enhancement

of sodium intake to levels similar to the AngII alone group (15.1±1.3 versus 10.9±1.3 ml,

p = 0.030). Likewise, a three-way ANOVA revealed that water intake differed between

treatment groups (F(1,51) = 5.8, p = 0.020). Post-hoc tests indicated that XC reduced

AngII-induced water intake (11.0±1.6 versus 6.9±1.0 ml, p = .030). However, when both

DOC and AngII were present, XC did not reduce water intake (8.7±1.1 versus 9.2±1.8

ml, p = 0.810). Taken together, these results suggests that IP3 signaling, possibly in the

OVLT, is necessary for in the cooperative actions of DOCA and AngII on sodium

appetite. Previous work suggested that DOC enhances AngII-induced sodium appetite

via the suppression of oxytocin activity in the PVN (Blackburn et al., 1992; Stricker &

Verbalis, 1987), and several findings reported here are consistent with that hypothesis.

Therefore, the next experiment was designed to manipulate PVN activity and measure

the effect on sodium appetite and neuronal activation in the OVLT.

 
62  
 
Sodium appetite and OVLT activation after suppression of PVN activity

Neural activity in the PVN was suppressed with bilateral injections of lidocaine

above the PVN. Behavioral analysis included only animals with cannula tract locations

verified to be above the PVN (depicted in Figure 3.4A). Animals were treated with

either vehicle or lidocaine into the PVN, followed by either vehicle or 20 ng AngII (icv). A

two-way ANOVA revealed a main effect for AngII and lidocaine, as well as an interaction

on sodium intake (F(1,20) = 21.5, p = 0.001; F(1,20) = 5.2, p = 0.035; F(1,20) = 6.7, p =

0.020; n = 10 rats/treatment group). As shown in Figure 3.4B, AngII increased sodium

intake compared to vehicle (5.6±1.0 versus 2.0±0.6 ml, p = 0.040). Additionally,

lidocaine enhanced AngII-induced sodium intake significantly (9.5±1.2 versus 5.5±1.4 ml

p = 0.045). Conversely, a two-way ANOVA revealed a main effect for AngII but not

lidocaine on water intake (F(1,23) = 28.8, p<0.0001; F(1,23) = 0.1, p = 0.740). Post-hoc

tests denoted AngII plus or minus lidocaine treated rats drank very similar amounts of

water (9.1±1.5 versus 8.6±1.0 ml, p = 0.760), but both groups drank significantly more

than vehicle treated rats (0.5±0.5 ml, p = .02 and p = 0.009). A subset of rats was

treated with oxytocin (10.0 µg, icv) in the lidocaine plus AngII condition. Oxytocin co-

administration decreased sodium intake to vehicle levels (9.5±1.2 versus 2.1±0.7 ml, p =

0.001). However, oxytocin treatment also partially decreased water intake in the

lidocaine plus AngII group (8.6±1.0 versus 4.7±1.2 ml, p = 0.030). In summary, lidocaine

in the PVN enhanced AngII-induced sodium intake, but not water intake, mimicking the

effects of DOC pretreatment.

 
63  
 
 After the behavioral studies, AngII-induced cFos immunohistochemistry was

performed on the brains of these animals to verify the efficacy of lidocaine administration

to the PVN and to test the hypothesis that PVN activity may restrain sodium appetite by

inhibiting neural activity in the OVLT (Figure 3.4C). A one-way ANOVA indicated that

cFos expression in the OVLT differed between treatment groups (F(2,21) = 34.47,

p<.001; n = 6 rats/treatment group, 4-6 sections per brain area). Post-hoc tests

indicated that lidocaine further enhanced 20 ng AngII-induced cFos expression in the

OVLT (33.29±3.0 versus 54.60±3.0, p = .001), emulating the effects seen with DOC

pretreatment. Additionally, cFos expression in the PVN was reduced with lidocaine

pretreatment, illustrating reduced PVN activity (140.6±15.6 versus 88.5±3.5, p = .010).

DISCUSSION
 

Classic studies showed that AngII and adrenal steroids work cooperatively to

elicit a robust salt appetite when elevated concurrently, as occurs during hypovolemia

and sodium depletion (Epstein, 1982; Stricker, 1966; Stricker & Wolf, 1966; Stricker,

1971; Toth, Stelfox, & Kaufman, 1987). This potentiation of sodium appetite has been

observed in rats, pigeons and baboons and is not secondary to natriuresis or diuresis

(Fluharty & Epstein, 1983; Massi & Epstein, 1990; Shade et al., 2002). Experiments with

agonists and antagonists confirmed the validity of the “synergy hypothesis” in situations

in which the appetite was associated with elevated endogenous levels of AngII and

aldosterone (Sakai et al., 1986). The present experiments aimed to reveal the cellular

mechanisms and brain sites employed by the combined actions of aldosterone-AngII to

potentiate sodium appetite. Our initial hypothesis was that DOC and AngII actions

 
64  
 
converge on the ERK1/2 signaling pathway, given that adrenal steroids can increase

AT1R expression (Harada et al., 2001; Mazak et al., 2004; Ullian et al., 1992) and

enhance their cellular signaling (Lemarie, Paradis, & Schiffrin, 2008; Lemarie et al.,

2009). However, the present behavioral and biochemical studies did not support this

hypothesis. Rather, a follow-up experiment found that IP3 signaling was necessary for

the behavioral cooperation, although it did not mediate the sodium appetite expressed

when either hormone alone. A subsquent functional neuroanatomy experiment showed

that the combined DOC and AngII treatment suppressed cFos expression in the PVN,

reduced neurosecretion of oxytocin, and augmented Fos expression in the OVLT. This

led to the novel hypothesis that DOC-induced suppression of oxytocin neural activity in

the PVN relieves inhibition in the OVLT to allow a robust sodium ingestions. Our results

with local lidocaine administration into the PVN, verified by a lack of AngII-induced cFos

expression in the PVN, support this proposal based on elevated sodium appetite and

cFos induction in the OVLT. Thus, the present studies implicate an inhibitory signal

arising from the oxytocin neurons in PVN which may target neurons in the OVLT.

Lack of support for an amplification of ERK1/2 signaling

As mentioned above, central AngII administration activates ERK1/2 in the SFO

and PVN (Wei et al., 2009). ERK1/2 signaling is specifically required for sodium

appetite after exogenous AngII treatment (Daniels et al., 2005) and treatment that trigger

endogenous AngII production (Felgendreger et al., 2013). Thus, we hypothesized that

ERK1/2 also would be critical for the cooperative effect of DOC and AngII on sodium

intake. Behavioral pharmacology and signaling biochemistry experiments, while

replicating the previous finding of AngII-induced ERK1/2 and ERK1/2-dependent sodium

 
65  
 
appetite, did not support a role for ERK1/2 in the combined effect of DOC and AngII.

The interaction of DOC and AngII on IP3-mediate cFos induction

The AT1R is coupled with intracellular IP3 formation (Beresford MJ, 1992;

Enjalbert et al., 1986; Smith et al., 1984; Taylor et al., 1990), which in turn acts on the

IP3 receptor on the endoplasmic reticulum to liberate intracellular calcium as a third

messenger (Capponi, Rossier, & Vallotton, 1988; Nabika, Velletri, Lovenberg, &

Beaven, 1985; Suarez et al., 2002). One consequence of calcium mobilization is

transcription of immediate early genes, including cFos (Clark et al., 1992; Daniels et al.,

2005; Jomphe et al., 2003; Templeton et al., 1998; W. Zhang et al., 2012). Central

administration of AngII activates cFos expression in several brain regions, including the

OVLT, SFO, PVN, and SON (Blackburn et al., 1992; Herbert, Forsling, Howes, Stacey,

& Shiers, 1992; McKinley et al., 1992), as replicated here. In agreement with previous

work (Blackburn et al., 1992), DOC pretreatment diminished AngII-induced cFos in the

PVN and oxytocin neurosecretion. The novel finding that DOC pretreatment increases

cFos expression in the OVLT represents a striking correlation with the behavioral effects

of DOC and AngII on sodium ingestion. The OVLT is included in the region referred to

as the anteroventral third ventricle (AV3V), and partial damage to this region disinhibits

sodium appetite (Andersson, Leksell, & Lishajko, 1975; Fitts, Tjepkes, & Bright, 1990;

Fitts, 1991; Gardiner, Jolley, Vagnucci, & Stricker, 1986). IP3 receptor activation was

necessary for the DOC and AngII cooperativity on sodium intake, without affecting the

sodium ingestion stimulated by either hormone alone. Taken together, 1) the IP3

signaling pathway is uniquely necessary for the DOC and AngII cooperative effect on

 
66  
 
sodium appetite; 2) its downstream effector cFos is uniquely correlated with sodium

appetite in the OVLT; 3) PVN activity and oxytocin release are suppressed during the

cooperative stimulation of sodium appetite. A parsimonious explanation of these results,

partially supported by previous studies, would be that DOC treatment inhibits oxytocin

neurons in the PVN, which in turn relieves inhibition on a subset of OVLT neurons, which

then augment the drive for sodium ingestion based on IP3 signaling.

The role of oxytocin in sodium appetite

A model has been proposed in which centrally acting AngII simultaneously

stimulates and inhibits sodium appetite, with the inhibition caused by oxytocin release in

the brain (Blackburn et al., 1992; Stricker & Verbalis, 1987; Stricker & Verbalis, 1996).

Other investigators also have observed a disinhibition of sodium appetite after central

administration of an oxytocin antagonist or in knockout mice (Chow, Sakai, Fluharty, &

Flanagan-Cato, 1997; Fitts et al., 2003; Puryear, Rigatto, Amico, & Morris, 2001),

although the enhancement of the sodium appetite by an oxytocin antagonist may be

somewhat dependent on the dose of AngII (Fitts et al., 2003; Stricker & Verbalis, 2004).

The present results replicate several key findings, including the dampening of AngII-

induced cFos activation of the PVN and oxytocin release by DOC pretreatment.

However, the site of oxytocin action to inhibit sodium appetite remained unknown.

The finding that DOC treatment uniquely enhanced the effect of AngII on cFos

expression in the OVLT suggests that it is the target of the oxytocin-ergic inhibition of

sodium appetite. Indeed, oxytocin receptor mRNA is localized to the OVLT (Yoshimura

et al., 1993), and oxytocin axons project to the OVLT from the PVN (Buijs, 1978). The

 
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OVLT includes osmosensitive neurons and is thought to function as the brain’s primary

osmostat (Denton, McKinley, & Weisinger, 1996; Johnson, Cunningham, & Thunhorst,

1996). Many neurons in the OVLT are excited by hypertonicity and inhibited by

hypotonicity (Honda, Negoro, Dyball, Higuchi, & Takano, 1990; Nissen, Bourque, &

Renaud, 1993; Sayer, Hubbard, & Sirett, 1984; Vivas, Chiaraviglio, & Carrer, 1990),

mediated by members of the transient receptor potential vanilloid proteins (Ciura &

Bourque, 2006). The OVLT sends both GABAergic and glutamatergic projections to a

variety of brain regions (Camacho & Phillips, 1981; Richard & Bourque, 1996), although

the functions of these projections remain ill-defined. The present results build on this

body of work by indicating that activation of the PVN normally inhibits a subpopulation of

OVLT neurons. Given that the natriorexic effect of PVN inhibition was reversed with

central oxytocin administration, oxytocin is likely the critical inhibitory signal arising from

the PVN. Thus, the OVLT, a key sensor of body fluid parameters appears to also be the

target of oxytocin modulation of sodium appetite.

Interpretational limitations

The present work focused on the OVLT, SFO, PVN and SON based on their

importance to DOC and AngII actions, based on previous studies. Brainstem, limbic,

gustatory, and other hypothalamic regions are certainly also involved, but their

contributions were beyond the scope of this study. Previous tracing studies have

demonstrated a direct connection between the PVN and OVLT; however, the present

work did not address whether the changes in PVN activity directly or indirectly affects the

OVLT. Future studies are needed to examine those questions. Likewise, the analysis of

cFos expression and the pharmacological disruption of neural activity are not informative

 
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about the cascade of information processing within the broader neural network. Given

that many hypothalamic areas are reciprocally connected, further studies will be needed

to disambiguate the roles that these brain regions play in promoting and inhibiting

sodium appetite.

CONCLUSIONS
 

The present analysis has yielded several new insights towards understanding the

cooperativity of DOC and AngII to elicit a robust sodium appetite. First, these results

rule out the ERK1/2 signaling pathway as an underlying mechanism. Second, the OVLT

and PVN display divergent patterns of activation, suggesting that DOC relieves an

inhibitory signal from the PVN to the OVLT. Third, IP3 signaling is required for the

cooperativity of DOC and AngII. Fourth, local inhibition of the PVN mimics the effect of

DOC to enhance sodium appetite and activation of the OVLT. Additional work is needed

to reveal the broader network controlling sodium appetite, including downstream target

of OVLT activity and the upstream signals to the oxytocin neurons.

ACKNOWLEDGEMENTS
 

This work was supported by the National Institutes of Health Grants R01

HL091314. Preliminary results were reported at the Society for Neuroscience meeting

(New Orleans, 2012) and the Society for Behavioral Neuroendocrinology (Madison,

2012). We gratefully acknowledge the dedicated assistance of Daris Olaleye. We also

greatly appreciate the expertise of Drs. Joseph Verbalis and Qin Xu at Georgetown

University in conducting radioimmunoassays for oxytocin.

 
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Figure 3.1. Erk1/2 is not necessary for the DOC/AngII potentiation of sodium appetite.

 
70  
 
Panel A. Bar graphs illustrating the potentiation of AngII-induced sodium but not water intake by
DOC (n = 12/group). Panel B. Bar graphs illustrating the effect of blocking ERK1/2 activation on
DOC and AngII-induced sodium and water intake (n=18/group). DOC potentiated 2 and 20 ng
AngII-induced sodium intake. This behavior was not reduced by the MEK inhibitor U0126,
however, U0126 reduced sodium intake with 20 ng AngII alone. AngII-induced water intake was
not potentiated by DOC. Panel C. Bar graphs illustrating phosphorylated ERK1/2 levels in the
OVLT/SFO and PVN/SON after either oil or DOC pretreatment followed by icv treatments with 2
or 20 ng AngII (n = 6/group). AngII treatment induced significant ERK1/2 phosphorylation
compared to vehicle in both sets of brain regions. DOC pretreatment did not enhance this
activation. Representative western blot images of ERK1/2 phosphorylation are shown above
each quantified bar. For the sake of clarity, only specific statistical differences are highlighted.
The symbol “A” indicates AngII is different from vehicle; “B” indicates DOC/AngII is different from
AngII; “C” indicates U0126/AngII is different from AngII; “D” indicates U0126/DOC/AngII is
different from DOC/AngII, p<0.05. Abbreviations: AngII= Angiotensin II, DOC =
deoxycorticosterone acetate, p-ERK1/2 = phosphorylated extracellular signal-regulated kinase 1
and 2, OVLT = Organum vasculosum of the lateral terminalis, PVN = paraventricular nucleus of
the hypothalamus, SFO = subfornical organ, SON = supraoptic nucleus of the hypothalamus, Veh
= vehicle.

 
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Figure 3.2. DOC pretreatment enhanced AngII-induced OVLT activation and reduced PVN
activity and OT release.

 
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Panel A. Bar graphs illustrating cFos cell counts in different brain areas (OVLT, SFO, PVN, and
SON) after either vehicle or DOC pretreatment followed by icv vehicle or 20 ng AngII (n =
6/group). In the OVLT, each treatment condition induces cFos immunostaining compared with
vehicle; DOC/AngII induced additional cFos immunostaining. In the SFO, both AngII alone and
DOC/AngII induced more cFos staining than either vehicle or DOC alone. cFos labeling in the
PVN was increased by AngII, but DOC reduced the AngII-induced cFos expression to control
levels. In the SON, AngII induced cFos staining. Representative images of the OVLT and PVN
(coronal plane, 10x) in each treatment condition are shown above the bar graphs. Representative
images of SFO and SON are not shown, but similar data were seen. Panel B. Bar graphs
illustrating OT and AVP plasma levels after either vehicle or DOC pretreatment followed by icv
vehicle or 20 ng AngII (n = 8/group). AngII treatment increased OT levels, and this effect was
reduced by DOC pretreatment. However, AngII treatment elevated AVP levels, regardless of
pretreatment. For the sake of clarity, only specific statistical differences are highlighted. The
symbol “A” indicates AngII is different from vehicle; “B” indicates DOC/AngII is different from
AngII. Abbreviations: AngII = Angiotensin II, AVP = vasopressin, DOC = deoxycorticosterone
acetate, OT= oxytocin, OVLT = Organum vasculosum of the lateral terminalis, PVN =
paraventricular nucleus of the hypothalamus, SFO = subfornical organ, SON = supraoptic
nucleus of the hypothalamus, Veh = Vehicle

 
73  
 
  
Figure 3.3. The IP3 receptor is required for DOC/AngII potentiation of sodium appetite.

 
74  
 
Panel A. Bar graphs illustrating the level of cFos expression in the OVLT and SFO after vehicle,
XC, 20 ng AngII, or XC plus 20 ng AngII icv (n = 6/group). Representative images of cFos
staining of OVLT coronal sections (10x) in each treatment group are shown above the graphs.
Representative images of the SFO are not shown, but similar data were seen. AngII induced
cFos staining in both the OVLT and SFO, and the effect was reduced by XC. Panel B. Bar
graphs illustrating the effect of XC on DOC and 20 ng AngII induced sodium and water intake (n =
10/group). DOC potentiates AngII-induced sodium intake; this effect was impaired by the IP3
receptor inhibitor XC. XC did not affect sodium intake induced by AngII alone. AngII-induced
water intake was reduced by XC. For the sake of clarity, only specific statistical differences are
highlighted. The symbol “A” indicates AngII is different from vehicle; “B” indicates DOC/AngII is
different from AngII; “C” indicates XC/AngII is different from AngII; “D” indicates XC/DOC/AngII is
different from DOC/AngII, p<0.05. Abbreviations: AngII = Angiotensin II, DOC =
deoxycorticosterone acetate, OVLT = Organum vasculosum of the lateral terminalis, SFO =
subfornical organ, Veh = vehicle, XC = Xestospongin-C

 
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Figure 3.4. PVN inactivation enhances AngII-induced sodium appetite.

 
76  
 
Panel A. Illustration of rat coronal brain slices depicting bilateral guide cannula placements (filled
circles) above the PVN for lidocaine administration. Each pair of dots represents data from one
animal. Panel B. Bar graphs illustrating the effect of intra-PVN lidocaine on 20 ng AngII-induced
sodium and water intake (n = 10/group). Lidocaine treated animals increased their AngII-induced
sodium intake, but not water intake, mimicking the effects of DOC. Panel C. Bar graphs
illustrating cFos expression in the OVLT and PVN after vehicle, lidocaine, 20 ng AngII, or
lidocaine plus 20 ng AngII icv (n = 6/group). Representative images of cFos staining of OVLT
and PVN coronal sections (10x) in each treatment group are shown above the graphs. AngII
induced cFos immunostaining was enhanced by lidocaine in the OVLT and reduced in the PVN.
For the sake of clarity, only specific statistical differences are highlighted. The symbol “A”
indicates AngII is different from vehicle; “B” indicates lidocaine/AngII is different from AngII.
Abbreviations: AngII = Angiotensin II, OT = oxytocin, OVLT = Organum vasculosum of the lateral
terminalis, PVN = paraventricular nucleus of the hypothalamus, Veh = Vehicle.

 
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CHAPTER 4: ALDOSTERONE AND ANGII-INDUCED SODIUM APPETITE IS

ASSOCIATED WITH MESOLIMBIC ACTIVATION

Laura A. Grafe1 and Loretta M. Flanagan-Cato1,2,3

Neuroscience Graduate Group1, Department of Psychology2, and the Mahoney Institute

of Neurological Sciences3, University of Pennsylvania,

Philadelphia Pennsylvania, USA

Abbreviated title: Aldo and AngII-induced sodium appetite

Key words: Aldosterone; Angiotensin; Dopamine; Reward; Sodium Appetite

 
 
78  
 
SUMMARY
 

Aldosterone and Angiotensin II (AngII) cooperate centrally to produce a robust

sodium appetite. Yet, it remains unknown how combined treatment with both hormones

affects mesolimbic circuits to selectively drive this ingestive behavior. We hypothesized

that treatment of aldosterone plus AngII enhances motivation for sodium, in part by

increasing mesolimbic activation. To test this, male rats pretreated with

deoxycorticosterone (DOC; a synthetic precursor of aldosterone) and central AngII were

trained to press levers for either sodium or water. In a progressive ratio reinforcement

schedule, rats treated with both hormones exhibited a breakpoint ratio of sodium to

water that was three times higher than animals given either hormone alone. However,

AngII treatment augmented cFos levels in the ventral tegmental area and nucleus

accumbens, regardless of DOC pretreatment. Lidocaine inactivation of the

paraventricular nucleus, a treatment known to augment sodium appetite, enhanced the

ability of central AngII to increase cFos in these mesolimbic areas. Lastly, DOC

increased levels of phosphorylated tyrosine hydroxylase immunoreactivity in the ventral

tegmental area, regardless of AngII treatment. Collectively, these results illustrate a

selective drive for sodium, prompted by the combination of AngII-induced mesolimbic

neural activity and DOC-induced dopamine synthesis.

 
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INTRODUCTION
 

Sodium homeostasis affects critical physiological functions such as extracellular

fluid volume, blood circulation, and neural activity (Weisinger et al., 1996). Several

physiological mechanisms minimize sodium loss, and when it must be replaced, a robust

sodium appetite develops (Andersson, 1977). Parallel mechanisms contribute to sodium

appetite, including the renin-angiotensin-aldosterone system, an endocrine mechanism

that acts both peripherally and centrally to correct for sodium imbalance (Johnson &

Thunhorst, 1997). Reduced sodium levels, blood volume, or blood pressure triggers

renin production, which subsequently leads to increased circulating AngII and

aldosterone levels (J. O. Davis & Spielman, 1974; Denton et al., 1996). AngII acts in

the brain to induce both water intake and sodium intake, whereas aldosterone induces

solely sodium appetite, replacing lost fluids (Epstein et al., 1969). Suppression of either

central AngII or aldosterone actions does not entirely reduce sodium appetite in several

different experimental preparations, but blocking central actions of both hormones

completely abolishes the behavior (Buggy & Jonklaas, 1984; Sakai et al., 1986).

Conversely, when both aldosterone and Angiotensin II (AngII) are given exogenously,

sodium appetite is potentiated beyond the additive effects of either hormone alone

(Fluharty & Epstein, 1983). This phenomenon has been observed in rats, pigeons and

baboons and is not secondary to natriuresis or diuresis (Massi & Epstein, 1990; Shade

et al., 2002). AngII and aldosterone bind to receptors in the subfornical organ and

nucleus of the solitary tract, respectively (Ganong, 1984; Geerling et al., 2006).

However, the neural circuits that integrate aldosterone and AngII actions to augment

sodium appetite remain undefined.

 
80  
 
 The interaction between aldosterone and AngII to potentiate sodium appetite

likely enhances activation of motivational circuits. Rats run faster towards a sodium

reward if both aldosterone and AngII are present compared to either hormone alone,

suggesting augmented behavioral drive (D. M. Zhang et al., 1984). Moreover, rats on a

sodium deficient diet, which increases aldosterone and AngII levels, choose sodium

ingestion over moderately reinforcing brain stimulation, indicating a common evaluative

mechanism (Conover, Woodside, & Shizgal, 1994; Stricker, Vagnucci, McDonald, &

Leenen, 1979).

Several reports indicate neuroanatomical connections between osmosensitive

brain areas and the mesolimbic system (Lucas, Grillo, & McEwen, 2007; Shekhtman et

al., 2007). The mesolimbic dopamine system processes natural rewards, including

sodium after sodium loss (Roitman et al., 2002). Dopamine neurons in the ventral

tegmental area project to the nucleus accumbens, which projects to brain regions such

as the ventral pallidum to generate goal directed movement (Carelli, 2002). Thus, it is

feasible that aldosterone and AngII integration may involve activation of the ventral

tegmental area and nucleus accumbens in potentiating sodium appetite. Moreover,

AngII and aldosterone elevate markers of dopamine release in the nucleus accumbens

(Axelrod, 1971; Roitman et al., 1999). For example, sodium depletion alters levels of

dopamine transporters and opioid peptides in the nucleus accumbens (Grondin, Gobeil-

Simard, Drolet, & Mouginot, 2011; Lucas, Grillo, & McEwen, 2003). Furthermore,

sodium appetite has been associated with changes in the dendritic arbor of ventral

striatum neurons (Roitman, Na, Anderson, Jones, & Bernstein, 2002). Though studies

have suggested a role for mesolimbic activity in sodium appetite, evidence for neural

activation and dopamine synthesis is lacking. Additionally, the effect of aldosterone and

 
81  
 
AngII cooperativity on mesolimbic activation has not been studied.

To examine the effect of combined aldosterone and AngII action in driving

sodium ingestion, rats were treated with either Vehicle/Vehicle, DOC/Vehicle,

Vehicle/AngII, or DOC/AngII and tested on a progressive ratio reinforcement schedule.

Whereas other studies measured reward by running speed or pressing a single lever for

sodium, which may be confounded with motor activation, our progressive ratio

experiment measured the motivational choice for sodium compared with water.

Mesolimbic neuronal activation was assessed by cFos expression in the ventral

tegmental area and nucleus accumbens after hormone treatments. In addition, levels of

tyrosine hydroxylase, a rate limiting enzyme for dopamine synthesis, was examined in

the ventral tegmental area and nucleus accumbens after hormone treatments. We

hypothesized that aldosterone and AngII cooperatively enhance neuronal activation and

tyrosine hydroxylase activation in the ventral tegmental area and nucleus accumbens,

augmenting motivation for sodium. Overall, these experiments determine if aldosterone

and AngII integrate information through mesolimbic circuitry to exert a robust sodium

appetite.

MATERIALS AND METHODS

 

Animals

Adult male Sprague-Dawley rats (n = 61) that weighed between 225-250 g were

obtained from Charles River Laboratories (Wilmington, MA, USA). Rats were pair-

housed in plastic tubs with standard bedding and with food and water available ad

libitum, except during experimental procedures. The temperature in the colony was
 
82  
 
maintained at 22 °C with a 12:12 h reversed light/dark cycle. Animals were allowed at

least one week to acclimate to the colony before any procedures were performed. The

Institutional Animal Care and Use Committee of the University of Pennsylvania approved

all procedures with animals.

Surgery

Surgeries were performed in aseptic conditions. Animals were anesthetized with

isofluorane gas before being fixed in a stereotaxic frame and implanted with a 26-gauge

guide cannulae (Plastics One, Roanoke, VA, USA) aimed at the lateral ventricle. For the

lateral ventricle surgeries, coordinates were: 0.48 mm caudal to bregma, 1.6 mm from

mid-line and 4.2 mm ventral to dura mater. For a subset of animals, an additional 26-

gauge double guide cannula (0.8 mm apart) was implanted into the paraventricular

nucleus (PVN). For PVN double cannula, coordinates were: 1.8 mm caudal to bregma,

0.4 mm from the mid-line and 7.7 mm ventral to dura mater. These coordinates were

chosen to allow an internal injection cannula to extend beyond the guide cannula into

either the ventricular space or the PVN itself. The cannulae were fixed in place with

dental cement and bone screws, and the animals were allowed at least five days to

recover before verification procedures were performed. After surgery, animals were

injected with yohimbine (0.11 mg/kg, Ben Venue Laboratories Bedford, OH). Upon

awakening, animals were singly housed.

Five days after surgery and prior to undergoing experimental treatments, animals

were tested for correct lateral ventricle cannula placement and patency. They were

given an icv injection of 20.0 ng of AngII diluted in aCSF via a Hamilton syringe

 
83  
 
connected with PE-10 tubing to an injector that terminated 1 mm beyond the guide

cannula. Animals were excluded from the experiment if they failed to demonstrate a

drinking response in less than 30 seconds, consuming at least 3 ml of water, in two

separate AngII challenges. Animals began behavioral testing three days after the icv

test injections.

Experimental Design

In all experiments, animals were assigned to one of four treatments in a 2 x 2

crossover design. Animals were first pre-treated twice daily (10 hours apart) for three

days with a subcutaneous injection of either sesame oil or Deoxycorticosterone Acetate

(DOC; .25 mg/.2 ml sesame oil; Sigma, St Louis, MO). DOC is typically used instead of

aldosterone because it penetrates the blood brain barrier more easily than Aldo due to

its low capacity for hydrogen bond formation (Kraulis, Foldes, Traikov, Dubrovsky, &

Birmingham, 1975). Animals were then injected icv with either artificial cerebrospinal

fluid (aCSF; R&D Systems, Minneapolis, MN) or 20.0 ng AngII in a volume of 2.0 ul

(Bachem, King of Prussia, PA). The treatment groups will be referred to as follows:

Vehicle/Vehicle, DOC/Vehicle, Vehicle/AngII, DOC/AngII.

Experiment 1. Progressive Ratio Task

Rats acclimated in wire mesh cages for one hour to two 25-ml bottles containing

tap water and 3% saline, each marked with 0.2 ml graduations. The saline bottle was

then removed, and rats were water restricted for 23 hours per day for the next six days.

 
84  
 
During the six days, rats underwent operant lever pressing training in conditioning boxes

for 30 minutes per day (Med Associates; MDPC IV Software, St. Albans, Vermont). The

conditioning boxes contained levers for both water (right lever) and 3% saline (left lever).

During the first two training days, a fixed ratio (FR1) autoshaping procedure was

employed (each lever press earned a 0.1 ml water or saline reward, depending on which

of the two levers was pressed; a free liquid reward was dispensed every 300 sec that

elapsed without reinforcement). The animals then received 2 training days of FR1

schedule without autoshaping, followed by 2 training days of FR3 training. After learning

the task, rats were given ad libitum access to water again. Rats were then assigned to

treatment groups as discussed above in Experimental Design, and given a break

operant lever pressing training while they received their three days of pretreatment

injections. After pretreatment was complete, rats were administered their assigned icv

injection, and immediately given a test with a progressive ratio (PR) reinforcement

schedule. The response requirement of the PR schedule increased progressively as

previously described (J. F. Davis et al., 2011). The breakpoint for each animal was

defined as the final completed requirement that preceded a 10 minute period without

earning a reinforcer, with a two hour limit total. Food was not available during testing.

Experiment 2: Hormone-Induced cFos expression

To observe brain activation after DOC and AngII treatments, rats were assigned

to treatment groups as discussed above in Experimental Design. To observe brain

activation after lidocaine and AngII treatment, rats were administered lidocaine into the

PVN (8.5 nmol/side of 2% lidocaine in a volume of 0.4 ul; Phoenix Pharmaceuticals,

 
85  
 
Burlingame, CA), followed five minutes later by 20 ng AngII icv. Sixty minutes after the

last icv injection in each study, each rat was anesthetized with 50 mg/kg ketamine and

20 mg/kg xylazine, intraperitoneally (ip). They were perfused transcardially with 100 mL

of heparinized saline followed by 200 mL 4% paraformaldehyde (Electron Microscopy

Sciences, Fort Washington, PA). The brains were isolated, post-fixed in

paraformaldehyde overnight at 4°C, then submerged in 20% sucrose in .1M phosphate

buffer for three days. Coronal sections were cut on a freezing microtome into three

serial sets of 40-um-thick sections. These slices encompassed the ventral tegmental

area as well ass the shell and core of the nucleus accumbens. One set from each animal

underwent immunohistochemical staining and analysis.

Sections were washed in Tris-buffered saline (TBS; pH 7.4) then incubated with

a cFos antibody (1:500, sc-52, rabbit; Santa Cruz Biotechnology, Santa Cruz, CA) in

TBS with .2% TritonX-100 and 3% normal donkey serum (Jackson Immunoresearch;

West Grove, PA) overnight at 4°C. After several washes, sections were incubated with a

Biotin-SP-conjugated AffiniPure Donkey Anti-rabbit IgG (1:100, Jackson

Immunoresearch) in TBS with .2% TritonX-100 and 3% normal donkey serum for 2

hours at room temperature. After several washes, sections were incubated with the

Vectastain ABC kit (Vector Laboratories, Burlingame, CA) for one hour. This was

followed by another set of washes before staining with 3’3’-diaminobenzidine (Sigma-

Aldrich) for 10 minutes. After a final set of washes, sections were mounted on slides,

air-dried, and cover slipped with DPX mounting media (Electron Microscopy Sciences:

Fort Washington, PA).

Images were acquired with a digital camera (Diagnostic Instruments, Sterling

Heights, MI, model RTKE), maintaining the same microscope and camera settings to
 
86  
 
ensure the same level of light and exposure for all images. Background was subtracted

from images using Photoshop, and images were thresholded to the same level (200).

Images were further analyzed in NIH Image J using standardized boxes for each brain

region (calculated using Paxinos & Watson Rat Brain Atlas), using the analyze particles

function. Pixel size minimum was 15 and circularity was set to 0.35.

Experiment 3: Hormone Regulation of Tyrosine Hydroxylase

Animals were assigned to treatment groups as described above in Experimental

Design. Five minutes after the last injections, animals were rapidly decapitated and

brains were flash frozen in hexane over dry ice. The five minute time point was selected

based on its correlation with a significant increase in lever presses for sodium in the

progressive ratio experiment. Using a cryostat, 1 mm punches of brain regions of

interest (ventral tegmental area, nucleus accumbens shell and core) were collected from

300 µm slices. Punches were immersed in lysis buffer containing 25 mM Tris-HCl (pH

8), protease inhibitors (pepstatin, leupeptin, aprotinin), and phosphatase inhibitors

(sodium pyrophosphate, sodium fluoride, sodium molybdate, phenylarsin oxide, and

sodium orthovanadate). The ventral tegmental and nucleus accumbens (shell and core)

punches were immersed in 50 and 100 uL of lysis buffer, respectively. Brain punches

were sonicated for three seconds followed by centrifugation at 14,000 rpm in 4°C for 15

minutes. Supernatant was collected and a Bicinchoninic acid (BCA) protein assay was

performed on five microliters of each sample. Based on the protein levels detected by

BCA, appropriate amounts of sample and sample buffer were loaded into wells of a 10%

sodium dodecyl sulfate polyacrylamide gel. Western blot for phospho- and total TH was

 
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performed using the LiCor Odyssey System. The following antibodies were used:

monoclonal anti-tyrosine hydroxylase antibody T2928 at 1:8000 (Sigma Aldrich); tyrosine

hydroxylase pS31 rabbit polyclonal antibody #36-9900 at 1:400 (Life Technologies,

Carlsbad, CA); IRDye 800CW Goat anti-Mouse IgG (H + L) 926-32210 at 1:2000 (Li-Cor

Biosciences, Lincoln, NE); IRDye 680LT Goat anti-Rabbit IgG (H + L) 926-68021 at

1:4000 (Li-Cor Biosciences).

Statistical Analysis

Data are presented as the mean ± the standard error of the mean. For all

experiments, comparisons were made between treatment groups with a two-way

ANOVA. When warranted, planned comparison post-hoc t-tests were performed. All

hypothesis tests used α=0.05 as the criterion level of significance. Statistical analyses

were conducted using Prism 2.0 software (La Jolla, CA) for one-way or two-way

ANOVAs.

RESULTS
 

Experiment 1: The Progressive Ratio Task

The progressive ratio task was used to determine if aldosterone and AngII shift

the effort rats are willing to exert for sodium ingestion. Rats were pretreated with either

oil or DOC, followed by icv treatments of vehicle or AngII and allowed to press levers:

one for access to 3% saline and the other for access to water. Figure 4.1A illustrates the

number of lever presses over time for 3% saline and water. Visual inspection suggests a
 
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difference based on treatment, with DOC/AngII treated rats pressing more for 3% saline

than all other treatment groups. Although the Vehicle/AngII group pressed more for

water, the DOC/AngII-treated rats pressed very little for water, and for the shortest

amount of time (Vehicle/Vehicle: 14.0±3.5 min, DOC/Vehicle: 18.6±3.7 min,

Vehicle/AngII: 23.4±5.7 min, DOC/AngII: 11.2±3.0 min). The total number of lever

presses for both 3% saline and water for each treatment group is quantified in bar

graphs in Figure 4.1B. A two-way ANOVA established a main effect for AngII (F(1,29) =

13.9, p<0.001) but not DOC (F(1,29) = 0.101, p = 0.752) on 3% saline lever presses,

and a trend for an interaction between the two treatments (F(1,29) = 2.3, p = 0.138; n =

6 rats/treatment group). Bonferonni post-hoc t tests revealed that the DOC/AngII group

pressed significantly more on the saline lever compared with the Vehicle/Vehicle and

DOC/Vehicle groups (Vehicle/Vehicle: 10.9±2.4 presses, DOC/Vehicle: 6.4±1.7 presses,

Vehicle/AngII: 19.1±6.4 presses, DOC/AngII: 26.0±2.8 presses; p<0.01). A two-way

ANOVA established a main effect for AngII (F(1,29) = 5.73, p = 0.023) but not DOC

(F(1,29) = 1.69, p = 0.203) on water lever presses, and a significant interaction between

the two hormones (F,29) = 8.35, p = 0.007). Bonferonni post-hoc t tests revealed that

rats treated with Vehicle/AngII pressed more for water than any other treatment group

(Vehicle/Vehicle: 16.9±5.2 presses, DOC/Vehicle: 29.5±4.9 presses, Vehicle/AngII:

58.9±10.7 presses, DOC/AngII: 25.5±8.9 presses; p<0.01).

With a progressive ratio schedule of reinforcement, the response requirement to

attain rewards increases according to the same rule throughout the test session until the

rat stops responding (Sclafani & Ackroff, 2003). The highest ratio completed is referred

to as the breakpoint ratio and provides a measure of reinforcement. Figure 4.1C

illustrates the breakpoint ratio of sodium to water for each treatment group, allowing us

 
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to compare the amount of effort exerted to receive a sodium reward versus a water

reward. A 2-way ANOVA established a main effect for AngII, DOC, and an interaction

between the two (F(1,29) = 4.59, p = 0.046; F(1,29) = 3.94, p = 0.0568; F(1,29) = 8.59, p

= 0.006). Bonferonni post-hoc t tests revealed that DOC plus AngII treated rats had a

significantly higher sodium to water breakpoint ratio than all other treatment groups

(Vehicle/Vehicle: 0.8±0.1, DOC/Vehicle: 0.3±0.1, Vehicle/AngII: 0.4±0.1, DOC/AngII:

2.7±0.9, p<0.05). Together, these data indicate that when both aldosterone and AngII

are present, rats are uniquely motivated to press for sodium compared to water.

Experiment 2: Hormone-Induced cFos expression

DOC/AngII cFos expression

Upon observing an increased sodium:water breakpoint ratio after DOC/AngII

treatment, we examined activation in key brain areas that may underlie this change in

motivation. As shown in Figure 4.2, the number of cFos labeled cells was quantified in

the ventral tegmental area and the nucleus accumbens after Vehicle/Vehicle,

DOC/Vehicle, Vehicle/AngII, and DOC/AngII treatment. A 2-way ANOVA revealed a

main effect for AngII and DOC, but not for an interaction between the two hormones in

the ventral tegmental area (F(1,8) = 49.63, p<0.001, F(1,8) = 6.25, p = 0.037, F(1,8) =

0.43, p = 0.527, respectively). Bonferonni corrected t tests indicated that Vehicle/AngII

and DOC/AngII increased cFos expression compared to Vehicle/Vehicle

(Vehicle/Vehicle: 72.6±12.3 versus DOC/Vehicle: 93.4±5.4, Vehicle/AngII: 129.0±4.3,

DOC/AngII: 143.5±11.5, p = 0.170, p<0.05, p<0.05, respectively.). However,

pretreatment with DOC did not significantly enhance AngII-induce cFos. In the nucleus
 
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accumbens core, a 2-way ANOVA revealed a main effect for AngII, but no effect for

DOC or their interaction on cFos expression (F(1,9) = 9.05, p = 0.014; F(1,9) = 4.17, p =

0.071; F(1,9) = 0.11, p = 0.749, respectively). Bonferonni post-hoc tests indicated that

Vehicle/AngII and DOC/AngII treatments increased cFos levels compared to

Vehicle/Vehicle (Vehicle/Vehicle: 14.5±4.2 versus DOC/Vehicle: 23.3±1.9, Vehicle

/AngII: 27.5±0.8, DOC/AngII: 34.9±8.8; p = 0.119, p<0.010, p<0.050, respectively).

Again, DOC pretreatment did not significantly enhance AngII-induced cFos expression in

the core. Lastly, a 2-way ANOVA for the nucleus accumbens shell revealed a main

effect for AngII, but no effect for DOC or the two-hormone interaction on cFos

expression (F(1,9) = 13.63, p = 0.005, F(1,9) = 2.89; p = 0.123; F(1,9) = 0.59, p = 0.462).

Bonferonni post-hoc t tests indicated that each treatment group induced significant cFos

expression compared to vehicle (Vehicle/Vehicle: 13.0±5.6, versus DOC/Vehicle:

23.5±0.6, Vehicle/AngII: 33.1±0.9, DOC/AngII: 38.9±10.1; p<0.05, p<0.01, p<0.05,

respectively). Similar to both the ventral tegmental area and core of the accumbens,

DOC pretreatment did not significantly enhance AngII-induced cFos expression in the

shell. To summarize, AngII induced significant cFos expression in the ventral tegmental

area and nucleus accumbens, but DOC pretreatment did not significantly enhance this

activation.

PVN-lido/AngII cFos expression

We next used another preparation of enhanced sodium appetite to examine its

effect on cFos activation in these key mesolimbic brain areas. Previous data from our

lab suggests that DOC enhances AngII-induced sodium appetite through inactivation of

 
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the PVN. In particular, inactivation of the PVN with lidocaine (PVN-lido) mimicked the

effect of DOC by enhancing AngII-induced sodium appetite. In Figure 4.3, cFos

expression was examined in the ventral tegmental area and nucleus accumbens after

PVN-lido/AngII treatment. cFos expression in the VTA differed between treatment

groups (F(2,8) = 19.02, p<0.001). AngII increased cFos expression compared to

Vehicle, and PVN-lido pretreatment further enhanced this activation (Vehicle/Vehicle:

68.0±7.0 versus Vehicle/AngII: 159.3±12.3, PVN-lido/AngII: 203.6±13.4; p<0.01,

p<0.05). cFos expression in the nucleus accumbens core also differed between

treatment groups (F(2,12) = 11.49, p<0.01). Analogous to the VTA, post-hoc tests

revealed that AngII induced significant cFos expression in the core compared to Vehicle.

Moreover, PVN-lido further enhanced this activation (Vehicle/Vehicle: 14.8±0.5 versus

Vehicle/AngII: 33.4±3.2, Lidocaine/AngII: 43.7±3.0; p<0.05, p<0.05). cFos expression in

the nucleus accumbens shell differed between treatment groups (F(2,12) = 9.87,

p<0.01). Post-hoc t tests indicated that AngII and PVN-lido/AngII both increased cFos

expression compared to Vehicle, however, PVN-lido did not further enhance levels

beyond AngII alone (Vehicle/Vehicle: 15.0±1.0, Vehicle/AngII: 36.3±3.0, Lidocaine/AngII:

42.3±3.1; p<0.01, p<0.01, p = 0.22). In summary, PVN-lido treatment enhances AngII-

induced cFos expression in the ventral tegmental area and nucleus accumbens core.

Experiment 3: Hormone Regulation of Tyrosine Hydroxylase

Hormone-induced increases in neural activation would logically also involve an

increase in dopamine activity in the ventral tegmental area and accumbens. To test this,

we quantified tyrosine hydroxylase activity, likely indicating dopamine release, in these

 
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brain areas. Rats were pretreated with vehicle or DOC followed by icv injections of

vehicle or AngII, and tissue was collected five minutes after the last injection. As shown

in Figure 4.4, tyrosine hydroxylase levels (panel A) and phosphorylated tyrosine

hydroxylase levels (panel B) were examined in the ventral tegmental and nucleus

accumbens. A 2-way ANOVA revealed a main effect for both DOC and AngII on

tyrosine hydroxylase expression in the ventral tegmental area (F(1,8) = 16.6, p<0.01;

F(1,8) = 6.0, p<0.05). Post hoc tests indicated each treatment group significantly

enhanced tyrosine hydroxylase expression compared to vehicle (Vehicle/Vehicle:

2.6±1.1, DOC/Vehicle: 13.5±1.1, Vehicle/AngII: 10.2±2.2, DOC/AngII: 15.8±3.0; p<0.05,

p<0.01, p<0.05). However, DOC pretreatment did not significantly enhance the effects

of AngII on tyrosine hydroxylase expression. In the core of the nucleus accumbens,

each treatment only resulted in trends for increasing tyrosine hydroxylase. However, in

the shell of the nucleus accumbens, DOC but not AngII had a main effect, and there was

a trend for their interaction on tyrosine hydroxylase expression (F(1,8) = 9.3, p<0.01;

F(1,8) = 1.0, p = 0.35; F(1,8) = 4.8, p = .059, respectively). Post-hoc t tests revealed

DOC increased tyrosine hydroxylase levels significantly (Vehicle/Vehicle: 8.4±1.8,

DOC/Vehicle: 13.9±0.2, Vehicle/AngII: 11.7±0.8, DOC/AngII: 12.7±0.7, p<.05). In

summary, DOC treatment induces tyrosine hydroxylase expression in the ventral

tegmental area and nucleus accumbens shell.

DOC may induce more tyrosine hydroxylase expression, but it is the level of

phosphorylation that indicates the amount of activated enzyme to synthesize dopamine.

In the ventral tegmental area, a 2-way ANOVA revealed a main effect for DOC, and not

AngII (F(1,8) = 23.5, p<0.01); F(1,8) = 0.8, p = 0.38) on tyrosine hydroxylase

phosphorylation. Post hoc t tests indicated DOC pretreatment increased tyrosine

 
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hydroxylase activation, but AngII treatment did not (Vehicle/Vehicle: 0.4±0.3,

DOC/Vehicle: 5.6±1.8, Vehicle/AngII: 1.6±0.8, DOC/AngII: 6.2±0.4; p<0.045, p = 0.23,

p<0.001). Similarly, DOC pretreatment conditions significantly increased tyrosine

hydroxylase phosphorylation compared to vehicle in the core of the nucleus accumbens

(Vehicle/Vehicle: 0.3±.3, DOC/Vehicle: 5.2±0.6, Vehicle/AngII: 4.0±1.1, DOC/AngII:

5.1±0.6, p<0.01, p = 0.08, p<0.01). However, a 2-way ANOVA revealed a main effect for

DOC, trend for AngII, and a significant interaction between the two hormones on tyrosine

hydroxylase phosphorylation in the accumbens shell (F(1,7) = 31.7, p<0.001; F (1,7) =

4.2, p = 0.08; F(1,7) = 19.8, p<0.01). Post hoc tests revealed that each treatment

increased phosphorylation of the enzyme compared to vehicle, but DOC pretreatment

did not significantly enhance phosphorylation compared to AngII alone (Vehicle/Vehicle:

0.7±0.6, DOC/Vehicle: 6.3±0.2, Vehicle/AngII: 4.4±0.5, DOC/AngII: 5.0±0.7; p<0.01,

p<0.05, p<0.05). In summary, DOC pretreatment increased phosphorylation levels of

tyrosine hydroxylase in the ventral tegmental area and accumbens, while AngII only had

an effect in the shell of the accumbens.

DISCUSSION
 

AngII and aldosterone increase the willingness to work for sodium. For example,

classic studies demonstrate that rats run faster to receive a sodium reward if both

aldosterone and AngII are present compared with either hormone alone (D. M. Zhang et

al., 1984). The mesolimbic dopamine system is known to be involved in generating goal

directed movement, including sodium ingestion after depletion (Carelli, 2002; Roitman et

al., 2002). These experiments aimed to define changes in motivation and mesolimbic

 
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activity underlying sodium appetite when aldosterone and AngII are present. Our

behavioral studies indicated that when both aldosterone and AngII are present, rats are

highly motivated to press levers for sodium compared to water. Functional

neuroanatomical studies showed increased activation in the nucleus accumbens and

ventral tegmental area after AngII treatment, but DOC pretreatment did not significantly

enhance this effect. However, PVN inactivation by lidocaine did enhance AngII-induced

mesolimbic activation in the ventral tegmental area and core of the accumbens.

Contrarily, tyrosine hydroxylase activation in the ventral tegmental area is mainly due to

DOC pretreatment. Thus, the present studies implicate a selective drive for sodium,

prompted by the combination of AngII-induced mesolimbic neural activity and DOC-

induced dopamine synthesis.

Increase in motivation for sodium

Under normal conditions, a rat will avoid concentrated sodium solutions

(Berridge, Flynn, Schulkin, & Grill, 1984). However, when stimulated by RAAS, rats will

exhibit robust sodium appetite for these same solutions, as demonstrated in our

progressive ratio studies. Progressive ratio schedules are widely used to measure

incentive value, as the response requirements to obtain reinforcements increases

according to the same rule until the rat stops responding; the highest ratio completed,

the breakpoint, provides a measure of reinforcer value (Sclafani & Ackroff, 2003).

Progressive ratio schedules emulate some aspects of foraging: resources are depleted

in a given area the more they are consumed, so they become increasingly more difficult

to find (Starr & Rowland, 2006). While some studies have used this method to examine

 
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sodium appetite after depletion, our experiments allowed us to compare how AngII and

aldosterone influence effort-value for both sodium and water. Our studies revealed a

dramatic shift in behavior with DOC/AngII treatment: the breakpoint ratio for sodium

compared to water increased significantly for those rats treated with both hormones

versus either hormone alone. Although AngII typically stimulates a high ratio of water to

saline intake (Prakash & Norgren, 1991), pretreatment with DOC considerably alters

behavior such that rats are more motivated to work for sodium than water when both

hormones are present. While the initial separate targets of aldosterone and AngII are

known, the downstream integration areas remain undefined. However, several reports

indicate neuroanatomical connections between osmosensitive brain areas and the

mesolimbic dopamine system (Lucas et al., 2007; Shekhtman et al., 2007). Although

the precise role of dopamine remains in question, studies depleting rats of dopamine

with 6-hydroxydopamine indicated that dopamine systems are necessary for motivation,

but do not participate in the hedonic pleasure (Berridge & Robinson, 1998).

The role of mesolimbic activity in sodium appetite

The mesolimbic dopamine system, which contains dopaminergic neurons in the

ventral tegmental area that project to the nucleus accumbens, is implicated in regulating

sodium appetite (Carelli, 2002; Roitman et al., 2002). Our finding that DOC/AngII

increased motivation for sodium, as demonstrated by lever pressing in the progressive

ratio experiment, further implicated the mesolimbic dopamine system. However, the

functional neuroanatomical studies revealed cFos activation in both the ventral

tegmental area and nucleus accumbens depended on AngII but not DOC treatment. In

 
96  
 
effect, these data suggest that treatment with both hormones does not enhance

neuronal excitability in these brain areas, and therefore cannot fully explain the increase

in motivation for sodium. Suprisingly, lidocaine in the PVN did enhance AngII-induced

activation in these brain regions, implying that this preparation of enhanced sodium

appetite does not in fact mimic that of DOC/AngII. It is possible that cFos expression is

not a good measure of DOC effects, as injections of DOC are given for days in advance,

and not perfectly timed to maximal cFos expression (Sagar, Sharp, & Curran, 1988).

This is in contrast to lidocaine injections, which took place minutes before AngII injection,

and thereby roughly an hour before cFos expression.

AngII-induced cFos expression in the mesolimbic areas is likely the result of

transynaptic activation. The ventral tegmental area does not have AngII receptors, so it

is not possible for this to be the primary site of activation (Brown, Steward, Ge, &

Barnes, 1996). In addition, lesions in initial brain targets that synapse onto the ventral

tegmental area ablate sodium appetite, indicating that upstream brain areas are

activated and required for this behavior (Fitts et al., 2004). Cells activated with

DOC/AngII treatment compared to AngII alone may be specific to motivation for sodium,

given that the combination of these hormones only potentates sodium intake. However,

concentrated nuclear staining on these brain sections makes this discrimination difficult.

Since cells were not double labeled for neural or dopaminergic markers, the cell types

activated are unknown; however, in the ventral tegmental area, the majority of neurons

present are dopaminergic (Oades & Halliday, 1987). Normally, the accumbens would

next integrate neural signals and organize the motivated behavior (Cardinal, Parkinson,

Hall, & Everitt, 2002), however, these rats did not ingest reinforcements before their

brains were examined. In studies that allowed rats access to salt after depletion, the

 
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nucleus accumbens shell rather than the core was more highly activated (Lucas et al.,

2003). As the rats did not have access to sodium, significant differences in cFos

activation between the shell and core of the accumbens were not observed.

The role of tyrosine hydroxylase in sodium appetite

An increase in neural activation in the mesolimbic system likely includes

enhanced dopaminergic activity. This may involve increases in tyrosine hydroxylase, a

rate-limiting enzyme for dopamine synthesis that is mainly located in the soma of

dopamine neurons in the ventral tegmental area, but can be axonally transported into

the terminals projecting to the nucleus accumbens (Pickel, Joh, & Reis, 1975). Our

results indicate that aldosterone treatment increases tyrosine hydroxylase expression in

the ventral tegmental area and nucleus accumbens shell. Aldosterone was injected for

three days before collecting tissue; this time frame is conducive to gene transcription,

explaining the increase in tyrosine hydroxylase expression. However, AngII, given only

five minutes before enzyme levels were quantified, significantly increased the enzyme in

the ventral tegmental area. Though the timeline may appear brief, our findings are in

agreement with a study examining tyrosine hydroxylase levels during conditioned place

preference for morphine-related cues: these authors demonstrate increased tyrosine

hydroxylase activity in the ventral tegmental area within five minutes of the task (Liang et

al., 2012).

Besides increases in tyrosine hydroxylase expression, there may be more Ser31

phosphorylation of the enzyme, which augments catalytic activity and allows synthesis of

dopamine (Colby, Thompson, & Patrick, 1989). Aldosterone treatment increases

 
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tyrosine hydroxylase phosphorylation in both the ventral tegmental area and nucleus

accumbens, likely indicating more dopamine production. Dopamine release in the

nucleus accumbens is correlated with both appetitive and consummatory aspects in

natural reward paradigms (Roitman, Stuber, Phillips, Wightman, & Carelli, 2004). Our

studies indicate that aldosterone primes the mesolimbic system by increasing tyrosine

hydroxylase activity, whereas AngII rapidly increases neuronal activity. Together, these

hormones may result in enhanced accumbal dopamine, which would correlate with the

increase in lever presses for sodium after DOC/AngII treatment in the progressive ratio

task. Through multiple techniques, we have demonstrated that aldosterone and AngII

together result in selective drive for sodium, which is associated with AngII-induced

increases in mesolimbic neural activity and aldosterone- induced increases in dopamine

synthesis.

The neural modules of sodium appetite

AngII acts initially on AT1R in circumventricular organs, such as the SFO and

OVLT (Lind, Swanson, & Ganten, 1984; McKinley et al., 1992; Tanaka, Kaba, Saito, &

Seto, 1986; Weiss & Hatton, 1990). The OVLT is known to project to the lateral

hypothalamus (Camacho & Phillips, 1981). Moreover, studies have revealed that

orexinergic neurons in the lateral hypothalamus project to and modulate dopaminergic

neurons in the ventral tegmental area, which then projects to the nucleus accumbens

(Fadel & Deutch, 2002; Narita et al., 2006). For instance, injection of a dopamine

receptor antagonist into the lateral hypothalamus abolishes sodium appetite, whereas

stimulation of the lateral hypothalamus stimulates dopamine release, enhancing sodium

 
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appetite (Hoebel, Hernandez, Schwartz, Mark, & Hunter, 1989; Liedtke et al., 2011). As

the VTA does not contain AT1R, these connections would explain how AngII enhances

the release of striatal dopamine in freely moving rats within minutes, which then is

associated with an increase in drinking behavior (Brown et al., 1996; Hoebel, Rada,

Mark, & Hernandez, 1994).

Although there are mineralocorticoid receptors present in the ventral tegmental

area and nucleus accumbens, it is unlikely that this is the mechanism by which

aldosterone affects mesolimbic activity. The available literature suggests that

mineralocorticoids act initially on NTS neurons, which contains HSD2 enzymes to

inactivate the much more highly concentrated glucocorticoids in the brain (Geerling &

Loewy, 2008). It is through both direct and indirect synaptic connections to the VTA that

aldosterone may affect dopaminergic activity. The indirect connections consist of an

NTS projection to the PVN, possibly via the BNST, which then projects to the OVLT,

where interaction with AngII may be possible. Whether it is at the OVLT, or VTA just

downstream, AngII and aldosterone eventually integrate their actions to cause this

potentiation in sodium appetite. Our data suggests that the combination of AngII and

aldosterone increases mesolimbic neuronal activation and tyrosine hydroxylase activity

prompting a selective drive for sodium.

 
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ACKNOWLEDGEMENTS
 

This work was supported by the National Institutes of Health Grants R01 HL091314. We

greatly appreciate the expertise of Dr. Scott Kanoski while conducting the progressive

ratio experiments. Additionally, we thank Harvey Grill for the use of his progressive ratio

conditioning boxes.

 
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Figure 4.1. DOC/AngII treatment increases motivation for sodium.

Panel A. Line graphs illustrating cumulative presses over time for 3% Saline and Water after
Vehicle, DOC, AngII, or DOC plus AngII treatments (n = 12/group). Panel B. Bar graphs
illustrating total lever presses for 3% saline and water after Vehicle, DOC, AngII, or DOC plus
AngII treatments (n=12/group). DOC plus AngII treatment increased sodium presses while AngII
alone increased water presses. Panel C. Bar graphs illustrating the breakpoint ratio of sodium to
water after Vehicle, DOC, AngII, or DOC plus AngII (n = 12/group). DOC plus AngII causes the
highest sodium to water breakpoint ratio. Abbreviations: AngII= Angiotensin II, DOC =
deoxycorticosterone acetate, Veh = vehicle.

 
102  
 
  
 

Figure 4.2. DOC/AngII treatment increases cFos expression in the ventral tegmental area
and nucleus accumbens.

Bar graphs illustrating cFos cell counts in the ventral tegmental area and nucleus accumbens
core and shell after either vehicle or DOC pretreatment followed by icv vehicle or AngII (n =
12/group). AngII and DOC/AngII treatment increased cFos expression in the ventral tegmental
area, with DOC/AngII inducing the most immunostaining. In the core and shell, each treatment
condition induces cFos immunostaining compared with vehicle; DOC/AngII induced the highest
amount of immunostaining. Representative images of the ventral tegmental area and nucleus
accumbens (coronal plane, 10x) in each treatment condition are shown above the bar graphs.
Abbreviations: AngII = Angiotensin II, DOC = deoxycorticosterone acetate, NuAcc= Nucleus
Accumbens, Veh = Vehicle, VTA= Ventral Tegmental Area

 
103  
 
  
Figure 4.3. PVN inactivation enhances AngII-induced cFos expression in the ventral
tegmental area and nucleus accumbens.

Bar graphs illustrating cFos cell counts in the ventral tegmental area and nucleus accumbens
core and shell after either vehicle or lidocaine pretreatment in the PVN followed by icv vehicle or
AngII (n = 12/group). AngII and DOC/AngII treatment increased cFos expression in the ventral
tegmental area, with DOC/AngII further enhancing this activation. In the core and shell, each
treatment condition induces cFos immunostaining compared with vehicle; DOC/AngII further
enhances activation in the core but not the shell. Representative images of the ventral tegmental
area and nucleus accumbens (coronal plane, 10x) in each treatment condition are shown above
the bar graphs. Abbreviations: AngII = Angiotensin II, NuAcc= Nucleus Accumbens, Veh =
Vehicle, VTA= Ventral Tegmental Area

 
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Figure 4.4. DOC/AngII treatment increases tyrosine hydroxylase activation in the ventral
tegmental area and nucleus accumbens.

Panel A. Bar graphs illustrating tyrosine hydroxylase levels in the ventral tegmental area and
nucleus accumbens (core and shell) after either oil or DOC pretreatment followed by icv
treatments with AngII (n = 3/group). Each treatment increased tyrosine hydroxylase expression
in the ventral tegmental area, but there was only a trend for increased tyrosine hydroxylase
expression in the nucleus accumbens core and shell compared to vehicle. Representative
western blot images of tyrosine hydroxylase are shown above each quantified bar. Panel B. Bar
graphs illustrating phosphorylated tyrosine hydroxylase levels in the ventral tegmental area and
nucleus accumbens (core and shell) after either oil or DOC pretreatment followed by icv
treatments with AngII (n = 3/group). Only DOC pretreatment significantly increased
phosphorylated tyrosine hydroxylase expression in the ventral tegmental area compared to
vehicle. However, each treatment increased phosphorylated tyrosine hydroxylase expression in
the nucleus accumbens core and shell. Representative western blot images of phosphorylated
tyrosine hydroxylase are shown above each quantified bar. Panel C. Bar graphs illustrating
phosphorylated tyrosine hydroxylase levels normalized to total tyrosine hydroxylase levels in the
ventral tegmental area and nucleus accumbens (core and shell) and after either oil or DOC
pretreatment followed by icv treatments with AngII (n = 3/group). Only DOC/AngII significantly
increased normalized phosphorylated tyrosine hydroxylase expression in the ventral tegmental
area compared to vehicle. However, each treatment increased normalized phosphorylated
tyrosine hydroxylase expression in the nucleus accumbens. Abbreviations: AngII = Angiotensin
II, NuAcc= Nucleus Accumbens, phospho-TH = phosphorylated tyrosine hydroxylase, TH =
tyrosine hydroxylase, Veh = Vehicle, VTA= Ventral Tegmental Area

 
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 CHAPTER 5: GENERAL CONCLUSIONS AND FUTURE DIRECTIONS
 

Sodium is a necessary part of our diet; we do not crave other minerals in the

same way we desire sodium (Geerling & Loewy, 2008). Sodium appetite is a crucial

participant in fluid balance, which determines one’s fluid volume, blood circulation, and

neuronal function (Andersson, 1977). Not only is this behavior vital to homeostasis, but

also it has been linked to an increased risk of hypertension (Vollmer et al., 2001). Yet,

the biological basis of sodium appetite remains undefined. With this in mind, the goal of

this thesis was to elucidate the cellular signaling and neural circuitry underlying sodium

appetite.

The previous chapters extend our knowledge of the etiology of sodium appetite,

identifying signaling proteins and brain areas required for this behavior. Specifically, in

Chapter 2, I described the role of MAPK signaling in sodium appetite using a preparation

of endogenous AngII production. This signaling protein was found to be specific to

sodium appetite, as its inhibition did not affect thirst or neurohypophyseal secretion.

Following these discoveries, I demonstrated in Chapter 3 that when both aldosterone

and AngII are present, IP3 becomes more important in modulating sodium appetite. We

next examined brain areas important for potentiation of sodium appetite by both

functional neuroanatomy and reversible lesions. Our data supported the hypothesis that

DOC suppresses oxytocin secretion in the PVN, disinhibiting the OVLT for further AngII

activation, and thereby potentiating sodium appetite. Chapter 4 targeted downstream

brain areas involved in aldosterone- and AngII-induced sodium appetite, identifying the

mesolimbic system as another possible integration site for these hormones to augment

the drive for sodium. Together, these experiments determine specific signaling proteins

 
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and brain sites required for aldosterone and AngII to induce sodium appetite. Below I

discuss the implications and important future directions supported by these studies.

Comparing Sodium Appetite Preparations

As mentioned in the Introduction, different preparations of sodium appetite

diverge in both mechanism and time course for development of the behavior. Taking

this into consideration, it is important to compare and contrast the two preparations I

utilized in my dissertation, as it may better explain my results. In Chapter 2, I

administered the diuretic furosemide, followed by a low dose of the ACE inhibitor

captopril, which increases plasma levels of both aldosterone and AngI (Thunhorst et al.,

1994). The resultant level of plasma aldosterone is high relative to control rats (B. Lu,

Yang, Chen, Yang, & Yan, 2009; Omouessi, Falconetti, Chapleur, Fernette, & Thornton,

2007). However, as aldosterone cannot penetrate the blood brain barrier efficiently, it is

likely that it remains in the plasma to counteract the effect of furosemide on the kidney

(Pardridge & Mietus, 1979). Concurrently, captopril blocks AngI conversion to AngII

peripherally, resulting in nearly ten times more plasma AngI than a control rat, allowing

for maximum conversion to AngII centrally (B. Lu et al., 2009; Thunhorst et al., 1994).

Importantly, my pharmacological data from Chapter 2 supports the key role of central

AngII in this preparation; the central AT1 receptor mediates the majority of the sodium

ingestion these studies.

On the other hand, our DOC/AngII preparation of sodium appetite in Chapter 3

and 4 allows central action of both hormones. Subcutaneous DOC easily crosses the

blood brain barrier due to its structure, giving it access to the hindbrain, where MR and

HSD2 reside (Pardridge & Mietus, 1979). Additionally, this preparation does not cause

natriuresis, so DOC action is not required peripherally. In fact, studies using both
 
107  
 
peripheral and central MR antagonists revealed that central aldosterone is of utmost

importance in this preparation of sodium appetite (Sakai et al., 1986; Sakai et al., 1996).

In contrast to the furo/cap preparation, I administered AngII directly into the lateral

ventricle of the brain, allowing it to act on its type 1 receptors in the forebrain. As the low

doses of each hormone in this preparation are not enough to stimulate sodium appetite

on their own, their interaction is required to elicit such a robust behavior (Epstein, 1982;

Fluharty & Epstein, 1983). To summarize, sodium appetite induced by furo/cap can be

attributed to actions of central AngII, whereas the behavior induced by low doses of

DOC and AngII requires central interaction of both hormones.

Cellular Signaling Underlying Sodium Appetite

The AT1 receptor and MR induce many signaling pathways, but the implications

of MAPK and IP3 have been of recent focus due to their demonstrated behavioral

consequences (Daniels D, Mietlicki EG, Nowak EL, Fluharty SJ, 2009; Fleegal &

Sumners, 2003). In agreement with previous findings administering exogenous central

AngII, I found a treatment inducing endogenous AngII utilized MAPK activation to prompt

sodium appetite (Daniels D, Mietlicki EG, Nowak EL, Fluharty SJ, 2009; Daniels D, Yee

DK, Faulconbridge LF, Fluharty SJ, 2005; Felgendreger, Fluharty, Yee, & Flanagan-

Cato, 2013b). However, when actions of central aldosterone and AngII were combined,

MAPK was not required for the potentiation of sodium appetite.

These opposing results can be explained pharmacologically or mechanistically.

Pharmacologically, it may be that the dose of the MAPK inhibitor used in the DOC/AngII

studies was simply not high enough to counteract an amplified MAPK signal that arises

when both hormones are present. While furo/cap-induced sodium appetite is attributed

to the AT1R, which may prompt a given level of MAPK signaling, DOC/AngII-induced
 
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sodium appetite may generate considerably higher levels of MAPK signaling. This could

be due to an upregulation of AT1R induced by DOC pretreatment, non-genomic MR

MAPK signals, or both (Sakai, McEwen, Fluharty, & Ma, 2000; Wilson, Sumners,

Hathaway, & Fregly, 1986). However, the western blot data from Chapter 3 does not

show further AT1R-MAPK activation with DOC pretreatment, so this explanation does

not seem likely. In contrast, DOC pretreatment may induce mechanistic changes that

substitute the role of MAPK. For example, perhaps the normal function of AT1R-MAPK

is to desensitize oxytocin receptors in the OVLT. If DOC inhibits oxytocin release from

the PVN during pretreatment days, oxytocin will not bind to its receptors in the OVLT, so

the actions of MAPK are no longer needed. Future studies should examine the relevant

cellular consequences of MAPK, and determine how DOC may interfere during

pretreatment.

Not only does the interaction of aldosterone and AngII no longer require MAPK

activation, but IP3 signaling contributes to the potentiation of sodium appetite. Another

study negated a role for PKC in exogenous AngII-induced sodium appetite; However, I

did not test the contribution of IP3 signaling in my furo/cap model of sodium appetite,

therefore it cannot be ruled out (Daniels D, Mietlicki EG, Nowak EL, Fluharty SJ, 2009;

Felgendreger et al., 2013). In support of a role for IP3 in sodium appetite, a previous

study used an analog of AngII that only induces AT1R-MAPK signaling, which does not

elicit as much sodium ingestion as AngII itself (Daniels D, Yee DK, Faulconbridge LF,

Fluharty SJ, 2005). Moreover, examination of sodium ingestion data from both Chapter

2 and 3 will reveal that inhibition of either MAPK or IP3 signaling does not completely

ablate the behavior. In effect, these data suggest that neither signaling protein is solely

responsible for the behavior.

 
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 After comparing the role of signaling proteins in two preparations of sodium

appetite, the hypothesis that divergent AT1R signaling causes two separate behaviors is

not well supported. In particular, the role of these signaling proteins changed when

aldosterone and AngII were both elevated. Moreover, these signaling pathways are

ubiquitous in the brain, making it unlikely that their function is specific to these behaviors.

The more apt explanation is that these molecules have specific functions within circuits

underlying thirst and sodium appetite.

Circuits for thirst and sodium appetite overlap in certain brain regions like the

OVLT. It is possible that there are subsets of cells within each brain region that use IP3

or MAPK signaling to contribute to each behavior. However, the subset of cells

important for each behavior may change depending on input from other brain areas in

the circuit, such as when aldosterone is present. My studies suggest that aldosterone

enhances the efficiency of neurons responsive to AngII, negating the need for MAPK

signaling in one subset of cells, and shifting to activation of IP3 in another set of cells to

contribute to the potentiation of sodium appetite. While parenchymal injections of

signaling inhibitors may more clearly delineate where signaling proteins exert their

effects on behavior, targeting subsets of cells within a small region like the OVLT may be

extremely difficult.

Neural Circuitry Underlying Sodium Appetite

Although I did not administer brain region-specific injections of signaling

inhibitors, I did examine protein activation in particular brain areas by both western blot

and immunohistochemistry. This allowed me to identify important brain regions involved

in the circuit underlying sodium appetite. In Chapter 2 and 3, MAPK activation was

induced by furo/cap treatment and icv AngII, respectively, in both the circumventricular
 
110  
 
organs and hypothalamus. Additionally, Chapter 3 revealed increased AngII-induced

cFos, used as a proxy for IP3 signaling, in these same brain areas. However, as AngII

causes two separate behaviors, namely, thirst and sodium appetite, it is difficult to

determine the contribution of these brain regions to sodium appetite alone. To

accomplish this, one would have to correlate brain activation with the effect of site-

specific inhibitors on behavior.

The Oxytocin Disinhibition Hypothesis

Conversely, one could examine brain activation after a treatment that specifically

enhances sodium appetite. Comparing AngII- with DOC/AngII-induced brain activation

gives us the opportunity to distinguish which brain regions play a significant role in

sodium appetite, as DOC pretreatment enhances only sodium appetite and not thirst. In

Chapter 3, DOC plus AngII uniquely enhanced OVLT activation and reduced PVN

activity, suggesting that these brain regions are important in potentiating sodium

appetite. Together with the oxytocin secretion data from Chapter 3, we hypothesized

that DOC suppresses oxytocin in the PVN, disinhibiting the OVLT to induce sodium

appetite. This would suggest that the OVLT contains oxytocin inputs from the PVN,

which is supported by the literature (Buijs, 1978; Kelly & Watts, 1996; Yoshimura et al.,

1993).

However, the OVLT may participate in circuits for both sodium appetite and thirst.

Supporting this concept, infusion of AngII into the OVLT induces both water and sodium

appetite (Fitts et al., 1990). Perhaps within the OVLT, a certain subset of cells is

involved in sodium appetite while another is involved in thirst. Taking our data into

account, the subset of cells receiving oxytocinergic input from the PVN would only be

involved in sodium appetite. I have acquired preliminary data labeling the OVLT with
 
111  
 
oxytocin after hormone treatments to determine the exact location of this subset of cells

(see Figure A.1 in the Appendix). This idea of behavior-specific cell clusters is

reinforced by previous studies lesioning the OVLT, which reveal different behavioral

consequences depending on the size and location of the cut (Johnson & Thunhorst,

1997). In particular, lesioning cells with oxytocin input from the PVN would enhance

sodium appetite, while destroying thirst or sodium appetite cells projecting downstream

would abrogate one or both behaviors. Further studies could classify locations of these

cell clusters within the OVLT after different hormone treatments to determine which are

important for thirst versus sodium appetite (Miller, Wang, Gray, Salkoff, & Loewy, 2013).

Beyond correlating brain activation, I used a reversible lesion to causally link

PVN inactivation and enhanced AngII-induced sodium appetite. I aimed to mimic the

actions of DOC in this experiment, but some key differences should be discussed.

Administration of lidocaine into the PVN globally inactivates action potentials in all cells

within the PVN, while DOC most likely targets the magnocellular oxytocin secreting

neurons. Therefore, it is possible that lidocaine may inhibit other PVN neuronal

phenotypes, such as preautonomic or vasopressinergic neurons, but as water intake

was unaffected, this is improbable. As a separate concern, the dose of lidocaine given

was based on studies examining pressor responses, and may or may not completely

inactivate all PVN neurons (Fernandes et al., 2007; Flanagan et al., 1992). Failure to

completely inactivate these PVN neurons might explain why sodium appetite was not as

robust compared to when DOC and AngII work together. On the other hand, perhaps

DOC has additional functions besides inhibiting oxytocin release, so recreating only one

of these functions in the lidocaine experiment does not fully potentiate sodium appetite.

Future experiments targeting inhibition of only the magnocellular oxytocin neurons,

perhaps with optogenetics, may help clarify which of these possibilities is true.
 
112  
 
 The path by which aldosterone may influence PVN oxytocin secretion remains

undefined, though some tracing studies identify the bed nucleus stria terminalis (BNST)

as the main candidate to transfer information from the NTS to the PVN (Geerling &

Loewy, 2009). The BNST contains GABAergic neurons that may receive input from the

NTS and project to the PVN, allowing for inhibition of oxytocin (Cullinan et al., 1993;

Dong et al., 2001). Preliminary data examining cFos activation revealed that DOC

increases BNST activity (See Figure A.2 in the Appendix). This would suggest that the

BNST is active when DOC is present, possibly sending an inhibitory signal to the PVN to

prevent oxytocin secretion. A more telling future experiment to determine these

connections would involve injecting a retrograde tracer into the PVN and an anterograde

tracer into the NTS and examining if they colocalize in the BNST.

Mesolimbic Activity Underlying Sodium Appetite

Upon discerning some of the forebrain regions important for sodium appetite, it

was logical to next examine brain regions that may be further downstream in the circuit

leading to a robust sodium appetite. The mesolimbic dopamine system, thought to be

involved in natural reward, was a good candidate for investigation. Chapter 4

demonstrated that the combination of DOC plus AngII resulted in a selective motivation

for sodium compared to water. However, DOC pretreatment only slightly enhanced

presses for sodium compared to AngII alone. Perhaps the doses of DOC and AngII

administered were only capable of stimulating the demonstrated amount of work for such

a highly concentrated saline solution, hitting a “ceiling”. Using a less concentrated saline

reward in the progressive ratio experiment could test this idea. Nevertheless, the

amount of 3% saline ingested in these studies was not sufficient to satisfy the daily

requirement for salt. In addition, the number of presses over time did not approach the
 
113  
 
limits of what is physically possible. Regardless, I think the most interesting data is not

the total presses for saline, but the shift from pressing water to saline when both

hormones are present. One cannot visualize this shift in behavior in Chapter 3 because

the intake data is reported at final time points. Differential circuit activation undoubtedly

underlies this shift in behavior.

Although the combination of DOC plus AngII induces a selective drive for

sodium, it is mainly AngII treatment that induces cFos activation in the VTA and nucleus

accumbens. It is possible that cFos expression is not a good measure of DOC effects

for two reasons: 1) injections of DOC are given for days in advance, and not perfectly

timed to maximal cFos expression (Sagar et al., 1988) and 2) the dose of DOC given is

too low to induce cFos expression (Pietranera et al., 2001). However, DOC and AngII

potentiation of cFos expression was seen in the OVLT in Chapter 3, discrediting these

possibilities. Similar to the number of sodium presses, DOC/AngII-cFos activation is not

significantly different from AngII alone. This, too, might be a result of a “ceiling”, as the

images of these brain areas show very intense cFos staining; it is feasible that the

maximum number of cells are activated in this condition. In contrast with the behavior,

we have only captured a snapshot of cell activation at one time point. Compared to

AngII treatment, we can attribute additional activated cells to motivation for sodium in the

combined hormone treatment, but concentrated nuclear staining makes this difficult.

Activity in these mesolimbic areas likely indicates motivation for both water and sodium;

Though DOC/AngII potentiates sodium intake, it also reduces water intake, so the total

activation in this brain region may not be further enhanced compared to AngII alone. It

might be informative to use treatments that induce only one behavior and examine the

pattern of activation in these brain areas, possibly identifying subregions of behavioral

specificity.
 
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 In contrast to DOC/AngII administration, lidocaine injected into the PVN in

combination with icv AngII enhanced mesolimbic activation. This negates the idea that

there is a “ceiling” for cFos activation in mesolimbic areas after DOC/AngII treatment.

Additionally, this indicates that lidocaine/AngII enhancement of sodium appetite does not

in fact mimic that of DOC/AngII treatment. On the other hand, the lidocaine/AngII data

does indicate that rapid inactivation of the PVN enhances mesolimbic activation, similar

to its effects on the OVLT in Chapter 3. Though the lateral hypothalamus is the most

likely candidate for transfer of information between the OVLT and VTA, I did not validate

this in my experiments (Camacho & Phillips, 1981; Fadel & Deutch, 2002). This would

be an important brain area to examine in future studies in order to have a more complete

picture of the circuit underling sodium appetite. While cFos activation in the lateral

hypothalamus after these treatments would be an important first step, tracing studies

would provide a more elegant demonstration of connections within this part of the circuit

for sodium appetite.

In accordance with previous literature, our preparation of sodium appetite was

associated with an increase in markers of dopamine synthesis, indicating the anticipation

of a reward (Roitman, Schafe, Thiele, & Bernstein, 1997; Sunsay & Rebec, 2008).

Specifically, aldosterone increased tyrosine hydroxylase expression in the ventral

tegmental area and accumbens. Aldosterone was injected for three days before

collecting tissue, a time frame conducive to gene transcription. However, AngII

increased tyrosine hydroxylase expression within five minutes in the ventral tegmental

area, which is in accordance with previous studies (Liang et al., 2012). This rapid

increase in tyrosine hydroxylase immunoreactivity may be due to: 1) translation of

preexisting mRNA or 2) a change in location or conformation of the protein that allows it

to become immunoreactive following hormone treatment (Cao et al., 1996). Future

 
115  
 
experiments could inspect tyrosine hydroxylase mRNA after different treatments,

including acute injections of DOC, to determine how these dynamic changes take place.

Aldosterone also increased tyrosine hydroxylase phosphorylation, which would

allow the enzyme to synthesize more dopamine, possibly enhancing motivation for

sodium. However, DOC treatment alone does not induce much sodium appetite, yet

marked increases in tyrosine hydroxylase activity were still observed. As a result, the

correlation between VTA tyrosine hydroxylase levels and sodium intake is weak (See

Figure A.3 in the Appendix). Nevertheless, DOC did induce water intake in our

progressive ratio studies, and as tyrosine hydroxylase activity would not discern between

motivation for water versus sodium, this may explain the disconnect. In contrast, the

correlation between OVLT activity and VTA tyrosine hydroxylase is strong, supporting

the idea that OVLT activity dictates downstream dopamine activity in the VTA. This

makes sense considering both brain areas are involved in thirst and sodium appetite, so

total activation levels are more likely to correlate. While tyrosine hydroxylase expression

indicates increased dopamine activity in sodium appetite, measuring dopamine levels

through microdialysis in real time would reveal much more detailed information on how

mesolimbic activity is affected during dynamic changes in motivation for sodium.

Thesis Contributions and the Future of Sodium Appetite

These studies advance our understanding of the central pathways controlling

sodium appetite, a behavior that is integral to our survival. I have identified signaling

proteins involved in the regulation of aldosterone- and AngII-induced sodium appetite,

but more importantly, revealed key brain areas and possible connections within the

circuitry underlying the potentiation of sodium appetite. In particular, my studies have

demonstrated that aldosterone relieves an inhibitory oxytocin signal from the PVN to the
 
116  
 
OVLT to enhance sodium appetite. Preliminary data suggests aldosterone may

inactivate oxytocin release through the BNST. In addition, my data suggest AngII and

aldosterone actions converge on the VTA to enhance sodium appetite. Nevertheless,

much work remains in delineating the complete circuit responsible for sodium appetite.

This would include tracing studies that show clear connections as demonstrated in the

diagram below (Figure 5.1). Moreover, it would be beneficial to perform optogenetic

studies activating or inactivating these key connections and observing the presence or

absence of the behavior. Lastly, it should be pointed out that this circuitry model of

sodium appetite is oversimplified, as there are other excitatory and inhibitory systems

underlying sodium appetite. Regardless, this thesis provides advances in understanding

this crucial behavior.

 
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Figure 5.1. Model Circuit Diagram for Aldosterone- and AngII-induced Sodium Appetite

Pictured above is a model circuit diagram explaining proposed connections in aldosterone- and
AngII-induced sodium appetite. Connections pictured in black have been established by my
data and previous literature. Brain areas in gray are expected connections between the
regions in black, based on my preliminary data and previous literature. Dotted lines indicate
multiple synaptic connections leading to expression of the behavior. Abbreviations: AngII =
Angiotensin II, BNST = Bed Nucleus Stria Terminalis, DA = Dopamine, DOC=
Deoxycorticosterone Acetate, H2O = Populations of cells responsive to signals for water intake
+
(thirst), LH = Lateral Hypothalamus, Na = Populations of cells responsive to signals for
sodium intake (sodium appetite), NuAcc = Nucleus Accumbens, NTS = Nucleus of the Solitary
Tract, OT= oxytocin, OVLT = Organum Vasculosum Lateral Terminalis, PVN = Paraventricular
Nucleus of the Hypothalamus, SFO = Subfornical Organ, VTA = Ventral Tegmental Area

 
118  
 
 APPENDIX

Figure A.1. AngII-induced oxytocin staining in the OVLT is reduced by DOC pretreatment.

Bar graphs illustrating the optical density for oxytocin immunohistochemical staining in the OVLT
after Vehicle, DOC, AngII, or DOC/AngII treatment (n = 3/group). Representative images of
oxytocin staining of OVLT coronal sections (10x) in each treatment are shown above the graphs.
AngII induced significant oxytocin staining, which was reduced by DOC pretreatment.
Abbreviations: AngII = Angiotensin II, DOC = Deoxycorticosterone Acetate

 
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Figure A.2. DOC increases cFos activation in the BNST.

Bar graphs illustrating cFos staining in the OVLT after Vehicle, DOC, AngII, or DOC/AngII
treatment (n = 5-6/group). Representative images of cFos staining of OVLT coronal sections
(10x) in each treatment are shown above the graphs. DOC increased cFos staining in the OVLT,
regardless of icv treatment. Abbreviations: AngII = Angiotensin II, DOC = Deoxycorticosterone
Acetate

 
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Figure A.3. Tyrosine Hydroxylase in the Ventral Tegmental Area correlates with cFos in the
OVLT, but not sodium intake.

Correlations of VTA Tyrosine Hydroxylase with both Sodium Intake (left) and cFos in the OVLT
(right) after Vehicle, DOC, AngII, and DOC/AngII treatments. Tyrosine Hydroxylase levels have a
weak correlation with sodium intake, but a strong correlation with OVLT cFos. Abbreviations:
AngII = Angiotensin II, DOC = Deoxycorticosterone Acetate, TH = tyrosine hydroxylase, VTA =
ventral tegmental area

 
121  
 
 REFERENCES

Abbott, W. E. (1946). A review of the present concepts on fluid balance. The American
Journal of the Medical Sciences, 211, 232-239.

Alderman, M. H. (2000). Salt, blood pressure, and human health. Hypertension, 36(5),
890-893.

Almli, C. R. (1970). Hyperosmolality accompanies hypovolemia: A simple explanation of

additivity of stimuli for drinking. Physiology & Behavior, 5(9), 1021-1028.

Anderson, N. S.,3rd, & Fanestil, D. D. (1976). Corticoid receptors in rat brain: Evidence
for an aldosterone receptor. Endocrinology, 98(3), 676-684.

Andersson, B. (1977). Regulation of body fluids. Annual Review of Physiology, 39, 185-
200.

Andersson, B., Eriksson, L., Fernandez, O., Kolmodin, C. G., & Oltner, R. (1972).
Centrally mediated effects of sodium and angiotensin II on arterial blood pressure
and fluid balance. Acta Physiologica Scandinavica, 85(3), 398-407.

Andersson, B., Leksell, L. G., & Lishajko, F. (1975). Perturbations in fluid balance
induced by medially placed forebrain lesions. Brain Research, 99(2), 261-275.

Avrith, D. B., & Fitzsimons, J. T. (1980). Increased sodium appetite in the rat induced by
intracranial administration of components of the renin-angiotensin system. The
Journal of Physiology, 301, 349-364.

Axelrod, J. (1971). Brain monoamines. biosynthesis and fate. Neurosciences Research

Program Bulletin, 9(2), 188-196.

Bader, M. (2010). Tissue renin-angiotensin-aldosterone systems: Targets for

pharmacological therapy. Annual Review of Pharmacology and Toxicology, 50, 439-
465.

Bakris, G. (2010). Are there effects of renin-angiotensin system antagonists beyond

blood pressure control? American Journal of Cardiology, 105, 21A.

Barnes, K. L., DeWeese, D. M., & Andresen, M. C. (2003). Angiotensin potentiates

excitatory sensory synaptic transmission to medial solitary tract nucleus neurons.
American Journal of Physiology.Regulatory, Integrative and Comparative
Physiology, 284(5), R1340-53.

Beresford MJ, F. J. (1992). Intracerebroventricular angiotensin II-induced thirst and

sodium appetite in rat are blocked by the AT1 receptor antagonist, losartan
(Dup753), but not by the AT2 antagonist, CGP 42112A. Experimental Physiology, ,
761-764.

 
122  
 
Berne, R., & Levy, M. (1988). Physiology (2nd Edition ed.). Washington D.C.: The CV
Mosby Company.

Berridge, K. C., Flynn, F. W., Schulkin, J., & Grill, H. J. (1984). Sodium depletion
enhances salt palatability in rats. Behavioral Neuroscience, 98(4), 652-660.

Berridge, K. C., & Robinson, T. E. (1998). What is the role of dopamine in reward:
Hedonic impact, reward learning, or incentive salience? Brain Research.Brain
Research Reviews, 28(3), 309-369.

Binart, N., Lombes, M., Rafestin-Oblin, M. E., & Baulieu, E. E. (1991). Characterization
of human mineralocorticosteroid receptor expressed in the baculovirus system.
Proceedings of the National Academy of Sciences of the United States of America,
88(23), 10681-10685.

Bisset, G. W., Clark, B. J., & Errington, M. L. (1971). The hypothalamic neurosecretory
pathways for the release of oxytocin and vasopressin in the cat. The Journal of
Physiology, 217(1), 111-131.

Blackburn, R. E., Demko, A. D., Hoffman, G. E., Stricker, E. M., & Verbalis, J. G. (1992).
Central oxytocin inhibition of angiotensin-induced salt appetite in rats. The
American Journal of Physiology, 263(6 Pt 2), R1347-53.

Blackburn, R. E., Stricker, E. M., & Verbalis, J. G. (1992). Central oxytocin mediates
inhibition of sodium appetite by naloxone in hypovolemic rats. Neuroendocrinology,
56(2), 255-263.

Bourgeais, L., Gauriau, C., & Bernard, J. F. (2001). Projections from the nociceptive
area of the central nucleus of the amygdala to the forebrain: A PHA-L study in the
rat. The European Journal of Neuroscience, 14(2), 229-255.

Bradbury, M. W. (1973). Physiology of body fluids and electrolytes. British Journal of

Anaesthesia, 45(9), 937-944.

Brennan, A. R., Dolinsky, B., Vu, M. A., Stanley, M., Yeckel, M. F., & Arnsten, A. F.
(2008). Blockade of IP3-mediated SK channel signaling in the rat medial prefrontal
cortex improves spatial working memory. Learning & Memory (Cold Spring Harbor,
N.Y.), 15(3), 93-96.

Brown, D. C., Steward, L. J., Ge, J., & Barnes, N. M. (1996). Ability of angiotensin II to
modulate striatal dopamine release via the AT1 receptor in vitro and in vivo. British
Journal of Pharmacology, 118(2), 414-420.

Buggy, J., & Jonklaas, J. (1984). Sodium appetite decreased by central angiotensin
blockade. Physiology & Behavior, 32(5), 737-742.

 
123  
 
Buijs, R. M. (1978). Intra- and extrahypothalamic vasopressin and oxytocin pathways in
the rat. pathways to the limbic system, medulla oblongata and spinal cord. Cell and
Tissue Research, 192(3), 423-435.

Camacho, A., & Phillips, M. I. (1981). Horseradish peroxidase study in rat of the neural
connections of the organum vasculosum of the lamina terminalis. Neuroscience
Letters, 25(3), 201-204.

Cao, Y., Wilcox, K. S., Martin, C. E., Rachinsky, T. L., Eberwine, J., & Dichter, M. A.
(1996). Presence of mRNA for glutamic acid decarboxylase in both excitatory and
inhibitory neurons. Proceedings of the National Academy of Sciences of the United
States of America, 93(18), 9844-9849.

Capponi, A. M., Rossier, M. F., & Vallotton, M. B. (1988). Production of inositol

trisphosphate isomers and release of intracellular calcium in cultured aortic vascular
smooth muscle cells. Journal of Cardiovascular Pharmacology, 12 Suppl 5, S92-5.

Cardinal, R. N., Parkinson, J. A., Hall, J., & Everitt, B. J. (2002). Emotion and motivation:
The role of the amygdala, ventral striatum, and prefrontal cortex. Neuroscience and
Biobehavioral Reviews, 26(3), 321-352.

Carelli, R. M. (2002). The nucleus accumbens and reward: Neurophysiological

investigations in behaving animals. Behavioral and Cognitive Neuroscience
Reviews, 1(4), 281-296.

Chai, S. Y., Allen, A. M., Adam, W. R., & Mendelsohn, F. A. (1986). Local actions of
angiotensin II: Quantitative in vitro autoradiographic localization of angiotensin II
receptor binding and angiotensin converting enzyme in target tissues. Journal of
Cardiovascular Pharmacology, 8 Suppl 10, S35-9.

Chow, S. Y., Sakai, R. R., Fluharty, S. J., & Flanagan-Cato, L. M. (1997). Brain oxytocin
receptor antagonism disinhibits sodium appetite in preweanling rats. Regulatory
Peptides, 68(2), 119-124.

Ciura, S., & Bourque, C. W. (2006). Transient receptor potential vanilloid 1 is required for
intrinsic osmoreception in organum vasculosum lamina terminalis neurons and for
normal thirst responses to systemic hyperosmolality. The Journal of Neuroscience :
The Official Journal of the Society for Neuroscience, 26(35), 9069-9075.

Clark, A. J., Balla, T., Jones, M. R., & Catt, K. J. (1992). Stimulation of early gene
expression by angiotensin II in bovine adrenal glomerulosa cells: Roles of calcium
and protein kinase C. Molecular Endocrinology (Baltimore, Md.), 6(11), 1889-1898.

Colby, K. A., Thompson, T. L., & Patrick, R. L. (1989). Tyrosine hydroxylase

phosphorylation in rat brain striatal synaptosomes. Brain Research, 478(1), 103-
111.

 
124  
 
Conover, K. L., Woodside, B., & Shizgal, P. (1994). Effects of sodium depletion on
competition and summation between rewarding effects of salt and lateral
hypothalamic stimulation in the rat. Behavioral Neuroscience, 108(3), 549-558.

Conrad, K. P., Gellai, M., North, W. G., & Valtin, H. (1993). Influence of oxytocin on renal
hemodynamics and sodium excretion. Annals of the New York Academy of
Sciences, 689, 346-362.

Cross, B. A., & Wakerley, J. B. (1977). The neurohypophysis. International Review of

Physiology, 16, 1-34.

Cullinan, W. E., Herman, J. P., & Watson, S. J. (1993). Ventral subicular interaction with
the hypothalamic paraventricular nucleus: Evidence for a relay in the bed nucleus of
the stria terminalis. The Journal of Comparative Neurology, 332(1), 1-20.

Daniels D, Yee DK, Faulconbridge LF, Fluharty SJ. (2005). Divergent behavioral roles of
angiotensin receptor intracellular signaling cascades. Endocrinology, 146(1), 5552-
5560.

Daniels, D., Mietlicki, E. G., Nowak, E. L., & Fluharty, S. J. (2009). Angiotensin II
stimulates water and NaCl intake through separate cell signalling pathways in rats.
Experimental Physiology, 94(1), 130-137.

Daniels, D., Yee, D. K., Faulconbridge, L. F., & Fluharty, S. J. (2005). Divergent
behavioral roles of angiotensin receptor intracellular signaling cascades.
Endocrinology, 146(12), 5552-5560.

Daniels, D., Yee, D. K., & Fluharty, S. J. (2007). Angiotensin II receptor signalling.
Experimental Physiology, 92(3), 523-527.

Davis, J. F., Choi, D. L., Schurdak, J. D., Fitzgerald, M. F., Clegg, D. J., Lipton, J. W., et
al. (2011). Leptin regulates energy balance and motivation through action at distinct
neural circuits. Biological Psychiatry, 69(7), 668-674.

Davis, J. O., & Spielman, W. S. (1974). The renin-angiotensin system in the control of
aldosterone secretion in the rat. Acta Physiologica Latino Americana, 24(5), 399-
404.

de Kloet, E. R., Van Acker, S. A., Sibug, R. M., Oitzl, M. S., Meijer, O. C., Rahmouni, K.,
et al. (2000). Brain mineralocorticoid receptors and centrally regulated functions.
Kidney International, 57(4), 1329-1336.

Del Giudice, A., Pompa, G., & Aucella, F. (2010). Hypertension in the elderly. Journal of
Nephrology, 23 Suppl 15, S61-71.

Dendorfer, A., Raasch, W., Tempel, K., & Dominiak, P. (1998). Interactions between the
renin-angiotensin system (RAS) and the sympathetic system. Basic Research in
Cardiology, 93 Suppl 2, 24-29.

 
125  
 
Denton, D. A., McKinley, M. J., & Weisinger, R. S. (1996). Hypothalamic integration of
body fluid regulation. Proceedings of the National Academy of Sciences of the
United States of America, 93(14), 7397-7404.

Dong, H. W., Petrovich, G. D., Watts, A. G., & Swanson, L. W. (2001). Basic
organization of projections from the oval and fusiform nuclei of the bed nuclei of the
stria terminalis in adult rat brain. The Journal of Comparative Neurology, 436(4),
430-455.

Dooley, R., Harvey, B. J., & Thomas, W. (2012). Non-genomic actions of aldosterone:
From receptors and signals to membrane targets. Molecular and Cellular
Endocrinology, 350(2), 223-234.

Eng, R., & Miselis, R. R. (1981). Polydipsia and abolition of angiotensin-induced drinking
after transections of subfornical organ efferent projections in the rat. Brain
Research, 225(1), 200-206.

Enjalbert, A., Sladeczek, F., Guillon, G., Bertrand, P., Shu, C., Epelbaum, J., et al.
(1986). Angiotensin II and dopamine modulate both cAMP and inositol phosphate
productions in anterior pituitary cells. involvement in prolactin secretion. The Journal
of Biological Chemistry, 261(9), 4071-4075.

Epstein, A. N. (1982). Mineralocorticoids and cerebral angiotensin may act together to

produce sodium appetite. Peptides, 3(3), 493-494.

Epstein, A. N., Fitzsimons, J. T., & Rolls, B. J. (1970). Drinking induced by injection of
angiotensin into the rain of the rat. The Journal of Physiology, 210(2), 457-474.

Epstein, A. N., Fitzsimons, J. T., & Simons, B. J. (1969). Drinking caused by the
intracranial injection of angiotensin into the rat. The Journal of Physiology, 200(2),
98P-100P.

Fadel, J., & Deutch, A. Y. (2002). Anatomical substrates of orexin-dopamine

interactions: Lateral hypothalamic projections to the ventral tegmental area.
Neuroscience, 111(2), 379-387.

Felder, R. B., Yu, Y., Zhang, Z. H., & Wei, S. G. (2009). Pharmacological treatment for
heart failure: A view from the brain. Clinical Pharmacology and Therapeutics, 86(2),
216-220.

Felgendreger, L. A., Fluharty, S. J., Yee, D. K., & Flanagan-Cato, L. M. (2013).

Endogenous angiotensin II-induced p44/42 mitogen-activated protein kinase
activation mediates sodium appetite but not thirst or neurohypophysial secretion in
male rats. Journal of Neuroendocrinology, 25(2), 97-106.

 
126  
 
Ferguson, A. V., Washburn, D. L., & Latchford, K. J. (2001). Hormonal and
neurotransmitter roles for angiotensin in the regulation of central autonomic
function. Experimental Biology and Medicine (Maywood, N.J.), 226(2), 85-96.

Fernandes, K. B., Tavares, R. F., Pelosi, G. G., & Correa, F. M. (2007). The
paraventricular nucleus of hypothalamus mediates the pressor response to
noradrenergic stimulation of the medial prefrontal cortex in unanesthetized rats.
Neuroscience Letters, 426(2), 101-105.

Findlay, A. L., & Epstein, A. N. (1980). Increased sodium intake is somehow induced in
rats by intravenous angiotnesin II. Hormones and Behavior, 14(1), 86-92.

Fitts, D. A. (1991). Effects of lesions of the ventral ventral median preoptic nucleus or
subfornical organ on drinking and salt appetite after deoxycorticosterone acetate or
yohimbine. Behavioral Neuroscience, 105(5), 721-726.

Fitts, D. A., Freece, J. A., Van Bebber, J. E., Zierath, D. K., & Bassett, J. E. (2004).
Effects of forebrain circumventricular organ ablation on drinking or salt appetite after
sodium depletion or hypernatremia. American Journal of Physiology.Regulatory,
Integrative and Comparative Physiology, 287(6), R1325-34.

Fitts, D. A., & Masson, D. B. (1990). Preoptic angiotensin and salt appetite. Behavioral
Neuroscience, 104(4), 643-650.

Fitts, D. A., Thornton, S. N., Ruhf, A. A., Zierath, D. K., Johnson, A. K., & Thunhorst, R.
L. (2003). Effects of central oxytocin receptor blockade on water and saline intake,
mean arterial pressure, and c-fos expression in rats. American Journal of
Physiology.Regulatory, Integrative and Comparative Physiology, 285(6), R1331-9.

Fitts, D. A., Tjepkes, D. S., & Bright, R. O. (1990). Salt appetite and lesions of the ventral
part of the ventral median preoptic nucleus. Behavioral Neuroscience, 104(5), 818-
827.

Fitzsimons, J. T. (1980). Angiotensin in the control of water and sodium intake. Society
of General Physiologists Series, 35, 99-106.

Fitzsimons, J. T. (1998). Angiotensin, thirst, and sodium appetite. Physiological Reviews,

78(3), 583-686.

Fitzsimons, J. T., & Stricker, E. M. (1971). Sodium appetite and the renin-angiotensin
system. Nature: New Biology, 231(19), 58-60.

Flanagan, L. M., Dohanics, J., Verbalis, J. G., & Stricker, E. M. (1992). Gastric motility
and food intake in rats after lesions of hypothalamic paraventricular nucleus. The
American Journal of Physiology, 263(1 Pt 2), R39-44.

Fleegal, M. A., & Sumners, C. (2003). Drinking behavior elicited by central injection of
angiotensin II: Roles for protein kinase C and Ca2+/calmodulin-dependent protein

 
127  
 
 kinase II. American Journal of Physiology.Regulatory, Integrative and Comparative
Physiology, 285(3), R632-40.

Fluharty, S. J., & Epstein, A. N. (1983). Sodium appetite elicited by

intracerebroventricular infusion of angiotensin II in the rat: II. synergistic interaction
with systemic mineralocorticoids. Behavioral Neuroscience, 97(5), 746-758.

Folkow, B. (1971). Regulation of the peripheral circulation. British Heart Journal, 33,
Suppl:27-31.

Fregly, M. J., Harper, J. M.,Jr, & Radford, E. P.,Jr. (1965). Regulation of sodium chloride
intake by rats. The American Journal of Physiology, 209, 287-292.

Funder, J. W., Pearce, P. T., Smith, R., & Smith, A. I. (1988). Mineralocorticoid action:
Target tissue specificity is enzyme, not receptor, mediated. Science (New York,
N.Y.), 242(4878), 583-585.

Gallinat, S., Busche, S., Raizada, M. K., & Sumners, C. (2000). The angiotensin II type 2
receptor: An enigma with multiple variations. American Journal of
Physiology.Endocrinology and Metabolism, 278(3), E357-74.

Ganong, W. F. (1977). The renin-angiotensin system and the central nervous system.
Federation Proceedings, 36(5), 1771-1775.

Ganong, W. F. (1984). The brain renin-angiotensin system. Annual Review of

Physiology, 46, 17-31.

Gardiner, T. W., Jolley, J. R., Vagnucci, A. H., & Stricker, E. M. (1986). Enhanced
sodium appetite in rats with lesions centered on nucleus medianus. Behavioral
Neuroscience, 100(4), 531-535.

Geerling, J. C., Chimenti, P. C., & Loewy, A. D. (2008). Phox2b expression in the
aldosterone-sensitive HSD2 neurons of the NTS. Brain Research, 1226, 82-88.

Geerling, J. C., Kawata, M., & Loewy, A. D. (2006). Aldosterone-sensitive neurons in the
rat central nervous system. The Journal of Comparative Neurology, 494(3), 515-
527.

Geerling, J. C., & Loewy, A. D. (2008). Central regulation of sodium appetite.

Experimental Physiology, 93(2), 177-209.

Geerling, J. C., & Loewy, A. D. (2009). Aldosterone in the brain. American Journal of
Physiology.Renal Physiology, 297(3), F559-76.

Gill, J. R.,Jr. (1979). Neural control of renal tubular sodium reabsorption. Nephron, 23(2-
3), 116-118.

 
128  
 
Gohlke, P., Kox, T., Jurgensen, T., von Kugelgen, S., Rascher, W., Unger, T., et al.
(2002). Peripherally applied candesartan inhibits central responses to angiotensin II
in conscious rats. Naunyn-Schmiedeberg's Archives of Pharmacology, 365(6), 477-
483.

Gomez-Sanchez, C. E., Zhou, M. Y., Cozza, E. N., Morita, H., Foecking, M. F., &
Gomez-Sanchez, E. P. (1997). Aldosterone biosynthesis in the rat brain.
Endocrinology, 138(8), 3369-3373.

Grondin, M. E., Gobeil-Simard, A., Drolet, G., & Mouginot, D. (2011). Na+ appetite
induced by depleting extracellular fluid volume activates the enkephalin/mu-opioid
receptor system in the rat forebrain. Neuroscience, 192, 398-412.

Grossmann, C., & Gekle, M. (2009). New aspects of rapid aldosterone signaling.
Molecular and Cellular Endocrinology, 308(1-2), 53-62.

Harada, E., Yoshimura, M., Yasue, H., Nakagawa, O., Nakagawa, M., Harada, M., et al.
(2001). Aldosterone induces angiotensin-converting-enzyme gene expression in
cultured neonatal rat cardiocytes. Circulation, 104(2), 137-139.

He, F. J., & MacGregor, G. A. (2010). Reducing population salt intake worldwide: From
evidence to implementation. Progress in Cardiovascular Diseases, 52(5), 363-382.

Herbert, J., Forsling, M. L., Howes, S. R., Stacey, P. M., & Shiers, H. M. (1992).
Regional expression of c-fos antigen in the basal forebrain following intraventricular
infusions of angiotensin and its modulation by drinking either water or saline.
Neuroscience, 51(4), 867-882.

Hines, J., Fluharty, S. J., & Yee, D. K. (2003). Structural determinants for the activation
mechanism of the angiotensin II type 1 receptor differ for phosphoinositide
hydrolysis and mitogen-activated protein kinase pathways. Biochemical
Pharmacology, 66(2), 251-262.

Hirono, Y., Yoshimoto, T., Suzuki, N., Sugiyama, T., Sakurada, M., Takai, S., et al.
(2007). Angiotensin II receptor type 1-mediated vascular oxidative stress and
proinflammatory gene expression in aldosterone-induced hypertension: The
possible role of local renin-angiotensin system. Endocrinology, 148(4), 1688-1696.

Hoebel, B. G., Hernandez, L., Schwartz, D. H., Mark, G. P., & Hunter, G. A. (1989).
Microdialysis studies of brain norepinephrine, serotonin, and dopamine release
during ingestive behavior. theoretical and clinical implications. Annals of the New
York Academy of Sciences, 575, 171-91; discussion 192-3.

Hoebel, B. G., Rada, P., Mark, G. P., & Hernandez, L. (1994). The power of integrative
peptides to reinforce behavior by releasing dopamine. Annals of the New York
Academy of Sciences, 739, 36-41.

 
129  
 
Honda, K., Negoro, H., Dyball, R. E., Higuchi, T., & Takano, S. (1990). The
osmoreceptor complex in the rat: Evidence for interactions between the supraoptic
and other diencephalic nuclei. The Journal of Physiology, 431, 225-241.

Huang, X. C., Richards, E. M., & Sumners, C. (1996). Mitogen-activated protein kinases
in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and
inhibited by angiotensin II type 2 receptors. The Journal of Biological Chemistry,
271(26), 15635-15641.

Jaffe, I. Z., & Mendelsohn, M. E. (2005). Angiotensin II and aldosterone regulate gene
transcription via functional mineralocortocoid receptors in human coronary artery
smooth muscle cells. Circulation Research, 96(6), 643-650.

Jalowiec, J. E. (1974). Sodium appetite elicited by furosemide: Effects of differential

dietary maintenance. Behavioral Biology, 10(3), 313-327.

Johnson, A. K., Cunningham, J. T., & Thunhorst, R. L. (1996). Integrative role of the
lamina terminalis in the regulation of cardiovascular and body fluid homeostasis.
Clinical and Experimental Pharmacology & Physiology, 23(2), 183-191.

Johnson, A. K., de Olmos, J., Pastuskovas, C. V., Zardetto-Smith, A. M., & Vivas, L.
(1999). The extended amygdala and salt appetite. Annals of the New York
Academy of Sciences, 877, 258-280.

Johnson, A. K., & Thunhorst, R. L. (1997). The neuroendocrinology of thirst and salt
appetite: Visceral sensory signals and mechanisms of central integration. Frontiers
in Neuroendocrinology, 18(3), 292-353.

Jomphe, C., Levesque, D., & Trudeau, L. E. (2003). Calcium-dependent, D2 receptor-

independent induction of c-fos by haloperidol in dopamine neurons. Naunyn-
Schmiedeberg's Archives of Pharmacology, 367(5), 480-489.

Jones, I. C., & Bellamy, D. (1964). Hormonal mechanisms in the homeostatic regulation
of the vertebrate body with special reference to the adrenal cortex. Symposia of the
Society for Experimental Biology, 18, 195-236.

Karppanen, H., & Mervaala, E. (2006). Sodium intake and hypertension. Progress in
Cardiovascular Diseases, 49(2), 59-75.

Keil, L. C., Rosella-Dampman, L. M., Emmert, S., Chee, O., & Summy-Long, J. Y.
(1984). Enkephalin inhibition of angiotensin-stimulated release of oxytocin and
vasopressin. Brain Research, 297(2), 329-336.

Kelly, A. B., & Watts, A. G. (1996). Mediation of dehydration-induced peptidergic gene

expression in the rat lateral hypothalamic area by forebrain afferent projections. The
Journal of Comparative Neurology, 370(2), 231-246.

 
130  
 
Kelly, A. B., & Watts, A. G. (1998). The region of the pontine parabrachial nucleus is a
major target of dehydration-sensitive CRH neurons in the rat lateral hypothalamic
area. The Journal of Comparative Neurology, 394(1), 48-63.

Khan, A. M., & Watts, A. G. (2004). Intravenous 2-deoxy-D-glucose injection rapidly

elevates levels of the phosphorylated forms of p44/42 mitogen-activated protein
kinases (extracellularly regulated kinases 1/2) in rat hypothalamic parvicellular
paraventricular neurons. Endocrinology, 145(1), 351-359.

King, S. J., Harding, J. W., & Moe, K. E. (1988). Elevated salt appetite and brain binding
of angiotensin II in mineralocorticoid-treated rats. Brain Research, 448(1), 140-149.

Kraulis, I., Foldes, G., Traikov, H., Dubrovsky, B., & Birmingham. (1975). Distribution,
metabolism and biological activity of deoxycorticosterone in the central nervous
system. Brain Research, 88(1), 1-14.

Lee, M. H., El-Shewy, H. M., Luttrell, D. K., & Luttrell, L. M. (2008). Role of beta-arrestin-
mediated desensitization and signaling in the control of angiotensin AT1a receptor-
stimulated transcription. The Journal of Biological Chemistry, 283(4), 2088-2097.

LeHoux, J. G., & Lefebvre, A. (2006). Novel protein kinase C-epsilon inhibits human
CYP11B2 gene expression through ERK1/2 signalling pathway and JunB. Journal
of Molecular Endocrinology, 36(1), 51-64.

Lemarie, C. A., Paradis, P., & Schiffrin, E. L. (2008). New insights on signaling cascades
induced by cross-talk between angiotensin II and aldosterone. Journal of Molecular
Medicine (Berlin, Germany), 86(6), 673-678.

Lemarie, C. A., Simeone, S. M., Nikonova, A., Ebrahimian, T., Deschenes, M. E.,
Coffman, T. M., et al. (2009). Aldosterone-induced activation of signaling pathways
requires activity of angiotensin type 1a receptors. Circulation Research, 105(9),
852-859.

Li, Z., Ji, G., & Neugebauer, V. (2011). Mitochondrial reactive oxygen species are
activated by mGluR5 through IP3 and activate ERK and PKA to increase excitability
of amygdala neurons and pain behavior. The Journal of Neuroscience : The Official
Journal of the Society for Neuroscience, 31(3), 1114-1127.

Liang, J., Ma, S. S., Li, Y. J., Ping, X. J., Hu, L., & Cui, C. L. (2012). Dynamic changes of
tyrosine hydroxylase and dopamine concentrations in the ventral tegmental area-
nucleus accumbens projection during the expression of morphine-induced
conditioned place preference in rats. Neurochemical Research, 37(7), 1482-1489.

Liedtke, W. B., McKinley, M. J., Walker, L. L., Zhang, H., Pfenning, A. R., Drago, J., et
al. (2011). Relation of addiction genes to hypothalamic gene changes subserving
genesis and gratification of a classic instinct, sodium appetite. Proceedings of the
National Academy of Sciences of the United States of America, 108(30), 12509-
12514.

 
131  
 
Lind, R. W., Swanson, L. W., & Ganten, D. (1984). Angiotensin II immunoreactivity in the
neural afferents and efferents of the subfornical organ of the rat. Brain Research,
321(2), 209-215.

Lombes, M., Kenouch, S., Souque, A., Farman, N., & Rafestin-Oblin, M. E. (1994). The
mineralocorticoid receptor discriminates aldosterone from glucocorticoids
independently of the 11 beta-hydroxysteroid dehydrogenase. Endocrinology,
135(3), 834-840.

Lu, B., Yang, X. J., Chen, K., Yang, D. J., & Yan, J. Q. (2009). Dietary sodium
deprivation evokes activation of brain regional neurons and down-regulation of
angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA
expression. Neuroscience, 164(3), 1303-1311.

Lu, J., Sherman, D., Devor, M., & Saper, C. B. (2006). A putative flip-flop switch for
control of REM sleep. Nature, 441(7093), 589-594.

Lucas, L. R., Grillo, C. A., & McEwen, B. S. (2003). Involvement of mesolimbic structures
in short-term sodium depletion: In situ hybridization and ligand-binding analyses.
Neuroendocrinology, 77(6), 406-415.

Lucas, L. R., Grillo, C. A., & McEwen, B. S. (2007). Salt appetite in sodium-depleted or
sodium-replete conditions: Possible role of opioid receptors. Neuroendocrinology,
85(3), 139-147.

Mann, S. J. (2013, Recent controversy over reducing sodium intake. Healthy Living,

Marc, Y., & Llorens-Cortes, C. (2011). The role of the brain renin-angiotensin system in
hypertension: Implications for new treatment. Progress in Neurobiology, 95(2), 89-
103.

Margolius, D., & Bodenheimer, T. (2010). Controlling hypertension requires a new

primary care model. The American Journal of Managed Care, 16(9), 648-650.

Massi, M., & Epstein, A. N. (1990). Angiotensin/aldosterone synergy governs the salt
appetite of the pigeon. Appetite, 14(3), 181-192.

Mazak, I., Fiebeler, A., Muller, D. N., Park, J. K., Shagdarsuren, E., Lindschau, C., et al.
(2004). Aldosterone potentiates angiotensin II-induced signaling in vascular smooth
muscle cells. Circulation, 109(22), 2792-2800.

McKelvie, R. S. (2011). Heart failure. Clinical Evidence, 2011, 0204.

McKinley, M. J., Allen, A. M., Burns, P., Colvill, L. M., & Oldfield, B. J. (1998). Interaction
of circulating hormones with the brain: The roles of the subfornical organ and the
organum vasculosum of the lamina terminalis. Clinical and Experimental
Pharmacology & Physiology.Supplement, 25, S61-7.

 
132  
 
McKinley, M. J., Badoer, E., & Oldfield, B. J. (1992). Intravenous angiotensin II induces
fos-immunoreactivity in circumventricular organs of the lamina terminalis. Brain
Research, 594(2), 295-300.

McKinley, M. J., Bicknell, R. J., Hards, D., McAllen, R. M., Vivas, L., Weisinger, R. S., et
al. (1992). Efferent neural pathways of the lamina terminalis subserving
osmoregulation. Progress in Brain Research, 91, 395-402.

McKinley, M. J., McAllen, R. M., Pennington, G. L., Smardencas, A., Weisinger, R. S., &
Oldfield, B. J. (1996). Physiological actions of angiotensin II mediated by AT1 AND
AT2 receptors in the brain. Clinical and Experimental Pharmacology & Physiology,
23 Suppl 3, S99-104.

Midgley, J. P., Matthew, A. G., Greenwood, C. M., & Logan, A. G. (1996). Effect of
reduced dietary sodium on blood pressure: A meta-analysis of randomized
controlled trials. JAMA : The Journal of the American Medical Association, 275(20),
1590-1597.

Miller, R. L., Wang, M. H., Gray, P. A., Salkoff, L. B., & Loewy, A. D. (2013). ENaC-
expressing neurons in the sensory circumventricular organs become c-fos activated
following systemic sodium changes. American Journal of Physiology.Regulatory,
Integrative and Comparative Physiology, 305(10), R1141-52.

Min, L. J., Mogi, M., Li, J. M., Iwanami, J., Iwai, M., & Horiuchi, M. (2005). Aldosterone
and angiotensin II synergistically induce mitogenic response in vascular smooth
muscle cells. Circulation Research, 97(5), 434-442.

Miselis, R. R. (1981). The efferent projections of the subfornical organ of the rat: A
circumventricular organ within a neural network subserving water balance. Brain
Research, 230(1-2), 1-23.

Mizuno, K., Tani, M., Niimura, S., Yamaguchi, M., Inagami, T., & Fukuchi, S. (1991). The
antihypertensive mechanism of delapril, a newly developed converting enzyme
inhibitor, is related to the suppression of vascular angiotensin II release in the
spontaneously hypertensive rat. American Journal of Hypertension, 4(1 Pt 2), 60S-
66S.

Nabika, T., Velletri, P. A., Lovenberg, W., & Beaven, M. A. (1985). Increase in cytosolic
calcium and phosphoinositide metabolism induced by angiotensin II and
[arg]vasopressin in vascular smooth muscle cells. The Journal of Biological
Chemistry, 260(8), 4661-4670.

Narita, M., Nagumo, Y., Hashimoto, S., Narita, M., Khotib, J., Miyatake, M., et al. (2006).
Direct involvement of orexinergic systems in the activation of the mesolimbic
dopamine pathway and related behaviors induced by morphine. The Journal of
Neuroscience : The Official Journal of the Society for Neuroscience, 26(2), 398-405.

 
133  
 
Nissen, R., Bourque, C. W., & Renaud, L. P. (1993). Membrane properties of organum
vasculosum lamina terminalis neurons recorded in vitro. The American Journal of
Physiology, 264(4 Pt 2), R811-5.

Numakawa, T., Yokomaku, D., Kiyosue, K., Adachi, N., Matsumoto, T., Numakawa, Y.,
et al. (2002). Basic fibroblast growth factor evokes a rapid glutamate release
through activation of the MAPK pathway in cultured cortical neurons. The Journal of
Biological Chemistry, 277(32), 28861-28869.

Oades, R. D., & Halliday, G. M. (1987). Ventral tegmental (A10) system: Neurobiology.
1. anatomy and connectivity. Brain Research, 434(2), 117-165.

Olsen, M. K., Reszka, A. A., & Abraham, I. (1998). KT5720 and U-98017 inhibit MAPK
and alter the cytoskeleton and cell morphology. Journal of Cellular Physiology,
176(3), 525-536.

Omouessi, S. T., Falconetti, C., Chapleur, M., Fernette, B., & Thornton, S. N. (2007).
Mineralocorticoid pretreatment enhances angiotensin II-induced neuronal excitation
but not salt drinking in male fischer rats. Journal of Neuroendocrinology, 19(2), 109-
115.

Oz, M., Yang, K. H., O'donovan, M. J., & Renaud, L. P. (2005). Presynaptic angiotensin
II AT1 receptors enhance inhibitory and excitatory synaptic neurotransmission to
motoneurons and other ventral horn neurons in neonatal rat spinal cord. Journal of
Neurophysiology, 94(2), 1405-1412.

Ozaki, Y., Soya, A., Nakamura, J., Matsumoto, T., & Ueta, Y. (2004). Potentiation by
angiotensin II of spontaneous excitatory postsynaptic currents in rat supraoptic
magnocellular neurones. Journal of Neuroendocrinology, 16(11), 871-879.

Pardridge, W. M., & Mietus, L. J. (1979). Transport of steroid hormones through the rat
blood-brain barrier. primary role of albumin-bound hormone. The Journal of Clinical
Investigation, 64(1), 145-154.

Peach, M. J. (1977). Renin-angiotensin system: Biochemistry and mechanisms of action.

Physiological Reviews, 57(2), 313-370.

Pickel, V. M., Joh, T. H., & Reis, D. J. (1975). Ultrastructural localization of tyrosine
hydroxylase in noradrenergic neurons of brain. Proceedings of the National
Academy of Sciences of the United States of America, 72(2), 659-663.

Pietranera, L., Saravia, F. E., McEwen, B. S., Lucas, L. L., Johnson, A. K., & De Nicola,
A. F. (2001). Changes in fos expression in various brain regions during
deoxycorticosterone acetate treatment: Relation to salt appetite, vasopressin mRNA
and the mineralocorticoid receptor. Neuroendocrinology, 74(6), 396-406.

 
134  
 
Prakash, M. R., & Norgren, R. (1991). Comparing salt appetites: Induction with
intracranial hormones or dietary sodium restriction. Brain Research Bulletin, 27(3-
4), 397-401.

Puryear, R., Rigatto, K. V., Amico, J. A., & Morris, M. (2001). Enhanced salt intake in
oxytocin deficient mice. Experimental Neurology, 171(2), 323-328.

Rafestin-Oblin, M. E., Farman, N., Cassingena, R., Ronco, P., & Vandewalle, A. (1993).
Mineralocorticoid receptors in SV40-transformed tubule cell lines derived from
rabbit kidney. The Journal of Steroid Biochemistry and Molecular Biology, 44(1), 45-
52.

Reagan L.P., Flanagan-Cato, L.M., Yee, D.K., Ma L.Y., Sakai, R.R., Fluharty, S.J.
(1994). Immunohistochemical mapping of angiotensin type 2 (AT2) receptors in rat
brain. Brain Research, 662(1-2), 45-59.

Reid, I. A. (1984). Actions of angiotensin II on the brain: Mechanisms and physiologic

role. The American Journal of Physiology, 246(5 Pt 2), F533-43.

Reid, I. A. (1985). The renin-angiotensin system and body function. Archives of Internal
Medicine, 145(8), 1475-1479.

Reis, W. L., Saad, W. A., Camargo, L. A., Elias, L. L., & Antunes-Rodrigues, J. (2010).
Central nitrergic system regulation of neuroendocrine secretion, fluid intake and
blood pressure induced by angiotensin-II. Behavioral and Brain Functions : BBF, 6,
64.

Renaud, L. P., & Bourque, C. W. (1991). Neurophysiology and neuropharmacology of

hypothalamic magnocellular neurons secreting vasopressin and oxytocin. Progress
in Neurobiology, 36(2), 131-169.

Rice, K. K., & Richter, C. P. (1943). Increased sodium chloride and water intake of
normal rats treated with desoxycorticosterone acetate. Endocrinology, 33, 106.

Richard, D., & Bourque, C. W. (1996). Atrial natriuretic peptide modulates synaptic
transmission from osmoreceptor afferents to the supraoptic nucleus. The Journal of
Neuroscience : The Official Journal of the Society for Neuroscience, 16(23), 7526-
7532.

Richter, C. (1936). Increased salt appetite in adrenalectomized rats. American Journal of

Physiology, 115, 155.

Riftina, F., Angulo, J., Pompei, P., & McEwen, B. (1995). Regulation of angiotensinogen
gene expression in the rat forebrain by adrenal steroids and relation to salt appetite.
Brain Research.Molecular Brain Research, 33(2), 201-208.

Rigatto, K., Puryear, R., Bernatova, I., & Morris, M. (2003). Salt appetite and the renin-
angiotensin system: Effect of oxytocin deficiency. Hypertension, 42(4), 793-797.

 
135  
 
Roesch, D. M., Blackburn-Munro, R. E., & Verbalis, J. G. (2001). Mineralocorticoid
treatment attenuates activation of oxytocinergic and vasopressinergic neurons by
icv ANG II. American Journal of Physiology.Regulatory, Integrative and
Comparative Physiology, 280(6), R1853-64.

Roitman, M. F., Na, E., Anderson, G., Jones, T. A., & Bernstein, I. L. (2002). Induction of
a salt appetite alters dendritic morphology in nucleus accumbens and sensitizes
rats to amphetamine. The Journal of Neuroscience : The Official Journal of the
Society for Neuroscience, 22(11), RC225.

Roitman, M. F., Patterson, T. A., Sakai, R. R., Bernstein, I. L., & Figlewicz, D. P. (1999).
Sodium depletion and aldosterone decrease dopamine transporter activity in
nucleus accumbens but not striatum. The American Journal of Physiology, 276(5 Pt
2), R1339-45.

Roitman, M. F., Schafe, G. E., Thiele, T. E., & Bernstein, I. L. (1997). Dopamine and
sodium appetite: Antagonists suppress sham drinking of NaCl solutions in the rat.
Behavioral Neuroscience, 111(3), 606-611.

Roitman, M. F., Stuber, G. D., Phillips, P. E., Wightman, R. M., & Carelli, R. M. (2004).
Dopamine operates as a subsecond modulator of food seeking. The Journal of
Neuroscience : The Official Journal of the Society for Neuroscience, 24(6), 1265-
1271.

Sadoshima, J., Qiu, Z., Morgan, J. P., & Izumo, S. (1995). Angiotensin II and other
hypertrophic stimuli mediated by G protein-coupled receptors activate tyrosine
kinase, mitogen-activated protein kinase, and 90-kD S6 kinase in cardiac myocytes.
the critical role of ca(2+)-dependent signaling. Circulation Research, 76(1), 1-15.

Sagar, S. M., Sharp, F. R., & Curran, T. (1988). Expression of c-fos protein in brain:
Metabolic mapping at the cellular level. Science (New York, N.Y.), 240(4857), 1328-
1331.

Sakai, R. R., Ma, L. Y., Zhang, D. M., McEwen, B. S., & Fluharty, S. J. (1996).
Intracerebral administration of mineralocorticoid receptor antisense oligonucleotides
attenuate adrenal steroid-induced salt appetite in rats. Neuroendocrinology, 64(6),
425-429.

Sakai, R. R., McEwen, B. S., Fluharty, S. J., & Ma, L. Y. (2000). The amygdala: Site of
genomic and nongenomic arousal of aldosterone-induced sodium intake. Kidney
International, 57(4), 1337-1345.

Sakai, R. R., Nicolaidis, S., & Epstein, A. N. (1986). Salt appetite is suppressed by
interference with angiotensin II and aldosterone. The American Journal of
Physiology, 251(4 Pt 2), R762-8.

 
136  
 
Sayer, R. J., Hubbard, J. I., & Sirett, N. E. (1984). Rat organum vasculosum laminae
terminalis in vitro: Responses to transmitters. The American Journal of Physiology,
247(2 Pt 2), R374-9.

Schenk, U., Menna, E., Kim, T., Passafaro, M., Chang, S., De Camilli, P., et al. (2005). A
novel pathway for presynaptic mitogen-activated kinase activation via AMPA
receptors. The Journal of Neuroscience : The Official Journal of the Society for
Neuroscience, 25(7), 1654-1663.

Schrader, L. A., Birnbaum, S. G., Nadin, B. M., Ren, Y., Bui, D., Anderson, A. E., et al.
(2006). ERK/MAPK regulates the Kv4.2 potassium channel by direct
phosphorylation of the pore-forming subunit. American Journal of Physiology.Cell
Physiology, 290(3), C852-61.

Schwartz, M. W., Woods, S. C., Porte, D.,Jr, Seeley, R. J., & Baskin, D. G. (2000).
Central nervous system control of food intake. Nature, 404(6778), 661-671.

Sclafani, A., & Ackroff, K. (2003). Reinforcement value of sucrose measured by

progressive ratio operant licking in the rat. Physiology & Behavior, 79(4-5), 663-670.

Shade, R. E., Blair-West, J. R., Carey, K. D., Madden, L. J., Weisinger, R. S., & Denton,
D. A. (2002). Synergy between angiotensin and aldosterone in evoking sodium
appetite in baboons. American Journal of Physiology.Regulatory, Integrative and
Comparative Physiology, 283(5), R1070-8.

Shekhtman, E., Geerling, J. C., & Loewy, A. D. (2007). Aldosterone-sensitive neurons of

the nucleus of the solitary tract: Multisynaptic pathway to the nucleus accumbens.
The Journal of Comparative Neurology, 501(2), 274-289.

Simpson, J. B., & Routtenberg, A. (1973). Subfornical organ: Site of drinking elicitation
by angiotensin II. Science (New York, N.Y.), 181(4105), 1172-1175.

Smith, J. B., Smith, L., Brown, E. R., Barnes, D., Sabir, M. A., Davis, J. S., et al. (1984).
Angiotensin II rapidly increases phosphatidate-phosphoinositide synthesis and
phosphoinositide hydrolysis and mobilizes intracellular calcium in cultured arterial
muscle cells. Proceedings of the National Academy of Sciences of the United
States of America, 81(24), 7812-7816.

Sofroniew, M. V., Weindl, A., Schrell, U., & Wetzstein, R. (1981). Immunohistochemistry
of vasopressin, oxytocin and neurophysin in the hypothalamus and
extrahypothalamic regions of the human and primate brain. Acta
Histochemica.Supplementband, 24, 79-95.

Starr, L. J., & Rowland, N. E. (2006). Characteristics of salt appetite in chronically

sodium-depleted rats using a progressive ratio schedule of procurement.
Physiology & Behavior, 88(4-5), 433-442.

 
137  
 
Stricker, E. M. (1966). Extracellular fluid volume and thirst. The American Journal of
Physiology, 211(1), 232-238.

Stricker, E. M. (1971). Effects of hypovolemia and-or caval ligation on water and NaCl
solution drinking by rats. Physiology & Behavior, 6(4), 299-305.

Stricker, E. M., Vagnucci, A. H., McDonald, R. H.,Jr, & Leenen, F. H. (1979). Renin and
aldosterone secretions during hypovolemia in rats: Relation to NaCl intake. The
American Journal of Physiology, 237(1), R45-51.

Stricker, E. M., & Verbalis, J. G. (1987). Central inhibitory control of sodium appetite in
rats: Correlation with pituitary oxytocin secretion. Behavioral Neuroscience, 101(4),
560-567.

Stricker, E. M., & Verbalis, J. G. (1996). Central inhibition of salt appetite by oxytocin in
rats. Regulatory Peptides, 66(1-2), 83-85.

Stricker, E. M., & Verbalis, J. G. (2004). Inhibition of salt appetite in rats by central
oxytocin. American Journal of Physiology.Regulatory, Integrative and Comparative
Physiology, 287(2), R487; author reply R487-8.

Stricker, E. M., & Wolf, G. (1966). Blood volume and tonicity in relation to sodium
appetite. Journal of Comparative and Physiological Psychology, 62(2), 275-279.

Suarez, C., Tornadu, I. G., Cristina, C., Vela, J., Iglesias, A. G., Libertun, C., et al.
(2002). Angiotensin and calcium signaling in the pituitary and hypothalamus.
Cellular and Molecular Neurobiology, 22(3), 315-333.

Sugaya, T., Nishimatsu, S., Tanimoto, K., Takimoto, E., Yamagishi, T., Imamura, K., et
al. (1995). Angiotensin II type 1a receptor-deficient mice with hypotension and
hyperreninemia. The Journal of Biological Chemistry, 270(32), 18719-18722.

Sumners, C., Zhu, M., Gelband, C. H., & Posner, P. (1996). Angiotensin II type 1
receptor modulation of neuronal K+ and Ca2+ currents: Intracellular mechanisms.
The American Journal of Physiology, 271(1 Pt 1), C154-63.

Sunsay, C., & Rebec, G. V. (2008). Real-time dopamine efflux in the nucleus
accumbens core during pavlovian conditioning. Behavioral Neuroscience, 122(2),
358-367.

Swank, M. W., & Sweatt, J. D. (2001). Increased histone acetyltransferase and lysine
acetyltransferase activity and biphasic activation of the ERK/RSK cascade in insular
cortex during novel taste learning. The Journal of Neuroscience : The Official
Journal of the Society for Neuroscience, 21(10), 3383-3391.

Swanson, L. W., Marshall, G. R., Needleman, P., & Sharpe, L. G. (1973).

Characterization of central angiotensin II receptors involved in the elicitation of
drinking in the rat. Brain Research, 49(2), 441-446.

 
138  
 
Tanaka, J., Kaba, H., Saito, H., & Seto, K. (1986). Subfornical organ efferents influence
the activity of median preoptic neurons projecting to the hypothalamic
paraventricular nucleus in the rat. Experimental Neurology, 93(3), 647-651.

Taylor, S. J., Smith, J. A., & Exton, J. H. (1990). Purification from bovine liver
membranes of a guanine nucleotide-dependent activator of phosphoinositide-
specific phospholipase C. immunologic identification as a novel G-protein alpha
subunit. The Journal of Biological Chemistry, 265(28), 17150-17156.

Templeton, D. M., Wang, Z., & Miralem, T. (1998). Cadmium and calcium-dependent c-
fos expression in mesangial cells. Toxicology Letters, 95(1), 1-8.

Thornton, S. N., & Nicolaidis, S. (1994). Long-term mineralocorticoid-induced changes in

rat neuron properties plus interaction of aldosterone and ANG II. The American
Journal of Physiology, 266(2 Pt 2), R564-71.

Thunhorst, R. L., & Johnson, A. K. (1994). Renin-angiotensin, arterial blood pressure,

and salt appetite in rats. The American Journal of Physiology, 266(2 Pt 2), R458-65.

Thunhorst, R. L., Morris, M., & Johnson, A. K. (1994). Endocrine changes associated
with a rapidly developing sodium appetite in rats. The American Journal of
Physiology, 267(5 Pt 2), R1168-73.

Toth, E., Stelfox, J., & Kaufman, S. (1987). Cardiac control of salt appetite. The
American Journal of Physiology, 252(5 Pt 2), R925-9.

Ullian, M. E., Schelling, J. R., & Linas, S. L. (1992). Aldosterone enhances angiotensin II
receptor binding and inositol phosphate responses. Hypertension, 20(1), 67-73.

Verbalis, J. G., McHale, C. M., Gardiner, T. W., & Stricker, E. M. (1986). Oxytocin and
vasopressin secretion in response to stimuli producing learned taste aversions in
rats. Behavioral Neuroscience, 100(4), 466-475.

Viengchareun, S., Le Menuet, D., Martinerie, L., Munier, M., Pascual-Le Tallec, L., &
Lombes, M. (2007). The mineralocorticoid receptor: Insights into its molecular and
(patho)physiological biology. Nuclear Receptor Signaling, 5, e012.

Vivas, L., Chiaraviglio, E., & Carrer, H. F. (1990). Rat organum vasculosum laminae
terminalis in vitro: Responses to changes in sodium concentration. Brain Research,
519(1-2), 294-300.

Vollmer, W. M., Sacks, F. M., Ard, J., Appel, L. J., Bray, G. A., Simons-Morton, D. G., et
al. (2001). Effects of diet and sodium intake on blood pressure: Subgroup analysis
of the DASH-sodium trial. Annals of Internal Medicine, 135(12), 1019-1028.

Wei, S. G., Yu, Y., Zhang, Z. H., & Felder, R. B. (2009). Angiotensin II upregulates
hypothalamic AT1 receptor expression in rats via the mitogen-activated protein

 
139  
 
 kinase pathway. American Journal of Physiology.Heart and Circulatory Physiology,
296(5), H1425-33.

Wei, S. G., Yu, Y., Zhang, Z. H., Weiss, R. M., & Felder, R. B. (2008). Angiotensin II-
triggered p44/42 mitogen-activated protein kinase mediates sympathetic excitation
in heart failure rats. Hypertension, 52(2), 342-350.

Weisinger, R. S., Blair-West, J. R., Burns, P., Denton, D. A., McKinley, M. J., & Tarjan,
E. (1996). The role of angiotensin II in ingestive behavior: A brief review of
angiotensin II, thirst and na appetite. Regulatory Peptides, 66(1-2), 73-81.

Weiss, M. L., & Hatton, G. I. (1990). Collateral input to the paraventricular and
supraoptic nuclei in rat. I. afferents from the subfornical organ and the anteroventral
third ventricle region. Brain Research Bulletin, 24(2), 231-238.

Wilkins, L., & Richter, C. P. (1940). A great craving for salt by a child with cortico-adrenal
insufficiency. The Journal of the American Medical Association, 114, 866.

Wilson, K. M., Sumners, C., Hathaway, S., & Fregly, M. J. (1986). Mineralocorticoids
modulate central angiotensin II receptors in rats. Brain Research, 382(1), 87-96.

Wolf, G. (1964). Effect of dorsolateral hypothalamic lesions on sodium appetite elicited

by desoxycorticosterone and by acute hyponatremia. Journal of Comparative and
Physiological Psychology, 58, 396-402.

Wolf, G. (1965). Effect of deoxycorticosterone on sodium appetite of intact and

adrenalectomized rats. The American Journal of Physiology, 208, 1281-1285.

Wolf, G. (1967). Hypothalamic regulation of sodium intake: Relations to preoptic and

tegmental function. The American Journal of Physiology, 213(6), 1433-1438.

Wolf, G., McGovern, J. F., & Dicara, L. V. (1974). Sodium appetite: Some conceptual
and methodologic aspects of a model drive system. Behavioral Biology, 10(1), 27-
42.

Wright, J. W., Morseth, S., Mana, M. J., LaCrosse, E., Petersen, E. P., & Harding, J. W.
(1984). Central angiotensin III-induced dipsogenicity in rats and gerbils. Brain
Research, 295(1), 121-126.

Xiao, F., Puddefoot, J. R., Barker, S., & Vinson, G. P. (2004). Mechanism for
aldosterone potentiation of angiotensin II-stimulated rat arterial smooth muscle cell
proliferation. Hypertension, 44(3), 340-345.

Xue, B., Beltz, T. G., Yu, Y., Guo, F., Gomez-Sanchez, C. E., Hay, M., et al. (2011).
Central interactions of aldosterone and angiotensin II in aldosterone- and
angiotensin II-induced hypertension. American Journal of Physiology.Heart and
Circulatory Physiology, 300(2), H555-64.

 
140  
 
Yoshimura, R., Kiyama, H., Kimura, T., Araki, T., Maeno, H., Tanizawa, O., et al. (1993).
Localization of oxytocin receptor messenger ribonucleic acid in the rat brain.
Endocrinology, 133(3), 1239-1246.

Zardetto-Smith, A. M., Beltz, T. G., & Johnson, A. K. (1994). Role of the central nucleus
of the amygdala and bed nucleus of the stria terminalis in experimentally-induced
salt appetite. Brain Research, 645(1-2), 123-134.

Zhang, D. M., Stellar, E., & Epstein, A. N. (1984). Together intracranial angiotensin and
systemic mineralocorticoid produce avidity for salt in the rat. Physiology & Behavior,
32(4), 677-681.

Zhang, W., Tingare, A., Ng, D. C., Johnson, H. W., Schell, M. J., Lord, R. L., et al.
(2012). IP3-dependent intracellular Ca2+ release is required for cAMP-induced c-
fos expression in hippocampal neurons. Biochemical and Biophysical Research
Communications, 425(2), 450-455.

 
141