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NEUROPEPTIDE PLASTICITY IN THE HYPOTHALAMIC PARAVENTRICULAR
NUCLEUS OF SALT-LOADED MALE MUS MUSCULUS
By
JUSTIN ANDREW SMITH
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2016
© 2016 Justin Andrew Smith
To those that struggle and persevere
4
ACKNOWLEDGMENTS
I would like to thank my mentor Dr. Eric Krause for providing the guidance and
resources necessary to perform this research. His drive and dedication to scientific investigation
are an inspiration. I would also like to thank my committee members Drs. Maureen Keller-
Wood, Joanna Peris, Jason Frazier and Debbie Scheuer, for their support during my graduate
training. I am also grateful to my lab mates Helmut Hiller, Lei Wang, and Dr. Annette de Kloet
who all helped with this project and many others. Finally, I am especially thankful for the love
and support of my mother Marcia, grandmother Virginia, brothers Shawn and Glenn, and long-
time friend Walter.
During my training I was supported by a Graduate Student Fellowship. This work was
funded in part by NIH grants HL096830 (EGK), HL122494 (EGK), and HL116074 (ADdK).
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ...............................................................................................................4
LIST OF TABLES ...........................................................................................................................8
LIST OF FIGURES .........................................................................................................................9
LIST OF ABBREVIATIONS ........................................................................................................11
ABSTRACT ...................................................................................................................................12
CHAPTER
1 INTRODUCTION ..................................................................................................................14
Paraventricular Nucleus of the Hypothalamus .......................................................................16
Central and Systemic Maintenance of Hydromineral Homeostasis .......................................21
Stress-Responsiveness During Osmotic Challenge ................................................................25
Chronic Salt-Loading ..............................................................................................................32
Aims ........................................................................................................................................39
Acute Salt-Loading ..........................................................................................................39
Chronic Salt-Loading ......................................................................................................40
2 MATERIALS AND METHODS ...........................................................................................46
Generation of CRH Reporter Mice .........................................................................................46
Validation of CRH Reporter ...................................................................................................46
Antibody Characterization ......................................................................................................47
Immunofluorescence ...............................................................................................................48
Image Capture and Analysis ...................................................................................................49
3 CENTRAL AND BEHAVIORAL EFFECTS OF ACUTE SALT-LOADING .....................53
Background .............................................................................................................................53
Materials and Methods ...........................................................................................................54
Animals ............................................................................................................................54
Restraint Stress and Blood Sampling ..............................................................................55
Immunohistochemistry ....................................................................................................55
Image Capture and Analysis ............................................................................................57
Behavioral Testing ...........................................................................................................57
Statistics ...........................................................................................................................58
Results .....................................................................................................................................58
Subcutaneous Delivery of 2.0 M NaCl Modestly Increases pNa+ but Blunts
Restraint-Induced Elevations in Plasma CORT ...........................................................58
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Acute Hypernatremia Inversely Affects Restraint-Induced Activation of CRH and
Oxytocin Neurons in the PVN .....................................................................................59
Slight Elevations in the pNa+ are Associated with Decreased Anxiety-Like
Behavior in the EPM ....................................................................................................59
Discussion ...............................................................................................................................59
4 CENTRAL AND BEHAVIORAL EFFECTS OF CHRONIC SALT-LOADING ................69
Background .............................................................................................................................69
Methods ..................................................................................................................................70
Animals ............................................................................................................................70
Procedure .........................................................................................................................70
Restraint Stress and CORT ..............................................................................................71
Image Capture and Analysis ............................................................................................72
Statistics ...........................................................................................................................73
Results .....................................................................................................................................73
Salt-Loaded Mice Exhibit Increased Fluid Intakes and pNa+ Concentrations, but
Maintain Normal Indices of Blood Volume ................................................................73
Salt-Loaded Mice Maintain Food Intake and Body Weight ...........................................74
Salt-Loading Does Not Increase Neuronal Activation in PVN Vasopressin Neurons
or the SON ...................................................................................................................74
Salt-Loading Increases Fos Expression in the Posterior PVN ........................................74
Salt-Loading does not Affect Restraint-Induced Fos Induction in tdTomato
Neurons ........................................................................................................................75
Salt-Loading Blunts Restraint-Induced Elevations in Plasma CORT During
Recovery ......................................................................................................................75
Discussion ...............................................................................................................................75
5 CHRONIC SALT-LOADING-ASSOCIATED NEUROPEPTIDE PLASTICITY ...............86
Background .............................................................................................................................86
Methods ..................................................................................................................................88
Animals ............................................................................................................................88
In situ Hybridization ........................................................................................................88
Image Capture and Analysis ............................................................................................89
Statistics ...........................................................................................................................92
Results .....................................................................................................................................92
Salt-Loading Reduces CRH mRNA Expression in the PVN ..........................................92
Fluorescent Tracking of CRH mRNA by tdTomato .......................................................93
Total tdTomato Unaffected by Salt-Loading or Restraint ...............................................93
Salt-Loading is not Associated with Alterations in the Extent of Oxytocin-
NP/tdTomato Colocalizations in the PVN ...................................................................94
Salt-Loading is Associated with an Increase in Vasopressin-NP/tdTomato
Colocalizations in the PVN ..........................................................................................95
Salt-Loading does not Affect CRH Expression in Vasopressin-NP or Oxytocin-NP
Expressing Neurons of the SON ..................................................................................95
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Fluorogold Injections were Localized to the Nucleus Ambiguous, Pre-Bötzinger
Complex, and Rostroventrolateral Medulla .................................................................96
Retrogradely-Labeled Neurons Increase Expression of tdTomato and Vasopressin-
NP in PVN ...................................................................................................................97
Retrogradely-Labeled Neurons in the Amygdala are Distinct from tdTomato-
Expressing Neurons .....................................................................................................98
Discussion ...............................................................................................................................98
6 GENERAL DISCUSSION AND FUTURE DIRECTIONS ................................................119
Closing Remarks ...................................................................................................................137
Future Directions ..................................................................................................................140
LIST OF REFERENCES .............................................................................................................144
BIOGRAPHICAL SKETCH .......................................................................................................170
8
LIST OF TABLES
Table page
2-1 Antibody information.........................................................................................................51
9
LIST OF FIGURES
Figure page
1-1 Schematic of the major systems that interact with neurons in the paraventricular
nucleus to maintain hydromineral homeostasis .................................................................43
1-2 Schematic of CRH pathways projecting from the PVN ....................................................44
1-3 Osmosensitive and stress-responsive circuits ....................................................................45
2-1 Representative images validating CRH-Cre induced tdTomato expression. .....................52
3-1 Plasma measurements from mice 60 min after 2.0 M or 0.15 M NaCl injections .............64
3-2 2.0 M NaCl administration reduces the CORT response to restraint stress relative to
0.15 M NaCl.......................................................................................................................65
3-3 2.0 M NaCl injection attenuates restraint-induced activation of CRH neurons ................66
3-4 2.0 M NaCl administration and restraint increases activation of oxytocin-producing
cells in the PVN .................................................................................................................67
3-5 Acute hypernatremia attenuates anxiety-like behavior ......................................................68
4-1 Fluid consumption and the effects on indices of hydration ...............................................80
4-2 Individual fluid and food intake values .............................................................................81
4-3 Correlation graphs for food and fluid intake ......................................................................82
4-4 Daily food intake and body mass measurements ...............................................................83
4-5 Stress induced neuronal activation .....................................................................................84
4-6 Chronic salt-loading reduces the CORT response to restraint ...........................................85
5-1 Representative images of PVN sections processed for in situ hybridization of
digoxigenin-conjugated probe and CRH mRNA .............................................................104
5-2 Analysis of tdTomato reporting for CRH mRNA expression in the PVN ......................105
5-3 Analysis of total tdTomato in the PVN ............................................................................106
5-4 Representative unilateral images and quantification of oxytocin-NP/tdTomato
colocalizations in the “neurosecretory” region of the PVN .............................................107
5-5 Representative unilateral images and quantification of oxytocin-NP/tdTomato
colocalizations in the “preautonomic” region of the PVN ...............................................108
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5-6 Representative unilateral images and quantification of vasopressin-NP/tdTomato
colocalizations in the “neurosecretory” region of the PVN .............................................109
5-7 Representative unilateral images and quantification of vasopressin-NP/tdTomato
colocalizations in the “preautonomic” region of the PVN ...............................................110
5-8 Average total colocalizations in the PVN ........................................................................111
5-9 Representative unilateral images used for quantification of colocalizations in the
supraoptic nucleus ............................................................................................................112
5-10 A summary of FG injections targeting the RVLM ..........................................................113
5-11 Representative images and quantification of PVN neurons labeled with FG. .................115
5-12 Representative images and quantification of FG/vasopressin-NP colocalization ...........116
5-13 Representative images and quantification of FG/tdTomato colocalization .....................117
5-14 Representative image of FG and tdTomato in the amygdala ...........................................118
6-1 Increased vesicular glutamate transporter 2 appositions on tdTomato neurons in the
PVN with salt-loading......................................................................................................142
6-2 Decreased labeling for the astrocytic marker glial fibrillary acidic protein with salt-
loading..............................................................................................................................143
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LIST OF ABBREVIATIONS
ACTH Adrenocorticotrophin hormone
ASD Autism spectrum disorder
AT1 Angiotensin type 1
BNST Bed nucleus of the stria terminalis
CORT Corticosterone
CRH Corticotrophin releasing hormone
DAPI 4',6-Diamidino-2-phenylindole, dihydrochloride
EDTA Ethylenediaminetetraacetic acid
FG Fluorogold
HSD Hydroxysteroid dehydrogenase
HPA Hypothalamus-pituitary-adrenal
mRNA Messenger ribonucleic acid
NaCl Sodium chloride
NP Neurophysin
OVLT Organum vasculosum of the lamina terminalis
PVN Paraventricular nucleus of the hypothalamus
pNa+ Plasma sodium
RAAS Renin-angiotensin-aldosterone system
RVLM Rostral ventrolateral medulla
SEM Standard error of the mean
SFO Subfornical organ
SON Supraoptic nucleus
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Abstract of Dissertation Presented to the Graduate School
of the University of Florida
Requirements for the Degree of Doctor of Philosophy
NEUROPEPTIDE PLASTICITY IN THE HYPOTHALAMIC PARAVENTRICULAR
NUCLEUS OF SALT-LOADED MALE MUS MUSCULUS
By
Justin Andrew Smith
August 2016
Chair: Eric G. Krause
Major: Pharmaceutical Sciences – Pharmacodynamics
The paraventricular nucleus of the hypothalamus (PVN) orchestrates neurohumoral
responses to dehydration that maintain body fluid homeostasis through the coordinated actions of
neuroendocrine and preautonomic neurons producing corticotrophin-releasing hormone (CRH),
vasopressin, and oxytocin. To evaluate the neuropeptide plasticity and neuronal activation
within the PVN resulting from acute and chronic salt-loading, we analyzed the central expression
of red fluorescent protein (tdTomato) driven by CRH transcription together with
immunohistochemistry. Acute salt-loading was accomplished via subcutaneous 2.0 M NaCl
injections while chronic salt-loading took place by replacing drinking water with 2% NaCl for 5
days. Acute salt-loading resulted in an attenuated neuroendocrine response to a psychogenic
stressor, as both restraint-induced Fos in tdTomato neurons of the PVN and plasma
corticosterone were reduced compared with controls. Moreover, acute salt-loading increased the
number of activated neurons producing oxytocin in the PVN and decreased anxiety-like behavior
assessed on the elevated plus maze. Chronic salt-loaded and control mice exhibited similar Fos
induction within vasopressin neurons and in response to restraint; however, chronic salt-loading
was associated with a blunted corticosterone response to restraint, but an increased activation in
the posterior preautonomic region of the PVN. While the number of oxytocin and tdTomato
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colocalizations did not differ between groups, chronic salt-loading did significantly increase the
colocalization of vasopressin and tdTomato in putative magnocellular PVN neurons. To
evaluate neuropeptide expression in preautonomic neurons, mice were injected with the
retrograde tracer Fluorogold targeting the rostroventrolateral medulla and then subjected to salt-
loading. Interestingly, salt-loading significantly increased the colocalization of vasopressin and
Fluorogold as well as tdTomato and Fluorogold in PVN neurons. These results indicate that the
effects of acute and chronic salt-loading include central effects mediated by diverse neuronal
phenotypes in the PVN that likely act in concert to maintain homeostasis as well as influence
behavior and stress-responsiveness.
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CHAPTER 1
INTRODUCTION
Maintenance of hydromineral homeostasis is a biological imperative observed across all
mammalian species representing fundamental and well conserved homeostatic mechanisms
(Mecawi Ade et al., 2015). These mechanisms ensure adequate hydration as a result of a
complex set of endocrine, neural, and behavior-based interactions with the external environment
that are well suited to experimental manipulation (Bernard, 1949). Experimental paradigms
involving exposure to water deprivation or excessive sodium chloride serve to provide a better
understanding of normal and pathological functions guarding against hypertonicity. The
extensive investigation of these functions has led to the better understanding of multiple central
and systemic signals that converge on the hypothalamus, are integrated there, and produce well-
characterized responses (Stellar et al., 1954; Teitelbaum and Stellar, 1954). Specifically,
challenges to osmoregulation include responses governed by specialized neurons of the
paraventricular nucleus of the hypothalamus (PVN) which, as shown in Figure 1-1, coordinate
neuroendocrine secretion of vasopressin and oxytocin (Bourque, 2008), stress-responsiveness of
the hypothalamic-pituitary-adrenal (HPA) axis (Roberts et al., 2011), aspects of the renin-
angiotensin-aldosterone system (RAAS) (Hwang et al., 1986), and neural control of autonomic
renal and cardiovascular regulation (Stocker et al., 2004; Bardgett et al., 2014). Experiments that
challenge hydration and sodium handling have been especially fruitful in a somewhat isolated
sense; that is, osmoregulatory responses controlled by neurons in the PVN can be induced by
manipulating water or salt intake with reliable results. However, discovering how the individual
subtypes of neurons in the PVN coordinate, interact, and alter their transcriptional products and
signaling modalities in relation to one another requires assessment of multiple systems.
Unraveling the complexities of these relationships may lead to a better understanding of affective
15
and cardiovascular disorders involving the PVN as well as the frequent comorbidity of these
disorders with dysregulated systems that normally serve to regulate hydromineral balance.
The RAAS and the neuropeptides oxytocin and vasopressin have physiological functions
that are coupled with stress-responsiveness and behavior. The physiological stressors of
hypotension and hypovolemia stimulate renin release to catalyze angiotensin-II formation and
aldosterone availability (Fitzsimons, 1998), as well as vasopressin release (Yao et al., 2011).
These hormones promote fluid retention to stabilize blood volume and pressure (Fitzsimons,
1998), but also act in the brain to modulate behavior and potentiate stress-responsiveness
(Krause et al., 2011b) through direct and indirect action in brain regions that innervate the PVN
(Englemann et al., 2000; Albrecht, 2010). Low pNa+ is alleviated by eliminating excess water
through diuresis and vasopressin inhibition (Wang et al., 2013b) as well as by increasing salt
intake and reducing salt excretion. Hypernatremia increases the pituitary release of vasopressin
and oxytocin to cause renal water reabsorption and salt excretion (Masaharu and Hiraki, 2013),
but the central release of these peptides is known to affect distant forebrain and hindbrain targets
(Antunes-Rodrigues et al., 2013) that also influence anxiety (Knobloch et al., 2012; Neumann
and Landgraf, 2012b; Benarroch, 2013b; Frazier et al., 2013). Together these signals represent a
constantly fluctuating background upon which the stress response may be potentiated or
inhibited depending on the magnitude, polarity, and duration of an osmotic challenge.
The neuroanatomical pathways and interactions between PVN neuronal subtypes that
govern stress-responsiveness, autonomic function, and adequate hydration are complex and
represent ongoing lines of research (Antunes-Rodrigues et al., 2013). While many studies have
focused on these interactions separately, a cohesive picture of the specific central pathways that
are modulated on a circuit, cellular, and molecular basis is lacking. The pathways outlined in
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Figure 1-1 represent the major systems that are potently activated during an osmotic challenge.
To provide a foundational understanding of these systems and how they are controlled, an
introduction to PVN anatomy and physiology, systemic systems that interact with the PVN, and
the salt-loading methods that are the basis of this dissertation are discussed.
Paraventricular Nucleus of the Hypothalamus
Neurons in general can be classified according to function by determining connectivity
and the types of signaling molecules they utilize, and neurons in the PVN are especially
amenable to this classification. The PVN is a bilateral nucleus situated on either side of the
anterior or rostral portion of the third ventricle (Swanson and Sawchenko, 1983). Its location at
the base of the brain is ideal for efficient connectivity with the pituitary gland, which acts as an
intermediary between the convergence of neuronal input to the hypothalamus and the dispersive
property of the blood (Swanson and Kuypers, 1980; Swanson and Sawchenko, 1980). The
neurons in the PVN that influence the pituitary are further divided into neurons that directly
release neuropeptide into the systemic circulation via the posterior pituitary (Vandesande and
Dierickx, 1975) and those that release into the portal circulation to act on neurons of the anterior
pituitary which then dispense their contents systemically (Vale et al., 1977; Vale et al., 1981;
Rivier et al., 1982; Vale et al., 1986). Neurons that project to the posterior pituitary are of two
major types, those that produce vasopressin and those that produce oxytocin (Poulain and
Wakerley, 1982); both of which are classified as magnocellular because of their large soma
compared with other the types of cells in the PVN. Magnocellular neurons are generally grouped
together although the degree to which they are segregated from other types of neurons in the
PVN is species-specific (Biag et al., 2012). Notably, the magnocellular population of neurons in
the rat is highly segregated while magnocellular neurons in the mouse are much more diffuse -
an important subject that will be revisited several times in this text. Typical grouping of
17
magnocellular neurons in rats is found in the dorsal and lateral aspects of the PVN, with axonal
projections extending laterally around the fornix and then ventrally before coursing medially
towards and through the internal zone of the median eminence (Swanson and Kuypers, 1980;
Swanson and Sawchenko, 1980; 1983). Projections of magnocellular neurons are not limited to
the pituitary, as axonal collaterals and neurons with dedicated projections innervate various areas
of the brain (Sofroniew, 1980; Knobloch et al., 2012). Additionally, magnocellular vasopressin
and oxytocin neurons are found in the second highest concentration within the supraoptic
nucleus, a bilateral nucleus located just above the laterodorsal aspects of the optic chiasm and
optic tracts (Swanson and Sawchenko, 1983). SON magnocellular neurons project to the
pituitary and are intimately involved in hydromineral balance (Swanson and Sawchenko, 1983;
Bourque, 2008). Magnocellular dendrites are capable of bi-directional communication as in
addition to postsynaptic reception, dendrites have the additional ability to release neuropeptide
into the extracellular fluid to signal presynaptically and via passive diffusion (Ludwig and Leng,
2006).
In contrast to magnocellular neurons, neurons that signal to the anterior pituitary do so
via release into the hypothalamic-hypophyseal portal circulation along the external zone of the
median eminence (Akmayev, 1971; Akmayev and Popov, 1977). These neurons are classified as
parvocellular due to a smaller and more evenly contoured soma profile. Parvocellular neurons
contain and release a variety of neuropeptides that signal corresponding release (or inhibition of
the release) of paired hormones (Schally et al., 1968; Schally et al., 1973; Schally, 1978). The
primary parvocellular neuroendocrine hormones with their respective systemically-released
hormones are: corticotrophin-releasing hormone (CRH) and adrenocorticotropic hormone
(ACTH) (Vale et al., 1981); thyrotropin-releasing hormone and thyroid-stimulating hormone
18
(Boler et al., 1969; Burgus et al., 1970); gonadotropin-releasing hormone/gonadotropin-
inhibiting hormone and luteinizing hormone or follicle-stimulating hormone (Clarke and
Cummins, 1982; Vale et al., 1986); prolactin-releasing peptide/prolactin-inhibiting factor and
prolactin (Freeman et al., 2000); as well as somatocrinin/somatostatin and growth hormone
(Burgus et al., 1973; Brazeau et al., 1982). Similar to magnocellular neurons, parvocellular
neurons individually project to regions of the brain other than the median eminence (Wittmann et
al., 2009). Furthermore, both magnocellular and parvocellular neuropeptides are found in
neurons residing in areas other than the PVN (Valentino et al., 1995; Rodaros et al., 2007; Guo et
al., 2013; Bosch et al., 2016), suggesting that each systemic function that is controlled by a
particular neuropeptide likely has a series of circuits in the brain that operates via similar
receptors; however, these coincidental actions are still an active area of research. Indeed, the
overall projection map of PVN neurons implicates that many central functions might be
coordinated precisely with the release of neuropeptide and suggests that the naming of certain
neuropeptides as “releasing hormones” or “releasing factors” may be unfortunate as it describes
only a fraction of what they do. In this sense, PVN neurons project directly to a multitude of
hindbrain (Geerling et al., 2010), midbrain (Rodaros et al., 2007), and forebrain targets
(Rivalland et al., 2006; Tilbrook et al., 2006) that mediate diverse functions including ingestive
behavior (Kirchgessner et al., 1988), sexual function (Tang and McKenna, 2001; Bancila et al.,
2002), anxiety-like behavior (Busnardo et al., 2013), memory processes (Wiedenmayer et al.,
2005; Lucas et al., 2013), as well as autonomic functions related to body temperature (Menendez
et al., 1990; Madden and Morrison, 2009) and cardiovascular (Loewy and McKellar, 1980;
Byrum and Guyenet, 1987; Coote, 1995; Benarroch, 2005; Coote, 2005; Li et al., 2006; Ferguson
et al., 2008; Kawabe et al., 2008; Pyner, 2009) regulation. Especially pertinent here, PVN
19
connectivity includes hindbrain regions controlling and interacting with cardiovascular and renal
circuits that promote normal maintenance of water and salt handling (Guyenet, 2006; Bourque,
2008). These “preautonomic” neurons are largely grouped in the posterior or caudal region of
the PVN although there is a small degree of overlap with pituitary-projecting or “neurosecretory”
regions (Biag et al., 2012). Thus, the anatomy of the PVN is indicative of a diverse population
of neurons; however, each has a potential for increased complexity above and beyond basic
connectivity and primary phenotypical marker, especially when considering the functional
implications of colocalized neuropeptide.
The anatomical and neuropeptide designations outlined above for neurons in the PVN are
important for an overall understanding of major functions controlled by the PVN, but are also
oversimplified as multiple neuropeptides have been localized to each neuronal subtype. It should
be noted that identification of a particular neuronal substance is dependent on the validity of the
method used. Magnocellular vasopressin-producing neurons additionally express galanin
(Melander et al., 1986a; Melander et al., 1986b; Rokaeus et al., 1988; Gaymann and Martin,
1989), dynorphin (Watson et al., 1982; Whitnall et al., 1983), [Leu]enkephalin (Martin and
Voigt, 1981; Martin et al., 1983), and angiotensin-II (Kilcoyne et al., 1980). Similarly,
magnocellular oxytocin-producing neurons colocalize with markers for cholecystokinin
(Beinfeld et al., 1980; Vanderhaeghen et al., 1981; Martin et al., 1983), galanin (Gaymann and
Martin, 1989), thyrotropin-releasing hormone (Tsuruo et al., 1988), renin (Fuxe et al., 1982),
and [Met]enkephalin (Martin et al., 1983; Vanderhaeghen et al., 1983; Adachi et al., 1985).
Furthermore, magnocellular vasopressin and oxytocin neurons in both the PVN and SON
colocalize with tyrosine hydroxylase (Swanson et al., 1981; Chan-Palay et al., 1984; Spencer et
al., 1985) and magnocellular oxytocin neurons in these same regions colocalized with CRH.
20
There is less diversity of colocalized neuropeptides in the parvocellular neurons of the PVN
compared with the magnocellular neurons, although the functionality of parvocellular
colocalization of CRH and vasopressin is perhaps better understood.
The PVN contains a heterogeneous population of neurons that respond to stressful stimuli
in part through activation of parvocellular cells producing CRH, vasopressin, and a subset of
cells containing both CRH and vasopressin (Lightman, 2008). A combination of steroid and
neurotransmitter influences relay information related to stressor type, novelty, and magnitude to
these neurons to induce corticotroph production and ACTH release (Armario, 2006). Several
factors are known to stimulate ACTH release from the anterior pituitary (Abousamra et al.,
1987). These factors include: oxytocin (Makara et al., 1996), norepinephrine and epinephrine
(Rivier and Vale, 1983; Labrie et al., 1984), and angiotensin-II (Gaillard et al., 1981) as well as
the better studied and more potently acting CRH and vasopressin (Gillies et al., 1982). While
CRH is the dominant ACTH secretagogue (Vale et al., 1981), CRH and vasopressin synergize to
greatly potentiate the release of ACTH (Gillies et al., 1982), thus attributing special interest to
neurons that colocalize these neuropeptides. CRH and/or vasopressin release from parvocellular
neurons initiates activation of the HPA axis (Arborelius et al., 1998; Jacobson, 2014) which
normally serves to mobilize energy reserves through adrenal glucocorticoid release in
cooperation with the autonomic nervous system (Ulrich-Lai and Herman, 2009). The efficiency
of the stress response is governed by 1) the ability to successfully integrate multiple lines of
central and peripheral information to induce a response (Ziegler and Herman, 2002) and 2) the
behavioral adaptation and glucocorticoid feedback mechanisms responsible for effecting a timely
resolution (Keller-Wood and Dallman, 1984). Moreover, CRH and vasopressin act as
neurotransmitters and neuromodulators in various regions of the brain to coordinate behavioral
21
and autonomic stress responses (Dunn and Berridge, 1990), and in this sense can be localized to
forebrain- and hindbrain-projecting neurons of the PVN as well (Fig. 1-2).
Central and Systemic Maintenance of Hydromineral Homeostasis
The RAAS plays a major role in the regulation of systemic blood pressure, urinary
sodium excretion, and renal hemodynamics. In experimental models, stimuli such as
pharmacologically-induced vasodilation (Stocker et al., 2000), hypovolemia induced by
polyethylene glycol administration (Stricker and Verbalis, 1986), or hemorrhage (Ken'ichi and
Hitoshi, 2010) are potent activators of the RAAS and elicit thirst. Furthermore, volume
depletion associated with sodium loss provokes a rise in angiotensin-II and aldosterone levels
(Badaue-Passos et al., 2007). These conditions will be briefly addressed followed by an account
of their relevance to the potentiation of stress-responsiveness.
Blood pressure is intrinsically bound to hydromineral balance and deficiency compels
several homeostatic mechanisms to action. Carotid sinus and aortic baroreceptors decrease tonic
firing frequency in response to a drop in pressure, activating neurons in the nucleus of the
solitary tract that increase sympathetic outflow while decreasing parasympathetic tone (Fisher
and Paton, 2012). If the drop in pressure is severe, afferent signals from the nucleus of the
solitary tract and forebrain induce vasopressin release which acts on vasopressin type 1 receptors
of the vasculature (Aoyagi et al., 2009) and vasopressin type 2 receptors in the kidney (Sampey
et al., 1999) to promote vasoconstriction and water reabsorption, respectively. Cardiopulmonary
baroreceptors also activate nucleus of the solitary tract neurons that increase sympathetic nervous
system activity. These neurons cause rostroventrolateral medulla and intermediolateral cell
column efferents to release norepinephrine onto β1 receptors of the juxtaglomerular apparatus,
thereby increasing renin release and plasma renin activity to promote the synthesis of
angiotensin-II (Hainsworth, 2014). Additionally, reduced perfusion pressure is sensed by
22
baroreceptors in the renal vasculature to increase plasma renin activity via prostaglandin
signaling (Schweda and Kurtz, 2010). Prostaglandin-mediated renin release is furthermore
induced by a reduction of NaCl circulation in the renal medulla (Schweda and Kurtz, 2010).
Thus, reduced extracellular fluid volume is monitored by the systemic vasculature and kidneys to
initiate activation of the RAAS as well as hypothalamic nuclei controlling endocrine release and
autonomic balance.
Increased circulating angiotensin-II affects both systemic and centrally-mediated
compensatory measures. Angiotensin-II binding to angiotensin type 1 (AT1) receptors is a
potent vasoconstrictive stimulus (Fitzsimons, 1998), and activation of AT1 receptors expressed
on the efferent arteriole of the kidney increases glomerular filtration rate (Hiroyuki et al., 2013)
during instances of low blood pressure. Furthermore, elevated levels of angiotensin-II
upregulate the synthesis and release of aldosterone in the zona glomerulosa of the adrenal gland
(Hattangady et al., 2012). Aldosterone binds to cytosolic mineralocorticoid receptors within
renal tubular cells of the cortical collecting ducts, increasing the reabsorption of sodium and
water (Biller et al., 2000). The effects of angiotensin-II in the periphery restore extracellular
fluid volume by conserving water and sodium, allowing normal function of vital organs.
Accessibility of blood-borne angiotensin-II to the brain however, is blocked by the blood-brain
barrier, except for in regions that have a specialized blood-brain barrier architecture (McKinley
et al., 2002). These regions, termed circumventricular organs, allow the brain to sample the
immediate systemic environment through specialized neurons (Duvernoy and Risold, 2007). In
particular, the subfornical organ (SFO) and organum vasculosum of the lamina terminalis
(OVLT) are circumventricular organs that express receptors for angiotensin-II and aldosterone
(De Luca et al., 2013). Activation of AT1 receptors in the core of the SFO and lateral margins of
23
the OVLT by angiotensin-II (Sunn et al., 2003) stimulates a neuronal network with nodes in
several brain regions.
The net result of angiotensin-II induced activation of this network is the enigmatic
behavioral and physiological phenomenon of thirst (Stricker and Sved, 2000). Thirst, which is
largely dependent on circumventricular organ afferent projections to the median preoptic nucleus
(Hollis et al., 2008), as well as complex regulation of neuropeptide release (McKinley et al.,
2004) and sympathetic activation (Stocker et al., 2008) designed to complement systemic
function. Moreover, it is now well-established that the brain is capable of producing all the
components of the RAAS (Phillips and Sumners, 1998) (Gomez-Sanchez et al., 2005), and
systemic angiotensin-II and aldosterone elevations may influence the activity of the local brain
RAAS to modulate limbic and neuroendocrine systems.
In regards to aldosterone, its central actions are not restricted by the blood-brain barrier,
but instead, are limited by competition with glucocorticoids for binding sites (Geerling and
Loewy, 2009). Because aldosterone circulates at concentrations ~1000 fold less than
glucocorticoids (Bledsoe et al., 2005), most mineralocorticoid receptors in the brain are not
activated by aldosterone. Exceptions include the PVN (Chen et al., 2014); amygdala and locus
coeruleus (Robson et al., 1998); and nucleus of the solitary tract (Geerling and Loewy, 2006),
where specialized subsets of cells express both mineralocorticoid receptors and 11β-
hydroxysteroid dehydrogenase (HSD), suggesting that these areas are susceptible to
physiological states that increase circulating angiotensin-II and aldosterone. However,
controversy exists over the ligand for the mineralocorticoid receptors in the PVN. 11β-HSD is
expressed in two isoforms: 11β-HSD1 reduces CORT in humans to cortisol which allows
activation of glucocorticoid receptors; while 11β-HSD2 oxidizes cortisol to prevent activation of
24
mineralocorticoid receptors. Chen et al. found that 11β-HSD1, but not 11β-HSD2 was expressed
in the caudal PVN, and that mineralocorticoid receptors were present and likely activated with
sodium deprivation to increase sympathetic output (Chen et al., 2014). Haque et al. found that
11β-HSD2 was present with mineralocorticoid receptors in magnocellular neurons of the PVN
and SON, but did not provide any functional data (Haque et al., 2015). Knowledge of the ligand
for mineralocorticoid receptors in the PVN and SON is important to understand the physiological
relevance of steroid signaling to the individual types of neurons in the PVN, especially during an
osmotic perturbation.
The acute elevation of plasma tonicity that occurs following hypertonic saline
administration creates an osmotic gradient which moves water from the intracellular fluid
compartment to the extracellular fluid, which in turn, increases blood volume and pressure
(Caeiro and Vivas, 2008). The loss of water from the intracellular fluid compartment causes cell
shrinkage that is thought to activate osmoreceptors located on the membrane of specialized
osmosensitive cells. Research investigating osmoreceptor function supports the notion that
changes in membrane tension elicit conformational changes in the osmoreceptor that lead to
increased membrane ion permeability, thereby transducing the change in osmolality into a
signaling mechanism that can affect other cells (Voisin and Bourque, 2002; Bourque, 2008).
Osmosensitive cells are located in strategic areas including the throat (Kuramochi and
Kobayashi, 2000), intestines (Dooley and Valenzuela, 1984), hepatic portal vein (Baertschi and
Vallet, 1981) and circumventricular organs in the brain (McKinley et al., 1999). In regards to
circumventricular organs, the OVLT conveys information regarding a change in osmolality
directly to vasopressin and oxytocin producing cells in the SON and PVN (Johnson and Gross,
1993). As discussed previously, the release of vasopressin into the systemic circulation counters
25
the increased plasma osmolality by promoting renal water retention. In rodents, the elevations in
systemic oxytocin that follows hypernatremia promotes the inhibition of renin release and
stimulates renal sodium excretion (Sjoquist et al., 1999). Thus, increased plasma osmolality due
to excess body sodium elicits activation of osmosensitive cells causing subsequent release of
neuropeptides into the systemic circulation that alter the renal handling of sodium and water to
alleviate hypernatremia.
In summary, neuropeptide-releasing PVN neurons and the RAAS are potently activated
by altered blood pressure, volume and tonicity. Through multiple sensory mechanisms, body
fluid homeostasis is maintained through cardiovascular and renal signals that converge on the
hypothalamus through circumventricular organ and hindbrain inputs, which in turn, are
reciprocally connected to limbic brain regions controlling mood and affect (Fig. 1-3). Thus,
brain regions controlling mood and affect promote cardiovascular and endocrine responses to
stress through hypothalamic and hindbrain nuclei. Consequently, overlapping neuronal networks
are positioned to influence cardiovascular and endocrine responses to hydromineral imbalance as
well as psychological stressors (Fig. 1-3).
Stress-Responsiveness During Osmotic Challenge
Stressors can be classified into the two broad categories of physiological or
psychological. Physiological stressors include those that threaten body fluid homeostasis, and
activation of the RAAS is an example of the body’s attempt to cope through endocrine,
autonomic, and behavioral adjustments. Stressors that are psychogenic in nature influence
anxiety level, sympathetic tone, and the HPA axis through anticipatory, memory, and fear-related
systems relayed to the hypothalamus via limbic brain regions (Jankord and Herman, 2008).
Although often unnecessary to the welfare of the individual, coping mechanisms that respond to
psychogenic stressors can include RAAS activation (Krause et al., 2011b). Both types of
26
stressors have the potential to activate the HPA axis via stimulation of PVN parvocellular CRH
neurons that project to the median eminence. As discussed above, the release of CRH into the
primary capillary plexus of the hypothalamo-hypophyseal portal vasculature induces anterior
pituitary ACTH release. The binding of ACTH to the zona fasciculata layer of the adrenal gland
causes glucocorticoid synthesis and release into the systemic circulation that bind to
glucocorticoid receptors throughout the body to mobilize energy reserves and down-regulate
processes not essential to the stress response. Negative feedback of glucocorticoid occurs at
several levels including the pituitary and PVN to bring about termination of HPA axis activity.
Exposure to psychogenic stress activates the RAAS in the absence of threats to
hydromineral balance to increase central and systemic angiotensin-II (Yang et al., 1993; Krause
et al., 2011b) as well as the expression of AT1 receptors in the brain (Aguilera et al., 1995a).
Stress-induced up-regulation of AT1 receptor mRNA occurs in the SFO, anterior pituitary, zona
glomerulosa and medulla of the adrenal gland (Leong et al., 2002), and on CRH cells in the PVN
(Aguilera et al., 1995b). Similarly, administration of dexamethasone, a glucocorticoid receptor
agonist, significantly increases AT1 receptor binding in the PVN and SFO (Shelat et al., 1999).
Taken together, these results suggest that glucocorticoids derived from stress-induced HPA axis
activation may feed forward to increase AT1 receptor expression to alter sensitivity to centrally
released angiotensin-II. Consistent with this notion, pretreatment with dexamethasone augments
water drinking stimulated by central administration of angiotensin-II (Ganesan and Sumners,
1989). Elevated glucocorticoids accompanied by increased angiotensin-II release in the PVN
reduces 11β-HSD effectiveness resulting in increased preautonomic stimulation (Gabor and
Leenen, 2012) and potentiation of sympathetic activity (de Kloet et al., 2005). Thus, induction
27
of the HPA axis may interact with an activated RAAS to sensitize subsequent behavioral and
autonomic responses to hydromineral imbalance.
The RAAS has gained considerable attention over the past decade as a potential
therapeutic target for treating stress-related disorders, anxiety, depression, alcoholism, cognitive
impairment as well as neuroinflammatory and neurodegenerative diseases (Saavedra and Pavel,
2005; Albrecht, 2010; Saavedra, 2012; Anil Kumar et al., 2014). Angiotensin receptor blockers
administered either peripherally or centrally, improve mood and cognition as well as attenuate
HPA axis and sympathetic responses to stressors having strong psychogenic components
(Saavedra and Pavel, 2005; Albrecht, 2010; Saavedra, 2012; Anil Kumar et al., 2014). While the
specific circuits that angiotensin-II receptor antagonists modulate to achieve these affects are not
fully understood, increased circulating angiotensin-II is known to influence hypothalamic
function through various pathways including those originating in circumventricular organs and
regions governing mood and affect.
The SFO and OVLT, together with the median preoptic nucleus represent key regulatory
areas that both share connectivity with one another and project to the hypothalamus. As
previously discussed, the SFO and OVLT are unique in their ability to sample the constituents of
the systemic circulation and promote activation of circuits to preserve homeostasis. Activation
has been shown to alter the balance between excitatory and inhibitory projections to the PVN
and SON which in turn strongly influences the activity of the HPA axis and behavior.
Recent investigation into the effects of hypertonic saline injection on humoral,
behavioral, and central measures of stress-responsiveness in male rats and mice revealed a
prominent role for activation of oxytocinergic pathways. Hypertonic (2.0 M) saline injected
subcutaneously followed by 60 min water deprivation resulted in decreased ACTH,
28
glucocorticoid, and plasma renin activity during a 60 min restraint period, but significantly
increased plasma oxytocin levels (Krause et al., 2011a). Cardiovascular recordings determined
that rats rendered mildly hypernatremic and subsequently exposed to restraint stress had mean
arterial pressure and heart-rate variability return to prerestraint levels faster than normonatremic
controls (Krause et al., 2011a). Consistent with previous reports (Pirnik et al., 2004), acute
hypernatremia increased Fos induction in vasopressin and oxytocin expressing neurons in the
PVN and SON, an effect that was predictive of increased social interactions but decreased
anxiety-like behavior in the elevated plus maze (Krause et al., 2011a). Acute hypernatremia and
restraint stress interacted to increase Fos expression in the OVLT, oval capsule of the BNST,
ventral lateral septum, and central nucleus of the amygdala (Frazier et al., 2013). However, the
increased neuronal activation that occurred subsequent to acute hypernatremia and restraint was
specific to these brain regions as the medial prefrontal cortex, lateral ventral BNST, and
dorsomedial hypothalamus showed no significant change in Fos expression (Frazier et al., 2013).
Whole cell patch clamp recordings revealed that acute hypernatremia caused inhibition of CRH
neurons in the PVN that was dependent on activation of oxytocin receptors (Frazier et al., 2013).
Several lines of investigation (Huang et al., 2014) are actively pursuing the neuronal
mechanism underlying the dampened HPA axis activation that occurs with acute hypernatremia.
Seminal studies conducted by Ludwig et al. discovered that subsequent to the systemic release of
vasopressin and oxytocin that follows peripheral salt-loading, brain levels of these neuropeptides
exhibit a robust and sustained increase (Ludwig et al., 1994). The implication is that
magnocellular neurons in the SON and PVN release neuropeptides from dendrites and these
neuropeptides may act as autocrine or paracrine signals at their site of origin. In this regard,
dendritic release of vasopressin serves as a powerful autocrine signal that modulates firing
29
patterns and activity of magnocellular vasopressin neurons in the SON and PVN (Ludwig and
Leng, 1997; Gouzenes et al., 1998). Recently, dendritically-released neuropeptide also was
found to signal in a paracrine fashion to influence the functional activity of neighboring neurons
of a differing neuronal phenotype. Specifically, the Stern laboratory discovered that dendritic
release of vasopressin stimulated neighboring presympathetic neurons in the PVN to drive
sympathoexcitation following a hyperosmotic stimulus (Son et al., 2013). Hyperosmotic stimuli
also trigger the dendritic release of oxytocin in the hypothalamus (Ludwig and Leng, 2006;
Mabrouk and Kennedy, 2012) and oxytocin neurons are in close proximity to CRH neurons that
express oxytocin receptor mRNA in the PVN (Dabrowska et al., 2011). Therefore, it is possible
that the inhibition of CRH neurons that accompanies acute hypernatremia maybe regulated by
dendritically released oxytocin that activates oxytocin receptors expressed on CRH neurons in
the PVN.
Identifying the oxytocinergic circuits underlying the anxiolytic effects of acute
hypernatremia may reveal neuronal mechanisms that quell excitation of neurons that facilitate
physiological and behavioral manifestations of fear and anxiety. The magnocellular neurons in
the PVN and SON are the major source of oxytocin but whether these neurons supplied oxytocin
to forebrain nuclei controlling mood and affect was unclear because their axons are known to
terminate in the posterior pituitary where they release oxytocin into the systemic circulation.
Consequently, endogenously generated oxytocin was hypothesized to activate oxytocin receptors
in forebrain nuclei by diffusion of dendritically released peptide (Ludwig and Leng, 2006) or via
axonal release from a subset of parvocellular oxytocinergic neurons residing in the PVN that do
not project to the pituitary (Swanson and Sawchenko, 1983). These hypotheses about the source
of centrally released oxytocin have undergone a paradigm shift as the result of recent research
30
utilizing advanced techniques in neuroanatomical tract-tracing. These elegant studies conducted
by Grinevich and colleagues discovered that oxytocin neurons in the PVN and SON that project
to the posterior pituitary also send axon collaterals to forebrain nuclei mediating stress-
responsiveness and anxiety-like behavior (Knobloch et al 2012). In other words, oxytocinergic
magnocellular neurons in the PVN and SON simultaneously project to the posterior pituitary and
the forebrain, presenting the possibility that systemic and central release of oxytocin maybe
accomplished by the same neurons. In rodents, magnocellular oxytocinergic neurons in the PVN
and SON are robustly excited by elevations in the pNa+ (Krause et al., 2011a; Frazier et al.,
2013). Therefore, it is possible that acute hypernatremia is a systemic stimulus that excites
hypothalamic osmosensing neurons to promote the release of oxytocin into systemic circulation,
and subsequently, stimulates the release of oxytocin from axons terminating in forebrain nuclei
known to mediate physiological and behavioral manifestations of fear and anxiety. Amygdalar
CRH neurons are implicated in the development of anxiety disorders and further research is
warranted to determine whether this potential oxytocinergic pathway is a viable therapeutic
target.
In addition to the central nucleus of the amygdala, the extensive mapping of
oxytocinergic afferents throughout the rat brain isolated dense innervation of the BNST
(Knobloch et al., 2012). The BNST is a complex set of at least a dozen sub-nuclei that mediate
the information gathered by higher-order perceptual and memory-based brain regions through an
integrating process involving the hindbrain and hypothalamus (Dong et al., 2001; Bota et al.,
2012). Because of this pivotal role, the BNST is rich in neurochemical diversity, both locally
and in terms of the phenotype of afferent and efferent projections (Dong and Swanson, 2006;
Bota et al., 2012). Rainnie and colleagues used viral anterograde tracing of PVN neurons to
31
reveal that oxytocin immunoreactive fibers terminating in the BNST originate from neurons in
the PVN (Dabrowska et al., 2011) and subsequent studies suggest that these cells are
osmosensing magnocellular neurons (Knobloch et al., 2012). Acute hypernatremia followed by
restraint-stress robustly activated oxytocin neurons in the PVN (Krause et al 2011a) as well as
neurons residing in the oval capsule portion of the BNST (Frazier et al., 2013). These studies
support the hypothesis that elevations in body sodium levels excite magnocellular oxytocin
neurons and subsequent psychogenic stress exposure may trigger the release of oxytocin from
axons terminating in the BNST, which in turn, activate neurons within this nucleus. The pattern
of neuronal activation that occurs after acute hypernatremia and restraint is predictive of
decreased anxiety-like behavior and increased social interactions (Krause et al., 2011a). In this
regard, Kim et al found that two BNST sub-regions have highly specific roles in mediating
separate features of anxiety such as risk-avoidance and respiratory rate (Kim et al., 2013). While
this study found that activation of the BNST increased anxiety, activation associated with acute
hypernatremia seemed to be related to a decrease in anxiety-like behavior. As mentioned, the
BNST is a highly diverse region in terms of cellular capabilities and connectivity that may be the
basis for understanding the apparent discrepancies in activation of this region. Ultimately, the
circuit level data that has produced valuable insights into the effects of activating cells in these
areas will have to be complemented by studies that take into consideration ligand-receptor
interactions.
An acute osmotic challenge induces anxiolytic and pro-social effects associated with an
increase in oxytocin-producing cell activation and a reduction in the HPA and behavioral
components of the stress response. As oxytocin cells in the PVN are both osmosensitive and
receive projections from osmosensitive areas of the brain, the increase in activation is likely due
32
to the pivotal role of oxytocin in rectifying the fluid imbalance. Through dendritic release and
collateralized axonal projections that target the posterior pituitary and limbic brain regions
influencing mood and behavior, oxytocin signaling orchestrates the humoral and behavioral
responses necessary to maintain hydromineral homeostasis specifically in the context of an acute
hyperosmotic stimulus.
Chronic Salt-Loading
Maintenance on a high-salt diet involves the replacement of standard rodent chow usually
composed of 0.3-0.6% NaCl with chow containing 3-8% NaCl (Yu et al., 1998; Fang et al.,
2000; Sanders, 2009). In contrast to other forms of salt-loading, high-salt diets generally allow
the experimental subject ad libitum access to water. As a result, the physiological and
psychological stress may be considered less severe than other salt-loading paradigms and in
certain cases may be an ideal model of hypertension in salt-sensitive individuals (Sanders, 2009).
High-salt diets increase blood pressure via volume expansion (Cowley, 1997), by increasing
pNa+ levels which activate central osmosensors to reflexively increase blood pressure (Johnson
and Gross, 1993), or by both mechanisms (Fang et al., 2000). Perhaps the most striking long-
term effects of a high-salt diet are those published by Meneely and Ball who found that in a
study of 679 male rats, survival rates decreased dramatically when dietary salt was increased
(Meneely and Ball, 1958). In this study, 75% of rats eating chow composed of 0.15%-2.0%
NaCl were still alive after 16 months, however after 5 months only 5% of rats eating 21% NaCl
chow were still alive. Gradations of NaCl content between 0.15% and 21% predicted mortality
as consumption of increased concentrations were positively correlated with increased death rates.
Death was usually preceded by a “nephrosis-like syndrome” and abrupt onset of massive edema.
Thus, even with access to water, high dietary salt intake can still significantly impact mortality
through vascular lesions (Tobian and Hanlon, 1990), albuminuria and collagen deposition (Yu et
33
al., 1998), cerebrovascular accidents (Tobian and Hanlon, 1990), and accelerated renal failure
(Sanders et al., 2001).
Chronic salt-loading can also be accomplished by replacing drinking water with sodium
chloride solutions generally consisting of 1.5%-3.0% NaCl. Perhaps the most severe
experimental paradigm of those described, chronic salt-loading both induces cellular dehydration
and with time depletes volume (Watts et al., 1995). Early reports (Duchen, 1962) of salt-loading
lasted up to 21 days after which point rats weighing 200-250g at the beginning of the study had
lost up to 60g. Additionally, rats lost all traces of what was then called “neurosecretory
material” in the posterior lobe of the pituitary which disappeared commensurate with vasopressin
secretion during the first week. This extensive remodeling of pituitary tissue morphology is
indicative of the intense involvement of pituitary hormones in the homeostatic response.
Comparison of the effects of rats eating 1% NaCl chow and rats drinking 1% NaCl saline found
that drinking saline resulted in a 50% reduction in life span compared with high-salt diet
consumption as well as a 66% rate in the development of hypertension (Koletsky, 1958; 1959).
Furthermore, it was determined that the vascular, renal, and cardiac lesions caused by drinking
saline were identical to those induced by pharmacologically activating mineralocorticoid
receptors via deoxycorticosterone treatment (Selye, 1943; Selye et al., 1943; Selye and Stone,
1943) which similarly overloads the body with sodium. The most common reason for death in
chronic salt loaded rats was pneumonia associated with renal dysfunction (Koletsky, 1961).
While the vascular injuries caused by salt-loading were very similar to those found in humans
with essential hypertension, they were not found to be identical (Koletsky, 1961). Essentially,
chronic salt-loading through forced saline imbibition is a potent stimulus with many
physiological correlates to the development of human hypertension especially in those that may
34
be salt-sensitive; therefore, the alterations in the brain that occur during chronic salt-loading may
also be mechanistically related to the development of essential hypertension. In pursuit of these
mechanisms, we designed our experiments in Chapters 4 and 5 to investigate changes that occur
in the early (Day 5) stages of these central alterations when peripheral causes for hypertension
have not yet fully developed.
The chronic salt-loading paradigm typically results in systemic osmoregulatory responses
that are pushed close to or beyond homeostatic capacity. The phases described by Watts et al
involve an initial short (~24-48 hr) successful adaptation that allows for normal food intake and
defense against intravascular volume loss that is then followed by a progressively increasing
hematocrit and plasma osmolality (Watts et al., 1995). The initial phase involving successful
homeostasis is also characterized by elevated CORT, which then gradually becomes suppressed
during the course of the salt-loading term. Furthermore, the end products of the RAAS,
angiotensin-II and aldosterone, have the potential to become involved in an adverse relationship
as angiotensin-II has both the effects of maintaining blood pressure in response to volume loss
and increasing aldosterone levels (Laragh et al., 1960), the latter of which is counterproductive in
the context of elevated pNa+. Regarding this adverse relationship, Husain et al. found that while
aldosterone was potently suppressed during chronic salt-loading, plasma angiotensin-II levels
were not different from controls (Husain et al., 1987). This was attributed to a sequestration of
circulating angiotensin-II in the adrenal gland by receptor mediated endocytosis which would
allow for the action of angiotensin-II to constrict the vasculature in response to increasing
hypovolemia without stimulating aldosterone release. Rats drinking hypertonic saline instead of
water develop hypertension, vascular and renal necrosis, and have shortened life spans which are
complications that take longer to develop in, but that are similar to those observed in the
35
deoxycorticosterone-salt model of cardiovascular disease (Koletsky, 1961). Thus, the highly
dysregulated humoral constituency is directed towards maintaining blood pressure and hydration
of each the body’s fluid compartments, but which also are involved with the development of
cardiovascular and renal damage.
During the course of a standard five- to seven-day experimental period of drinking
hypertonic saline, the continued threat of long-term intracellular dehydration and hypotension
results in behavioral adaptations characterized by drinking large volumes of fluid, but limiting
food intake (Watts et al., 1995). However, increased saline intake has not always been the
observed behavior, as rats were found to exhibit highly variable fluid intakes in response to salt-
loading (Watts et al., 1995). It is possible that in this case, the behavioral response variability
may reflect a conflict between goal-directed motor responses aimed towards increased water-
seeking behavior and altered salt-appetite which may vary with each individual.
As discussed above, the paradigm of chronic salt-loading results in several deviations
from corresponding homeostatic set points, requiring an appropriately altered stress response to
promote survival in the face of a maligned internal environment. Stressful psychological events
experienced in the context of the physiological stress of forced hypertonic saline administration
have shown that HPA axis sensitivity is augmented during hydromineral imbalance. Rats
maintained on 2% NaCl for seven days and then rehydrated presented with sensitization of HPA
axis measures upon rehydration (Amaya et al., 2001). These rats had decreased CORT during
dehydration, but exaggerated their basal and restraint-induced ACTH responses after one week
of rehydration. Parvocellular CRH decreased with salt-loading, but increased during rehydration
while vasopressin increased during both periods. Interestingly, plasma vasopressin levels
increased with restraint during dehydration, but CRH did not suggesting that vasopressin has an
36
important role in stress-responsiveness specifically during the course of long-term osmotic
challenge when ACTH secretion is inhibited.
Chronic salt-loading is known to modulate the expression of CRH in the PVN through
circuits originating in the lamina terminalis. Kovacs and Sawchenko found that rats maintained
on 2% NaCl solution instead of water for seven days exhibited a shift in CRH mRNA expression
from a parvocellular region to a magnocellular distribution (Kovacs and Sawchenko, 1993).
This shift was abolished with knife cuts dissociating the connectivity between the OVLT and
SON, but not when pathways between the median preoptic nucleus and SFO or median preoptic
nucleus and SON were severed, promoting the idea that the OVLT is the primary osmosensitive
region influencing CRH plasticity in the PVN of rats (Kovacs and Sawchenko, 1993). This
study also provided evidence that the increased magnocellular expression of CRH was specific to
oxytocin cells.
In support of these findings, another study found increased CRH mRNA in oxytocin cells
of the PVN and SON in rats using the same protocol (Imaki et al., 2001), but also found
increased CRH type 1 receptors and increased urocortin (a CRH type-2 receptor ligand) in both
oxytocin and vasopressin magnocellular cells of the PVN and SON (Imaki et al., 2001). Salt-
loading also decreased CRH type 1 receptors in parvocellular neurons of the PVN, but increased
their expression in the intermediate pituitary (Imaki et al., 2001). The purpose of an increased
influence of CRH in the intermediate pituitary is unclear; however, it has been suggested that
corelease of CRH with magnocellular neuropeptides potentiates the release of the latter (Bondy
and Gainer, 1989). Overall, the relationship between where CRH synthesis goes up relative to
where receptor expression increases following osmotic stimulation is not exactly known.
However, CRH expression does increase with salt-loading in neurons that project to the PVN.
37
These regions include the fusiform nucleus of the BNST, periventricular nucleus, and lateral
hypothalamic area (Watts et al., 1995). Furthermore, CRH expression is increased in the
amygdala with salt-loading, and the amygdala contains a dense population of neurons projecting
to the BNST (Sun et al., 1991). Interesting enough, it seems that concomitant increases in CRH
and its receptor is specific to PVN neurons (Rivest et al., 1995). Together, the changing CRH
system in the brain during salt-loading may reflect a concert of mechanisms working through the
PVN to alter the pituitary release of ACTH and posterior pituitary hormones.
The components of the HPA axis and osmoregulatory mechanisms are conserved across
mammalian species; however, it is possible that salt-related plastic changes in CRH and CRH
type 1 receptor gene expression in rats may not be generalized to the mouse model. In a
comparative study, restraint-induced CRH mRNA upregulation and Fos expression occurred in
the PVN of both rat and mouse (Imaki et al., 2003). However, restraint- and hypertonically-
induced increases in CRH type 1 receptors as well as hypertonically-stimulated CRH expression
in PVN magnocellular neurons were all found to be specific to rats with no significant changes
noted in mice (Imaki et al., 2003). In mice, CRH type 1 receptor expression was mainly
colocalized with CRH-positive cells and very few oxytocin or vasopressin cells suggesting an
autoregulatory or positive feedback mechanism as well as a potential species-specific
intermediary between salt-balance and stress-responsiveness (Imaki et al., 2003). The increase
in CRH expression found in rat SON with salt-loading was found to not occur in mice, a
difference confirmed by others (Hinks et al., 1995). Further investigation into the apparent
differences in mouse and rat neuroplastic responses to salt-loading are indeed warranted.
The alteration in components of stress-responsive neurons in the PVN is predictive of
altered autonomic stress-responsiveness. A year-long experiment was carried out to test the
38
effects of conflict stress on baboons that were maintained on a normal or high-salt diet (Turkkan,
1994). In this endeavor, it was found that a five-month period elapsed before high-salt diet alone
significantly impacted a rise in mean arterial pressure of 6+/-5 mmHg (Turkkan, 1994) .
Following twice daily food/shock conflict sessions, stress and a high-salt diet combined had
almost three times the effect on blood pressure compared with high-salt diet alone with a rise in
mean arterial pressure of 17+/-3 mmHg. Individual representative cases revealed indices of
stress-sensitivity that positively correlated with the impact high-salt diet had on increased mean
arterial pressure. These include urinary free cortisol, degree of indecision during conflict stress,
as well as pressure diuresis and natriuresis. It is therefore evident that the physiological stress
brought about by high sodium intake can exacerbate the autonomic response to psychological
stress, thereby contributing to the development of hypertension and associated cardiovascular
disease.
Parvocellular neurons in the PVN likely modulate autonomic function through the release
of CRH in the rostroventrolateral medulla (RVLM). As with any long-term challenge involving
blood pressure regulation, the core network of barosensitive sympathetic efferents composed of
connectivity between the RVLM, nucleus of the solitary tract, spinal cord, and PVN is poised to
be altered to counter chronic hyperosmotic conditions. Regarding the RVLM, barosensitive
neurons residing in this region are hyperactive in spontaneously hypertensive, salt-sensitive, and
renal hypertensive rat strains (Guyenet, 2006) and PVN neurons projecting to the RVLM induce
this hyperactivity during water deprivation (Stocker et al., 2005; Stocker et al., 2006). Milner et
al. found that CRH-releasing parvocellular PVN neurons project to the RVLM where CRH
binding to the CRH receptor caused a sustained increase in mean arterial pressure (Milner et al.,
1993). Furthermore, in addition to local production of CRH by neurons in the RVLM, afferent
39
input from CRH-positive PVN neurons forms excitatory asymmetric synapses on RVLM
neurons. Ultrastructural localization of these synapses were found most frequently innervating
distal dendrites suggesting that CRH release in the RVLM serves more of a modulatory role
rather than a direct influence on neuronal activity. Taken together, the PVN to RVLM circuit
composed of parvocellular CRH containing neurons is situated to coincidentally influence the
HPA axis and autonomic response to stress. Thus, CRH reconfiguration in the PVN during
chronic salt-loading may involve increased CRH production in neurons projecting to the RVLM.
Aims
Acute Salt-Loading
Previously published research found that acute salt-loading is anxiolytic and dampens
stress-responsiveness in laboratory rats (Krause et al., 2011a; Frazier et al., 2013). While these
effects were consistently replicated in rats, whether these effects generalize to mice, and whether
acute salt-loading decreases neuronal activation in PVN neurons genetically modified to
fluorescently report for CRH transcription has yet to be determined. Furthermore, methods to
identify CRH mRNA in the brain have been limited by poor spatial resolution suggesting that
utilizing an advanced method to detect CRH in the brain will be a significant contribution to this
area of research.
The aims of Chapter 3 were to determine whether acute salt-loading decreases anxiety-
like behavior and restraint-induced HPA axis activation in mice and to evaluate the associated
neuronal activation in the PVN. Mice rendered mildly hypernatremic via systemic
administration of 2.0 M NaCl were subjected to restraint or behavioral testing on the elevated
plus maze. Quantification of Fos-induction in both CRH- and oxytocin-positive neurons in the
PVN served to provide insight towards neural mechanisms contributing to the HPA axis-
dampening and anxiolytic effects of acute mild hypernatremia.
40
Chronic Salt-Loading
The anxiolytic and HPA axis suppressing effects of acute salt-loading may have
reinforcing value leading to an over-consumption of salt. High sodium intake leads to
hypertension in salt-sensitive individuals, and elevated blood pressure is a risk factor for
developing a heart, vascular, and renal pathologies. Chronic salt-loading causes many of the
physiological manifestations associated with cardiovascular and renal disease, but also induces
relevant alterations in neuropeptide expression in the PVN. These alterations involve the same
PVN neuropeptides active in the response to acute hypernatremia, and include rearrangement of
CRH expression and increased systemic release of vasopressin and oxytocin. However, the
majority of chronic salt-loading experiments have been performed in rats and experiments in
mice have not always supported data using a rat model. Given the increased usage of mice in
research, substantiation of the central and behavioral effects of salt-loading in mice is a general
goal of these experiments. Collectively, the following aims serve to identify specific changes in
behavior and neuropeptide expression associated with a known progression of physiological
alterations that result in hypertension, cardiovascular and renal damage, and attenuated life-span.
The first and second aims are covered in Chapter 4 while the third and fourth aims are covered in
Chapter 5.
The first aim was to evaluate ingestive behavior and the effects of drinking 2% NaCl on
pNa+, hematocrit, and plasma proteins. One of the goals of Chapter 4 was to determine if
transgenic reporter mice drinking 2% NaCl for five days would alter ingestive behavior to
increase fluid intake and decrease food intake. The literature conflicts as to whether volume of
2% NaCl consumed is highly variable or only increases substantially. Additionally, feeding
behavior in the mouse is comparatively less well studied than the rat, meaning that these data
will serve to substantiate the murine response to salt-loading. Subsequent experiments
41
investigated changes in neuronal activation and neuropeptide expression that were to be
interpreted with respect to the changes in ingestive behavior and plasma constituency found in
Aim 1 i.e. the changes in the brain viewed in the context of anorexia, polydipsia, hypovolemia,
etc.
The second aim included evaluation of neuronal activation and the HPA axis response to
restraint. While the literature indicates that in the rat PVN and SON neurons are activated and,
more specifically, vasopressin neurons are activated in these regions, similar experiments in mice
have yielded conflicting results. Moreover, the rostro-caudal organization of the PVN is such
that neuroendocrine neurons are largely parceled out from preautonomic neurons suggesting that
evaluation of total neuronal activation will indicate the degree to which these functions are
involved in the regulatory response. Therefore, the goals of the second aim were to determine if
1) vasopressin neurons were activated 2) restraint or salt-loading had an effect on total Fos in
neuroendocrine or preautonomic subregions of the PVN 3) restraint or salt-loading had an effect
on PVN neurons reporting for CRH transcription and 4) the CORT response to restraint was
suppressed. Based on the literature and our acute salt-loading experiments, we hypothesized that
vasopressin neurons would be activated to conserve water, but activation of CRH neurons would
be suppressed as ACTH and CORT in salt-loaded rats are attenuated. We hypothesized that the
surge in CORT in response to restraint would similarly be suppressed in mice. Furthermore,
activation of preautonomic neurons located mainly in the posterior region of the PVN has to our
knowledge never been addressed following chronic salt-loading. As substantial changes are
known to occur centrally to accommodate high salt-intake, we hypothesized that preautonomic
neurons in the PVN would increase their activity. The data obtained from these experiments
42
serve to provide a foundational perspective of the cell types and therefore the functions that are
active to mediate the central response to salt-loading.
The third aim was to determine the extent of CRH colocalizations with vasopressin and
oxytocin neurons in the PVN and SON. This aim necessitated the detailed evaluation of CRH
mRNA expression using both a conventional method and genetic reporting for CRH
transcription. Salt-loading induced rearrangement of CRH expression in the rat has not always
been confirmed by similar experiments performed in mice. Furthermore, the limitations inherent
in using colchicine pretreatment to utilize antibodies directed against the CRH peptide make
colocalization experiments difficult. Moreover, the use of more than one method to isolate CRH
transcript represents an important methodological investigation into a novel technique to isolate
CRH positive neurons. The experiments in Chapter 5 included immunohistochemical
investigation with antibodies for vasopressin and oxytocin in brain tissue from CRH reporter
mice to compare increases in CRH induced by salt-loading within these particular cell types in
the PVN and SON. These results indicate the neuropeptides involved with the central response
to salt-loading.
The final aim was to determine the extent of salt-loading induced CRH and vasopressin
expression in PVN preautonomic neurons projecting to the RVLM. The PVN coordinates both
neuroendocrine and autonomic functions. Preautonomic neurons were identified using the
retrograde tract-tracer Fluorogold (FG) injected into the RVLM. The ultimate goal of Chapter 4
was to test the hypothesis that CRH and vasopressin increase expression in a defined circuit
composed of PVN projections to the RVLM, thereby coordinating neuroendocrine control of the
response to salt-loading with centrally-mediated autonomic regulation.
43
Figure 1-1. Schematic of the major systems that interact with neurons in the paraventricular
nucleus of the hypothalamus to maintain hydromineral homeostasis. Image is of a
coronal section taken through the brain of a CRH reporter mouse (see Chapter 2 for
description). The image includes a unilateral portion of the PVN with fluorescent
reporting for CRH (red), immunohistochemical labeling of vasopressin neurons
(green), and neurons retrogradely-labeled with tract tracer injected into the
rostroventrolateral medulla (magenta).
44
Figure 1-2. Schematic of CRH pathways projecting from the PVN. While the effects of CRH
released from neurons residing in the PVN is well characterized for pituitary-
projecting neurons, the functions of CRH released in other areas are less well
understood. However, the presence of CRH receptors and functional studies
microinjecting CRH in these areas suggests a role in autonomic regulation.
45
Figure 1-3. Osmosensitive and stress-responsive circuits. Circuits adapted in part from Ulrich-
Lai and Herman, 2009 and Bourque, 2008 demonstrating key hypothalamic circuits
that mediate the behavioral, endocrine, and autonomic stress-response (shown in red
and green). Many of these circuits are also activated when the plasma osmolality is
elevated above a threshold set point (shown in blue and red). The circuits shown in
red represent stress-responsive pathways that may be altered during an osmotic
challenge such as hypernatremia. This figure is not exhaustive and does not represent
circuits that are necessarily monosynaptic.
46
CHAPTER 2
MATERIALS AND METHODS
Generation of CRH Reporter Mice
Adult male CRH reporter mice were generated as previously described (Taniguchi et al.,
2011). Briefly, induction of tdTomato red fluorescent protein to indicate CRH transcription in
neurons was accomplished by the generation of B6(Cg)-Crhtm1(cre)Zjh/J knockin mice (Jackson
Laboratory Stock # 012704) expressing a Cre recombinase coding region immediately after the
STOP codon terminating CRH transcription. These mice were then crossed with
Gt(ROSA)26Sortm14(CAG-tdTomato)Hze congenic mice (Jackson Laboratory Stock # 007914)
expressing a loxP-flanked STOP cassette preventing tdTomato transcription. CRH reporter mice
were 8-12 weeks old at the beginning of the experiments and were maintained on a 12:12 h
light/dark cycle in clear plastic ventilated cages with ad libitum access to pelleted chow (Harlan
Teklad) and water (except where otherwise noted). All procedures were approved by the
Institutional Animal Care and Use Committee at the University of Florida.
Validation of CRH Reporter
RNAscope in situ hybridization (ISH) was performed on brain tissue collected from
CRH-reporter mice to determine the extent to which CRH mRNA colocalizes with tdTomato in
the PVN. Mice were overdosed with sodium pentobarbital, transcardially perfused with 0.9%
saline followed by 4% paraformaldehyde (PFA). Subsequently, brains were extracted, coronally
sectioned at 20 µm into 6 series and then immediately rinsed and mounted onto Superfrost Plus
Gold slides. Tissue collection, sectioning and mounting of sections were performed in RNase-
free conditions. Slides were allowed to air dry for 20-30 min and then were stored at -80°C until
processing for in situ hybridization. Three slides containing separate series of sections through
the PVN were allowed to reach room temperature for 30 min prior to performing the
47
manufacturer’s protocol (Advanced Cell Diagnostics; Hayward, CA). RNAscope ISH was
performed using the following probes: (1) Negative Control, DapB, (2) Positive control, Ubc, (3)
CRH. All images were captured at 40x magnification and the exposure time was adjusted for
each image using the best-fit feature in Axiovision. Subsequently, the min-max feature was
utilized to minimize background fluorescence and provide optimal visualization of RNA signal.
All images were processed using the same automated parameters.
Figure 2-1 (A-C) depicts a unilateral PVN fluorescent photomicrograph from a coronal
section taken from the brain of a CRH-reporter mouse and processed for in situ hybridization.
Highly specific single-strand fluorescent labeling of CRH mRNA revealed consistent
colocalization of CRH probe and tdTomato fluorescence (Fig. 2-1C). Figure 2-1D shows a
qualitative image of the ventral portion of a coronal brain section taken from the hypothalamus
of a CRH-reporter mouse and processed for immunofluorescent labeling of oxytocin. Consistent
with hypothalamic-pituitary axonal projections, green oxytocin fibers and red tdTomato fibers
are seen in the internal and external zones of the median eminence, respectively.
Antibody Characterization
The details of the antibodies used in this study are given in Table 2-1. Both the PS-38
mouse anti oxytocin-neurophysin (oxytocin-NP) and PS-41 mouse anti vasopressin-neurophysin
(vasopressin-NP) antibodies were previously screened for specificity and lack of cross-reactivity
by Ben-Barak and colleagues (Ben-Barak et al., 1985). Following initial harvesting, solid phase
ELISA was used to isolate posterior pituitary-positive hybridoma supernatant from anterior
pituitary- or cerebellar-positive supernatant. An immunohistochemistry assay was then
performed to determine which antibodies selectively stained hypothalamic and pituitary cells,
and those with the predicted pattern were cloned. Liquid phase RIA results using iodinated rat
neurophysin from both normal and Brattleboro rats showed that PS-38 reacted potently to normal
48
and Brattleboro tissue, and PS-41 failed to produce any signal in Brattleboro tissue.
Furthermore, iodinated vasopressin-NP was highly reactive with vasopressin-NP antibody, but
did not react with PS-38 and iodinated oxytocin-NP performed similarly (Ben-Barak et al.,
1985). In our experiments, labeling with PS-38 and PS-41 resulted in clear neuronal profiles
with distinctive patterns in the PVN and SON which were consistent with previously published
studies (Castel and Morris, 1988). Omission of the primary antibody during the
immunofluorescent labeling protocol outlined below resulted in a complete loss of detectable
fluorescence.
Fluorogold could readily be identified at the site of injection. Labeling was also apparent
unilaterally in PVN cells without the use of an antibody by imaging with a DAPI filter set.
However, labeling intensity was improved with primary incubation in AB153, which was
previously validated in mouse brain tissue (Dimitrov et al., 2013).
Two anti-c-Fos antibodies were used to identify activated neurons. Both antibodies
produced a nuclear staining pattern indicative of activated neurons in the PVN following
restraint, but produced little to no reactivity in the PVN of unrestrained mice. Furthermore, both
antibodies revealed bands in western blots in the range of 50-65 kDa.
Immunofluorescence
Immunohistochemical identification of oxytocin/vasopressin neurophysin, Fos and FG in
the PVN was performed using indirect fluorescence. Rinses and solutions were made using 50
mM potassium phosphate buffered saline and took place at room temperature on an orbital
shaker unless otherwise noted. Single labeling began by rinsing free-floating sections 5 X 5 min
to remove cryoprotectant. Blocking consisted of 2% normal donkey serum (Jackson
ImmunoResearch) with 0.2% Triton-X (Sigma) for 1 h followed by incubation with primary
antibody in blocking solution overnight at 4° C. The next day, sections were rinsed 5 X 5 min
49
and incubated in blocking solution with either one or two secondary antibodies. After a final
series of rinses, sections were mounted on Superfrost Plus slides (Fisher) in KPB, allowed to air
dry, and then coverslipped using polyvinyl alcohol mounting medium with DABCO (Sigma).
Image Capture and Analysis
Images of the PVN were captured using an AxioImager M.2 fluorescent microscope
(Carl Zeiss, Thornwood, New York) equipped with an Apotome.2 and connected to a PC
running Axiovision 4.8. Using a Plan-Apochromat 20x/0.8 M27 objective, z-stacks were
captured from sections corresponding to various rostral-caudal levels through the PVN or SON.
Each image was assigned to a specific atlas figure by an individual experimenter that analyzed
the shape of the PVN proper and matched it with the shapes of adjacent anatomical landmarks
found in The Mouse Brain in Stereotaxic Coordinates 3rd Ed (Franklin and Paxinos, 2008).
Image size was approximately 1388 X 1040 pixels and each z-step was 1 µm with an average of
20 optical sections per PVN image. When capturing images from sections processed for
immunohistochemistry, exposure time was determined based on an overexposure reporting
function automatically set by the software. Exposure time during the imaging process varied and
are reported in each chapter. Images were adjusted using the brightness and contrast settings
available in Axiovision to increase clarity.
Quantification of colocalizations took place using a method similar to the “direct three-
dimensional counting” process (Williams and Rakic, 1988). Each z-stack was opened in
Axiovision 4.8 or Zen lite 2012 (Carl Zeiss) and then each image was inspected while alternately
turning the respective channels on and off to identify potential colocalizations. Candidate cells
were then further analyzed as necessary by removing the area of the image containing the cell
into a separate region of interest (ROI) image to generate a 3D reconstruction. If, as a 3D
reconstruction, the cell contained fluorescent labeling on each individual channel throughout the
50
z-plane of the image, then the cell was counted. The number of bi-lateral colocalizations were
determined for each atlas-matched section containing the PVN. An identical procedure was
repeated for image capture and analysis of the SON. Statistical analyses were performed by
grouping according to atlas region and in a separate analysis by grouping all images obtained
from each subject.
51
Table 2-1. Antibody information.
Antibody Antigen Species Dilution Manufacturer
Sc-52 c-Fos Rabbit Polyclonal 1:1000 Santa Cruz
MCA-2H2 c-Fos Mouse Monoclonal 1:2000 Encor
Biotechnology
PS-38 Oxytocin
Neurophysin
Mouse Monoclonal 1:400 Dr. Gainer,
NIH
PS-41 Arginine-
Vasopressin
Neurophysin
Mouse Monoclonal 1:400 Dr. Gainer,
NIH
AB153 Fluorogold Rabbit Polyclonal 1:1000 Millipore
Alex Fluor 488 Mouse IgG (H+L) Donkey 1:500 Jackson
Alexa Fluor 647 Rabbit IgG (H+L) Donkey 1:500 Jackson
Alkaline
Phosphatase, Fab
Fragments
Digoxigenin Sheep Polyclonal 1:1500 Roche
52
Figure 2-1. Representative images validating CRH-Cre induced tdTomato expression. A-C: A
unilateral coronal section through the PVN depicting two images of the same set of
neurons labeled for (A) CRH mRNA probe amplification (green) and (B) tdTomato
(red soma). A merged image (C) illustrates a high degree of CRH mRNA and
tdTomato colocalization. Scale bars = 20 m. D: tdTomato expression adjacent to
oxytocin fibers in the median eminence. Scale bar = 100 m.
53
CHAPTER 3
CENTRAL AND BEHAVIORAL EFFECTS OF ACUTE SALT-LOADING
Background
In mammals the pNa+ is regulated by neural, humoral and behavioral mechanisms that
maintain blood tonicity at levels that allow normal physiological function. Sodium deficiency
causes hyponatremia and increases circulating levels of angiotensin-II and aldosterone which
activate receptors in the kidney and brain to restore the pNa+ to homeostatic levels by promoting
the retention and consumption of sodium. Conversely, excess sodium causes hypernatremia and
suppresses angiotensin-II and aldosterone but causes the secretion of vasopressin and oxytocin
into the systemic circulation which alleviates the elevated pNa+ by promoting renal water
retention and sodium excretion. Thus, the pNa+ is tightly regulated by neurohumoral
compensatory responses that act in the brain and periphery to maintain blood tonicity at
homeostatic levels when challenged with sodium deficiency or excess.
The neuropeptides and hormones that maintain the pNa+ are also known to influence
mood, affect, and stress-responsiveness. For example, studies conducted in humans and animals
have found that elevated circulating levels of angiotensin-II and aldosterone are predictive of
affective disorder (Murck et al., 2003b; Grippo et al., 2005; Niebylski et al., 2012; Häfner et al.,
2013), and that oxytocin and vasopressin are mediators of stress-responsiveness and anxiety
(Neumann and Landgraf, 2012a; Benarroch, 2013a). As mentioned, the secretion of angiotensin-
II, aldosterone, vasopressin, and oxytocin are heavily influenced by the pNa+, and therefore, it is
possible that alterations in body sodium levels affect stress responding and anxiety-like behavior
through manipulation of these neuroendocrine signals. In this regard, previous results indicate
that sodium depletion is anxiogenic (Leshem, 2011), but acute salt-loading is anxiolytic and
dampens stress-responsiveness in laboratory rats (Krause et al., 2011a; Frazier et al., 2013).
54
While the changes in the pNa+ are found to influence mood and stress responding in rats,
whether these effects generalize to mice, and therefore, allow the use of the genetic
manipulations that mouse models afford to investigate central mechanism(s) underlying the
stress limiting effects of acute hypernatremia has not been evaluated.
The goal of the present study was to determine whether acute modest increases in the
pNa+ affect anxiety-like behavior and HPA axis activation in laboratory mice. Mice were
rendered mildly hypernatremic via systemic administration of 2.0 M NaCl, and subsequently,
were subjected to psychogenic stress or tests of anxiety-like behavior. Administration of 2.0 M
NaCl produced a modest but significant increase in the pNa+ relative to control injection of 0.15
M NaCl. This modest rise in the pNa+ was associated with attenuated anxiety-like behavior and
decreased stress-induced HPA activation. Both quantitative and qualitative neuroanatomical
studies were conducted to provide insight towards neural mechanisms contributing to the
anxiolytic effects of acute mild hypernatremia. Collectively, the results demonstrate that the
stress limiting and anxiolytic effects of slight elevations in the pNa+ also occur in mice. The
implication is that acutely increasing the pNa+ may trigger interactions between neurons
expressing oxytocin and CRH to limit responding to psychological stress.
Materials and Methods
Animals
Studies examining the effects of acute hypernatremia on anxiety-like behavior and HPA
activation used adult male C57BL/6 mice obtained from Harlan. Neuroanatomical studies
utilized CRH reporter mice generated and housed as described in Chapter 2. Standard mouse
chow (Harlan) was suspended in a wire rack that also supported an accessory water bottle
allowing ad libitum access to both food and water except where otherwise noted. All procedures
were approved by the Institutional Animal Care and Use Committee of the University of Florida.
55
Restraint Stress and Blood Sampling
Mice were injected subcutaneously with 0.1 mL of either 2.0 M (n=10) or 0.15 M NaCl
(n=10) and returned to their home cages where water was made unavailable. Saline injections
were preceded by 2% lidocaine (~0.01 mL) to minimize discomfort. Sixty-minutes after saline
injections, mice were placed in clear plastic ventilated tubes to initiate a stress response in the
context of normal or elevated pNa+. Tail blood samples (~20 µL) were collected in chilled
EDTA-coated plastic collection tubes immediately at the onset of restraint and again after 30 min
of immobilization in plastic restrainers. Mice were then released and allowed to recover in their
home cages where two more blood samples were taken at 60 min and 120 min relative to the
initiation of restraint. Blood samples were kept on ice until centrifuging at 4° C at 6500 rpm for
15 min. Microcapillary samples were measured for hematocrit, and plasma was extracted and
stored at -80° C until pNa+, plasma proteins, and CORT analyses took place. Plasma sodium
levels were determined for the blood sample taken at the onset of restraint using an auto flame
photometer as previously described (Frazier et al., 2013) (Instrumentation Laboratory,
Lexington, Massachusetts). Plasma CORT was determined for each time point a blood sample
was taken using an 125I RIA kit (MP Biomedicals, Santa Ana, California) as previously described
(Frazier et al., 2013). Plasma proteins and hematocrit were determined for the blood sample
taken at the onset of restraint using a handheld refractometer (VET 360, Reichert) and
microcapillary reader, respectively.
Immunohistochemistry
Two cohorts of CRH-reporter mice (n = 6 and 8) were each further divided into groups
given either 2.0 M NaCl or 0.15 M NaCl and then restrained 60 min later as described above.
Mice were sacrificed 120 min after the onset of restraint (180 min after injections) and
stimulated Fos induction, a marker of neuronal activity, is known to peak during this time
56
(Hoffman et al., 1993). Mice were overdosed with sodium pentobarbital inducing a level of
anesthesia that rendered them unresponsive to toe-pinch before transcardial perfusion with 0.9%
saline. Following the clearing of blood, mice were perfused with 4% PFA and brains were
carefully extracted then postfixed for 2 hours in 4% PFA before cryoprotection in 30% sucrose.
Four series of coronal 30 µm brain sections were cut on a Leica CM3050 S cryostat (Leica,
Buffalo Grove, Illinois) and then stored in cryoprotective solution at -20° C.
Rinses and solutions were made using 50 mM potassium phosphate buffered saline and
took place at room temperature on an orbital shaker unless otherwise noted. Immunofluorescent
labeling of Fos in brain sections from CRH-reporter mice began by rinsing free-floating sections
5 X 5 min to remove cryoprotectant. Blocking consisted of 2% normal donkey serum (Jackson
ImmunoResearch, West Grove, Pennsylvania) with 0.2% Triton-X (Sigma) for 1 h followed by
primary antibody incubation with rabbit anti-Fos (sc-52 1:1000; Santa Cruz) in blocking solution
overnight at 4° C. The second day consisted of rinses 5 X 5 min and incubation for 2 h in
blocking solution with donkey anti-rabbit Alexa-Fluor 647 (1:500; Jackson ImmunoResearch,
West Grove, Pennsylvania). After a final series of rinses, sections were mounted on Superfrost
Plus slides (Fisher) in KPB, allowed to air dry, and then coverslipped using polyvinyl alcohol
with DABCO (Sigma).
CRH-reporter mice were used for double-immunofluorescent labeling of oxytocin and
Fos. The protocol used was identical to the one used above to label Fos except for the addition
of primary antibody (mouse anti-oxytocin neurophysin, PS-38 1:400; generously provided by Dr.
Gainer, NIH) (Ben-Barak et al., 1985) to the blocking solution containing Fos antibody on day
one. Additionally, the secondary antibody, donkey anti-mouse Alexa-Fluor 488 (1:500; Jackson
57
ImmunoResearch, West Grove, Pennsylvania), was mixed with donkey anti-rabbit Alexa-Fluor
647 in blocking solution on day two.
Image Capture and Analysis
All images were captured using an AxioImager M.2 fluorescent microscope (Carl Zeiss,
Thornwood, New York) connected to a PC running Axiovision 4.8. Using a Plan-Apochromat
10x/0.45 M27 objective, z-stacks of tdTomato and Fos expression were captured through the
PVN (from bregma -0.46mm to -1.22mm) using anatomical landmarks found in The Mouse
Brain in Stereotaxic Coordinates 3rd Ed.(Franklin and Paxinos, 2008). Image size was 1388 X
1040 pixels and each z-step was 1 µm with an average of 20 optical sections per PVN image.
Excitation/emission spectra used to image tdTomato and Fos were 540/580 nm and 649/670 nm,
respectively. Exposure time was automatically set by the software and varied from 400-600 ms
for the channel capturing tdTomato images and 1-3 s for the channel capturing Fos images.
Quantification of Fos positive tdTomato neurons took place by first opening each z-stack in
Axiovision 4.8 and then marking colabeled neurons using an event marker function. To verify
that Fos-positive nuclei were within tdTomato neurons, z-stacks were scrolled through and
channels were turned on and off as needed. Fos counts were performed by personnel blind to
treatment conditions on matched sections from both sides of the PVN and then averaged for
animals injected with 0.15 M NaCl and 2.0 M NaCl. Image capture and analysis of Fos
positive oxytocin neurons was performed using the same methods. Visualization of oxytocin
used an excitation/emission of 470/509 and exposure of ~700 ms while parameters for Fos were
similar to those described above.
Behavioral Testing
Naïve C57BL/6 mice were injected with 2.0 M NaCl (n=13) or 0.15 M NaCl (n=12) and
water deprived for 60 min before assessment of anxiety-like behavior on an EPM. During the
58
light phase, mice were brought one at a time into a procedure room with a black curtain which
separated the EPM from the experimenters. Testing began by placing a mouse in the center of an
orthogonally-oriented two plank maze (62 cm X 62 cm) facing an open arm. A 5 min period of
exploration was recorded by a ceiling-mounted video camera connected to a PC running
TopScan software (TopScan; CleverSys, Reston, Virginia). Simultaneous video tracking of the
mouse’s position allowed for the automated scoring of time spent in the open arms and total
distance traveled. The testing apparatus was cleaned with 30% ethanol between subjects.
Statistics
All data presented as mean +/- SEM. Plasma sodium, hematocrit, plasma proteins, EPM
data, Fos positive oxytocin cells and Fos positive CRH cells were assessed with a 2-tailed t test.
Plasma CORT was analyzed using a 2-factor ANOVA using GraphPad Prism version 5.04 for
Windows (GraphPad Software; San Diego, California).
Results
Subcutaneous Delivery of 2.0 M NaCl Modestly Increases pNa+ but Blunts Restraint-
Induced Elevations in Plasma CORT
Analysis of blood samples taken from mice given injections of 2.0 M NaCl found a slight
but significant increase in pNa+ relative to controls injected with 0.15 M NaCl (Fig. 3-1A).
Hematocrit and plasma protein measurements were similar between hypernatremic and control
mice.
Plasma CORT analysis revealed a significant time X condition interaction [Fig. 3-2,
F(3,39) = 3.48, (P<0.05)]. Restraint increased CORT at 30 min compared to baseline
concentrations in both groups; however, mice injected with 2.0 M NaCl had an attenuated rise at
30 and 60 min compared with mice injected with 0.15 M NaCl. This attenuation was specific to
these time points as both baseline and 120 min CORT concentrations were not significantly
59
different between groups. With respect to interpolated time points, the area under the curve was
also decreased relative to controls [Fig. 3-2 inset, (P<0.05)].
Acute Hypernatremia Inversely Affects Restraint-Induced Activation of CRH and
Oxytocin Neurons in the PVN
Figure 3-3 shows photomicrographs of atlas matched sections through the PVN
containing tdTomato reporting of CRH positive neurons and immunofluorescent labeling of Fos.
Whereas control mice (Fig. 3-3A) expressed robust restraint-induced Fos expression in CRH
neurons, this same region displayed fewer activated CRH neurons following 2.0 M NaCl
injection (Fig. 3-3B). Compared with mice injected with 0.15 M NaCl, mice injected with 2.0 M
NaCl had significantly less (P<0.05) restraint-induced Fos positive CRH neurons (Fig. 3-3C).
Conversely, mice injected with 2.0 M NaCl and restrained (Fig. 3-4B) exhibited a significant
[Fig. 3-4C, (P<0.05)] increase in Fos induction in oxytocin neurons relative to controls (Fig. 3-
4A).
Slight Elevations in the pNa+ are Associated with Decreased Anxiety-Like Behavior in the
EPM
Mice injected with 2.0 M NaCl spent more time exploring the open arms of an EPM than
mice injected with 0.15 M NaCl [Fig. 3-5A, (P<0.05)]. This increased exploration was not due
to an overall increase in locomotion as the total distances traveled were similar between groups
(Fig. 3-5B).
Discussion
The goal of the current study was to determine whether the anxiolytic and stress
dampening effects of acute mild hypernatremia occur in laboratory mice. To this end, mice
systemically delivered 2.0 M NaCl had a modest but significant increase in the pNa+ relative to
control mice treated with 0.15 M NaCl. The slight rise in the pNa+ that was observed in mice
administered 2.0 M NaCl was associated with an attenuated restraint-induced activation of the
60
HPA axis and increased time spent in the open arms of an EPM. We conducted neuroanatomical
studies to evaluate how acute hypernatremia may interact with restraint-stress to affect Fos
induction, a marker of neuronal activation, in PVN neurons expressing oxytocin or CRH. Acute
hypernatremia elicited robust Fos induction within oxytocin neurons but significantly decreased
Fos within CRH neurons. Our results, in conjunction with previous studies, suggest that acute
hypernatremia dampens stress-responsiveness, in part, by promoting oxytocin-mediated
inhibition of CRH neurons. The implication is that increased salt intake may be used as a coping
strategy to alleviate the impact of psychological stressors.
Mice rendered hypernatremic via systemic administration of 2.0 M NaCl had a slight
(≈2%) but significant increase in the pNa+ relative to control mice delivered 0.15 M NaCl.
While this increase stimulates oxytocin and vasopressin release as well as osmotically driven
water intake (Stricker and Verbalis, 1986; Krause et al., 2008), the elevation in the pNa+
observed in our study is modest relative to the order of magnitude (10% increase) that is required
to increase brain osmolytes to defend the CNS against the damaging effects of severe
hypernatremia (Heilig et al., 1989). Our experimental paradigm, does however, produce an
increase in the pNa+ that is strikingly similar to that which occurs in humans and animals given
excess dietary sodium to cause increases in blood pressure or the development of salt-sensitive
hypertension, respectively (He et al., 2005) (de Wardener et al., 2004) (Fang et al., 2000;
O'Donaughy and Brooks, 2006; O'Donaughy et al., 2006). Thus, our experimental model of
hypernatremia produces a modest increase in the pNa+ that likely affects cardiovascular function
as well as endocrine and behavioral responses to psychogenic stress.
Activation of the HPA axis is an endocrine measure of stress-responsiveness that is
initiated by excitation of CRH neurons in the PVN which stimulate ACTH secretion from the
61
anterior pituitary, which in turn, promotes CORT release from the adrenal cortex. Relative to
isonatremic controls, mice rendered mildly hypernatremic had decreased plasma CORT
subsequent to the onset of restraint. These results are in agreement with our previous studies
demonstrating that rats subjected to a similar degree of hypernatremia have decreased plasma
CORT subsequent to a restraint challenge (Krause et al., 2011a; Frazier et al., 2013). Our
previous study also determined that acute hypernatremia decreases the secretion of ACTH into
the systemic circulation (Krause et al., 2011a). Because the release of ACTH is controlled by
excitation of CRH neurons in the PVN, we hypothesized that acute hypernatremia may attenuate
Fos induction in PVN neurons producing tdTomato, a marker for CRH expression. Importantly,
mRNA for CRH was present in ≈ 95% of all tdTomato producing neurons in the PVN (Fig. 2-1),
thereby demonstrating the validity of CRH-reporter mice. Consistent with our hypothesis,
restraint-evoked Fos induction of tdTomato producing neurons was significantly decreased in
mice subjected to acute hypernatremia relative to controls. Taken together, these results suggest
that acute hypernatremia dampens stress-induced activation of the HPA axis by inhibiting CRH
neurons in the PVN.
Hypernatremic mice had strong Fos induction in oxytocin neurons in the PVN relative to
isonatremic controls and these results are consistent with those from our previous research using
rats to demonstrate that acute hypernatremia combined with restraint more than tripled the
number of Fos positive oxytocin neurons in the PVN compared to restraint alone (Krause et al.,
2011a). Work conducted by Ludwig and colleagues established that systemic hypernatremia
triggers the dendritic release of oxytocin, which produces robust and sustained elevations in
central levels of this peptide (Ludwig and Leng, 2006). Given that exogenous administration of
oxytocin decreases stress-induced activation of the HPA axis (Windle et al., 1997; Windle et al.,
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2004), it is possible that acute hypernatremia triggers endogenous release of oxytocin within the
CNS, which inhibits activation of CRH neurons, and consequently, blunts HPA activity. In
support of this, electrophysiological studies conducted by Frazier and colleagues (Frazier et al.,
2013) revealed that acute hypernatremia created an inhibitory tone on putative CRH neurons that
was dependent on activation of oxytocin receptors. Therefore, elevating the pNa+ may cause
activation of osmosensitive oxytocin neurons in the hypothalamus, which influence excitation of
neurons responsive to psychogenic stress by releasing oxytocin into the CNS.
Hypernatremia and restraint likely utilize different neural circuits to activate the PVN and
both are known to elicit robust Fos induction in this nucleus (Sharp et al., 1991; Herman and
Cullinan, 1997). Our previous study found that hypernatremia followed by restraint significantly
increased Fos in oxytocin and vasopressin containing neurons; however, the total number of cells
expressing Fos within the PVN was similar to that of isonatremic animals subjected to restraint
(Krause et al., 2011a). These results suggested that the effects of hypernatremia and restraint
were not additive. Given that hypernatremia blunted restraint-induced activation of the HPA
axis (Krause et al., 2011a), we hypothesized that hypernatremia activated oxytocin and
vasopressin containing neurons, but inhibited CRH containing neurons in the PVN. Consistent
with this hypothesis, the present study determined that hypernatremia followed by restraint
significantly decreased Fos induction in tdTomato expressing neurons in the PVN. Taken
together, the result suggest that hypernatremia selectively activates oxytocin and vasopressin
containing neurons in the PVN but inhibits those that express CRH, which accounts for the
similarities in total Fos induction observed between hypernatremic and isonatremic animals
subjected to restraint.
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In addition to attenuating stress-induced activation of the HPA axis, the present study
found that acute hypernatremia decreased anxiety-like behavior in the EPM relative to that of
isonatremic controls. Once again, these results are consistent with those from our previous
studies using rats that found that slight elevations in the pNa+ are anxiolytic, especially in social
situations (Krause et al., 2011a; Frazier et al., 2013). Central administration of oxytocin
decreases anxiety-like behavior in the EPM (Mak et al., 2012) and oxytocin efferents are found
in limbic brain regions (Knobloch et al., 2012) like the amygdala, that are heavily implicated in
the expression of fear and anxiety-like behavior (Davis et al., 1994). Of relevance, some of the
oxytocin efferents in the amygdala arise from magnocellular oxytocinergic neurons in the PVN
and supraoptic nucleus (Knobloch et al., 2012). Magnocellular oxytocinergic neurons are
osmosensitive and become excited by elevations in the pNa+ (Hattori et al., 1990). Therefore, it
is possible that the anxiolytic effects of acute hypernatremia result from excitation of
osmosensitive oxytocin neurons with axonal projections to limbic brain nuclei controlling the
expression of fear and anxiety-like behavior.
Collectively our results demonstrate that the anxiolytic and stress dampening effects of
acute hypernatremia extend to mice. Consequently, these results allow the use of mice and the
genetic manipulations that this animal model affords to further investigate the neural and
humoral mechanism(s) underlying the stress limiting effects of acute hypernatremia. From a
broader perspective, our results may provide insight as to why some patients with salt-sensitive
hypertension and high levels of life-stress (Calhoun, 1992; Calhoun and Oparil, 1995; Barnes et
al., 1997) also have difficulties complying with restricted dietary sodium intake (Epstein et al.,
2012). These more far-reaching conclusions are discussed further in Chapter 6.
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Figure 3-1. Plasma measurements from mice 60 min after 2.0 M or 0.15 M NaCl injections. (A)
Injections of 2.0 M NaCl significantly increased pNa+ relative to control injections of
0.15 M NaCl, but had no effect on (B) hematocrit or (C) plasma proteins. *p<0.05
Error bars indicate SEM.
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Figure 3-2. 2.0 M NaCl administration reduces the CORT response to restraint stress relative to
0.15 M NaCl. The integrated CORT response was also significantly reduced.
*p<0.05 0.15 M NaCl vs 2.0 M NaCl. AUC=Area Under the Curve. Error bars
indicate SEM.
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Figure 3-3. 2.0 M NaCl injection attenuates restraint-induced activation of CRH neurons. (A)
Representative photomicrograph of a unilateral coronal section through the PVN
depicting Fos induction (cyan nuclei) in tdTomato (red soma) containing neurons
following 0.15 M NaCl and restraint. (B) Representative photomicrograph of a
unilateral coronal section through the PVN depicting Fos induction in tdTomato
containing neurons following 2.0M NaCl and restraint. (C) The group mean for Fos
induction in tdTomato containing neurons was significantly less for mice subjected to
2.0 M NaCl injection and restraint relative to control. *p<0.05 Scale bars = 50 m.
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Figure 3-4. 2.0 M NaCl administration and restraint increases activation of oxytocin-producing
cells in the PVN. (A) Representative photomicrograph of a unilateral coronal section
depicting Fos induction (red nuclei) in oxytocin (green cell bodies) containing
neurons following 0.15 M NaCl and restraint. (B) Representative photomicrograph
of a unilateral coronal section depicting Fos induction in oxytocin neurons following
2.0 M NaCl and restraint. (C) The group mean for Fos induction in oxytocin-
producing cells was significantly more for mice subjected to 2.0 M NaCl injection
and restraint relative to control. *p<0.05 Scale bars = 50 m.
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Figure 3-5. Acute hypernatremia attenuates anxiety-like behavior. (A) Mice treated with 2.0 M
NaCl spent a greater proportion of time exploring the open arms of an EPM than mice
treated with 0.15 M NaCl. (B) Overall locomotion was unaffected by 2.0 M NaCl as
total distance traveled was similar for each group. * p < 0.05. Error bars indicate
SEM.
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CHAPTER 4
CENTRAL AND BEHAVIORAL EFFECTS OF CHRONIC SALT-LOADING
Background
Chronic salt-loading through the replacement of drinking water with concentrated sodium
chloride solution has been used experimentally in rats and mice to investigate factors that
accompany persistently hyperactive osmoregulatory mechanisms. Specifically, an initial period
of intracellular dehydration during the first 24 hrs stimulates a variety of physiological
alterations to maintain normal fluid intake and plasma volume with increased urinary output
(Watts et al., 1995; Polito et al., 2006). These alterations along with increased central and
systemic vasopressin (Amaya et al., 1999) secretion promote water retention and the excretion of
excessive amounts of sodium. However, following three days of drinking 2% NaCl, rats fail to
maintain adequate hydration of the extracellular fluid compartment which is apparent by an
increasing hematocrit (Watts et al., 1995). Without sufficient hydration rats become increasingly
aphagic and lose weight although fluid intakes range from hyperdipsic to adipsic as homeostasis
continues to be unattainable (Watts et al., 1995). Furthermore, after an initial period of
heightened CORT (Watts, 1992a), ACTH and CORT are suppressed to lower than control levels
and restraint-induced CORT is suppressed as well (Amaya et al., 2001). Neuronal activation
increases in diverse brain areas and neuronal phenotypes as a result of chronic salt-loading
(Watts, 1992a; b; Watts et al., 1995; Watts, 1996). Overall Fos activation increases in the
OVLT, median preoptic nucleus, SFO, SON, and PVN as these regions sense and relay
hyperosmotic plasma conditions to brain areas responsible for hydromineral homeostasis
(Kovacs and Sawchenko, 1993). However, there have been inconsistencies reported across
species as Fos expression in the SON of the rat (Kovacs and Sawchenko, 1993) was not found to
be expressed in similarly salt-loaded mice (St-Louis et al., 2012). Additionally, accounting for
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Fos expression throughout the entire rostral-caudal extent of the PVN has not been done, nor has
Fos expression in the PVN been assessed with the combined stressors of chronic salt-loading and
restraint. Finally, in contrast to several studies in rats, restraint-induced CORT has yet to be
determined in chronically salt-loaded mice. Thus, during the final period of chronic salt-loading
a variety of neural, humoral, and autonomic processes caused by prolonged hyperosmotic
conditions and which are putatively involved in the pathogenesis of hypertension and
cardiovascular disease can be characterized.
Methods
Animals
Studies examining the effects of chronic hypernatremia on HPA activation used adult
male C57BL/6 mice obtained from Harlan. Neuroanatomical studies utilized adult male CRH
reporter mice which were generated as detailed in Chapter 2. All mice either continued ad
libitum access to water (control group; Water) or had water replaced with a 2% NaCl solution for
five days (experimental group; 2% NaCl). All procedures were approved by the Institutional
Animal Care and Use Committee at the University of Florida.
Procedure
Assessment of food and fluid intake was performed at the same time each day for the
duration of the 5 d study. Weights of both chow and water- or saline-filled bottles were recorded
by an individual experimenter and are presented as mean daily intakes corrected for body weight.
Body weights were assessed during the same monitoring session and are presented as daily
averages. Tail blood samples (~20 µL) were collected from mice in both groups using chilled
EDTA-coated plastic collection tubes immediately before water was replaced with saline.
Furthermore, microcapillary tubes were used to sample blood which was immediately
centrifuged to measure hematocrit. After the fifth consecutive day of salt-loading, a final tail
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blood sample was taken from mice in both groups upon initiation of restraint and hematocrit was
again assessed. Restraint was accomplished by placing mice in clear plastic ventilated restraint
devices constructed of 50 mL conical tubes for 30 min and allowed a 60 min recovery period.
Mice were then overdosed with sodium pentobarbital and transcardially perfused with 0.9%
saline (30-50 mL) followed by ice-cold 4% PFA (30-50 mL). Brains were carefully extracted
and postfixed for 4 h in 4% PFA before cryoprotection in 30% sucrose. Four series of coronal
30 µm sections were cut on a Leica CM3050 S cryostat (Leica, Buffalo Grove, Illinois) and then
stored in cryoprotective solution at -20° C. Blood samples were kept on ice until centrifuging at
4° C at 6500 rpm for 15 min. Plasma was extracted and stored at -80° C until pNa+ levels were
determined using an auto flame photometer (Instrumentation Laboratory, Lexington,
Massachusetts) and plasma protein could be determined using a hand-held refractometer
(Reichert, Depew, New York).
Restraint Stress and CORT
Mice were placed in clear plastic ventilated tubes to initiate a stress response in the
context of normal or elevated pNa+. Tail blood samples (~20 µL) were collected in chilled
EDTA-coated plastic collection tubes immediately at the onset of restraint and again after 30 min
of immobilization in plastic restrainers. Mice were then released and allowed to recover in their
home cages where two more blood samples were taken at 60 min and 120 min relative to the
initiation of restraint. Blood samples were kept on ice until centrifuging at 4° C at 6500 rpm for
15 min and stored at -80° C CORT analyses took place. Plasma CORT was determined for each
time point a blood sample was taken using an 125I RIA kit (MP Biomedicals, Santa Ana,
California) as previously described in Chapter 3.
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Image Capture and Analysis
Images of the PVN were captured using an AxioImager M.2 fluorescent microscope
(Carl Zeiss, Thornwood, New York) equipped with an Apotome.2 and connected to a PC
running Axiovision 4.8. Using a Plan-Apochromat 20x/0.8 M27 objective, z-stacks of tdTomato
and fluorescently-labeled vasopressin-NP or Fos expression were captured from sections
corresponding to various rostral-caudal levels through the PVN (from Bregma -0.46 mm to -1.22
mm). Each image was assigned to a specific atlas figure by an individual experimenter that
analyzed the shape of the PVN proper and matched it with the shapes of adjacent anatomical
landmarks found in The Mouse Brain in Stereotaxic Coordinates 3rd Ed (Franklin and Paxinos,
2008). Image size was approximately 1388 X 1040 pixels and each z-step was 1 µm with an
average of 20 optical sections per PVN image. When capturing images from sections processed
for immunohistochemistry, exposure time was determined based on an overexposure reporting
function automatically set by the software. Exposure time during the imaging process varied
from 30-40 ms for the channel capturing tdTomato images, 125-175 ms for vasopressin-NP
images, and 400-800 ms for Fos images. Images were adjusted using the brightness and contrast
settings available in Axiovision to increase clarity.
Quantification of colocalized tdTomato or vasopressin-NP with Fos positive nuclei took
place using a method similar to the “direct three-dimensional counting” process (Williams and
Rakic, 1988). Each z-stack was opened in Axiovision 4.8 or Zen lite 2012 (Carl Zeiss) and then
each image was inspected while alternately turning the respective channels on and off to identify
potential colocalizations. Candidate cells were then further analyzed as necessary by removing
the area of the image containing the cell into a separate region of interest (ROI) image to
generate a 3D reconstruction. If, as a 3D reconstruction, the cell contained fluorescent labeling
on each individual channel throughout the z-plane of the image, then the cell was counted. The
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number of bi-lateral colocalizations were determined for each atlas-matched section containing
the PVN. Statistical analyses were performed by grouping according to atlas region and in a
separate analysis by grouping all images obtained from each subject.
Statistics
All grouped data presented as mean +/- SEM. Fluid intake, food intake, body weight,
total Fos, percentage of Fos positive tdTomato neurons, and plasma CORT were assessed with a
two-factor analysis of variance. Main effects or interactions (P<0.05) were assessed post hoc
with the Bonferroni method. Within group (Pre vs post salt-loading) and between group pNa+,
hematocrit, and plasma protein percentage were each assessed with a paired and unpaired 1-
tailed t-test, respectively. Significance for all analyses was set at P<0.05. Statistical analyses
were performed and graphs were created using GraphPad Prism 5.
Results
Salt-Loaded Mice Exhibit Increased Fluid Intakes and pNa+ Concentrations, but Maintain
Normal Indices of Blood Volume
Daily monitoring of fluid intake revealed that mice in the control condition drank an
average of 5.27+/-0.10 mL of water each day and intake did not significantly change over the
course of the study. In contrast, mice with 2% NaCl as a sole source of fluid drank significantly
more after 48 h of salt exposure (time x treatment interaction; [F(4, 115) = 3.14, (P<0.05)]) and
intake remained significantly (P<0.0001 on days 2, 3, and 4; P<0.001 on day 5) higher than
controls for each day thereafter (Fig. 4-1, A). Consumption of 2% NaCl for five days
significantly elevated the pNa+ relative to Day 1 and compared with water drinking controls
(P<0.05) (Fig. 4-1, B). Volume of fluid consumed after the first day was similar between
groups, however, mice drinking 2% NaCl exhibited highly variable intakes after the second day
(Fig. 4-2, A). Despite the challenge to hydromineral homeostasis, hematocrit and plasma protein
74
concentrations were similar between groups both at the beginning and the end of the experiment
(Fig. 4-1, C & D).
Salt-Loaded Mice Maintain Food Intake and Body Weight
Analysis of chow consumption revealed a main effect of 2% NaCl [F(1, 115) = 9.53,
(P<0.05)]; however, post hoc analysis did not reveal significant differences during any particular
24 period (Fig. 4-4, A). During the first 24 hs, salt-loaded mice showed no correlation
(R2=0.0065, P>0.05) between food and fluid intake (Fig. 4-3, A). In contrast, there was a
positive correlation (R2=0.5393; P <0.05) observed in water-drinking controls after the first 24
hrs. Following the second 24 hrs of salt-loading this trend changed (Fig. 4-3, B) with a
significant positive correlation emerging in salt-loaded mice (R2=0.3283; P <0.05). Water-
drinking controls failed to show any correlation between food and fluid intake for the remainder
of the experiment. Figure 4-4B shows both groups maintained stable daily average body weights
throughout the five-day period.
Salt-Loading Does Not Increase Neuronal Activation in PVN Vasopressin Neurons or the
SON
Figure 4-5 depicts the Fos immunoreactivity and vasopressin-NP expression in the PVN
of Water (Fig. 4-5, A) and 2% NaCl (Fig. 4-5, B) mice subjected to restraint. Fos expression
was not evident in vasopressin-NP neurons in the PVN of control mice and only 7.35 +/- 4.52%
of vasopressin-NP neurons were Fos positive in the PVN of mice drinking 2% NaCl (P=0.12).
Furthermore, within the SON neither group was found to express Fos (Fig. 4-5; C, D).
Salt-Loading Increases Fos Expression in the Posterior PVN
Total Fos counts for PVN “neurosecretory” regions (Bregma -0.70mm and -0.82mm) and
“preautonomic” regions (Bregma -0.94mm and -1.06mm) were averaged and compared for
groups of mice drinking water, 2% NaCl, water + restraint, and 2% NaCl + restraint (Fig. 4-5,
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E). In the “neurosecretory” regions, restraint increased the number of Fos-positive nuclei
independent of saline treatment [F(1,24) = 58.13, P <0.05; Fig. 4-5E]. In “preautonomic” regions,
there was an interaction between salt-loading and restraint [F(1,24) = 44.09, P <0.05; Fig. 4-5E].
In addition to restraint-induced Fos increases, mice subjected to salt-loading alone exhibited
increased Fos immunoreactivity [Main effect of 2% NaCl: F(1,24) = 4.53, P <0.05; Fig. 4-5E]
compared to water-drinking controls.
Salt-Loading does not Affect Restraint-Induced Fos Induction in tdTomato Neurons
Figure 4-5F shows the average percentage of Fos-positive tdTomato neurons in the PVN.
The average percentage of Fos-positive tdTomato neurons in the PVN was similar in
unrestrained mice drinking water or 2% NaCl. The percentage of Fos-positive tdTomato neurons
in both PVN regions was significantly increased by restraint [F(1,44) = 65.89, P<0.05] in both
euhydrated and salt-loaded mice. There was no main effect of salt-loading or interaction
between salt-loading and restraint.
Salt-Loading Blunts Restraint-Induced Elevations in Plasma CORT During Recovery
Plasma CORT analysis revealed a significant time X treatment interaction [Fig. 4-6,
F(3,54) = 3.32, (P<0.05)]. Restraint increased CORT at 30 min compared to baseline
concentrations in both groups; however, salt-loaded mice recovered faster as CORT at 120 min
was significantly reduced (P<0.05). Attenuation was specific to this time point as both baseline
and 60 min CORT concentrations were not significantly different between groups. With respect
to interpolated time points, the area under the curve was not different relative to controls (Fig. 4-
6 inset).
Discussion
We evaluated how salt-loading caused by consumption of 2% NaCl changes ingestive
behavior, neuronal activation, and restraint-induced CORT. The behavioral adaptations that
76
occurred to counter the osmotic stress included significant increases in daily saline intake that
correlated with the maintenance of chow intake. Relative to water drinking controls, salt-loading
significantly increased the pNa+ but had no effect on hematocrit or plasma protein
concentrations. Fos induction within vasopressin-NP and tdTomato expressing neurons was
similar between salt-loaded and control mice and no Fos induction was evident in the SON;
however, total Fos increased in the posterior preautonomic region of the PVN with salt-loading
alone. These measures of neuronal activation did not predict similar HPA axis functionality as
salt-loaded mice had CORT levels significantly lower at 120 min after the onset of restraint.
In the current study, drinking 2% NaCl instead of water robustly increased fluid intake
but did not substantially affect food consumption. The behavioral adaptations of salt-loaded
mice were accompanied by a significant rise in pNa+, but did not elicit changes in body weight,
hematocrit or plasma proteins. In contrast to our results here using a mouse model, rats drinking
saline instead of water exhibit highly variable fluid intake and decreased food consumption
resulting in significantly reduced blood volume and body weight (Watts, 1992a; Watts et al.,
1995; Yue et al., 2008; Boyle et al., 2012). The renal handling of sodium and water seems to be
more efficient in mice relative to rats (Zhai et al., 2006; Christensen et al., 2014). Whereas rats
respond to forced saline consumption by limiting food intake, mice quickly adapt to maintain
intake levels by drinking and excreting large volumes of fluid. Consistent with this, the higher
ratio of short to long loop nephrons in the mouse kidney (81:18, (Zhai et al., 2006)) compared
with rats (70:30, (Kriz, 1967)) is predictive of the lower rise in plasma osmolality observed in
mice (3.3%, (Polito et al., 2006)) compared with rats (6.5%, (Watts et al., 1995)) following five
days of drinking 2% NaCl. Interestingly, the ratio of short to long loop nephrons in humans
(81:15, (Peter et al., 1927)) is closer to the mouse kidney than that of the rat, suggesting more
77
similarities between the human and murine renal handling of sodium than with the rat. Thus,
relative to rats, it seems that mice are less susceptible to the hypernatremia that occurs with salt-
loading which may explain why dehydration anorexia was limited in the present study.
During the first 24 hrs, mice drinking 2% NaCl instead of water did not have fluid intake
that correlated with food consumption, but subsequently, a positive correlation emerged
suggesting the engagement of compensatory mechanisms that allow normal food intake by
alleviating intracellular dehydration. Our experiments evaluating Fos induction may provide
insight towards such mechanisms. We found that Fos induction within vasopressin neurons was
not affected by five days of salt-loading and these results are consistent with those of (St-Louis et
al., 2012) which determined that magnocellular vasopressin neurons are activated after two days
of salt-loading but this activation subsided six days later. Taken together, these results suggest
that in mice the activity of vasopressin neurons may return to basal levels earlier than previously
thought and activation of neurosecretory vasopressin neurons may be transient while vasopressin
mRNA and hnRNA continue to rise with enduring hypernatremia (Ozaki et al., 2004; St-Louis et
al., 2012).
The differences in Fos expression observed in the PVN of chronic salt-loaded mice may
be indicative of neuroendocrine and autonomic adaptations aimed at coping with chronic osmotic
stress. The results from Chapter 3 indicate that acute hypernatremia blunts HPA axis activation
elicited by psychogenic stress, an effect that was observed here 90 min after chronically salt-
loaded mice were released from restraint tubes. Basal CORT levels were not affected by acute
or chronic salt-loading. Our results in Chapter 3 also indicate that acute salt-loading is
associated with decreased Fos-induction within CRH neurons in the PVN; however, our results
in the chronic salt-loading paradigm indicate that this decrease does not occur in CRH neurons of
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the PVN. Specifically, salt-loaded and control mice exhibited similar restraint-induced Fos
expression within tdTomato neurons in the PVN. Interestingly, salt-loading alone increased total
Fos in the posterior preautonomic region of the PVN; however, this effect also cannot be
attributed to increased activation of tdTomato neurons within the posterior preautonomic region
because colocalization of Fos and tdTomato was similar in salt-loaded and water-drinking mice.
These results suggest that the increased sympathetic drive that occurs with chronic salt-loading
may not derive from an increase in the number of active preautonomic CRH neurons as
detectable by Fos, but rather, may be the result of altered hypothalamic expression of
neuropeptides, like CRH and vasopressin, which allows modulation of neuronal activity in
hindbrain nuclei that control sympathetic outflow.
Collectively these experiments evaluated in a broad way the murine response to salt-
loading. In agreement with previous studies, mice respond to the presentation of only saline as a
source of water by drinking large volumes after an initial period of somewhat normal intakes.
However, in stark contrast to similar studies in rats, mice maintained food intake and body
weights with consumptions of fluid and food that were positively correlated with one another
after the first day. Furthermore, while the acute salt-loading experiment in Chapter 3 indicated
that restraint-induced CORT is suppressed at 30 and 60 min following the onset of restraint, the
chronic salt-loading experiment here indicated no effects at these time points and suppression
with salt-loading at 120 min. Analysis of the neuronal activation in the PVN and SON that may
have contributed to the adaptive ingestive behaviors included lack of evidence for magnocellular
vasopressin activity at the time of tissue collection, but rather an indication that the activity of
preautonomic neurons may be involved in the response to salt-loading. Further investigation into
79
the potential for neuropeptide plasticity and preautonomic involvement are explored in Chapter
5.
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Figure 4-1. Fluid consumption and the effects on indices of hydration. Mice with access to 2%
NaCl (shaded circles) instead of water (open squares) drank significantly more (time
x treatment interaction; [F(4, 115) = 3.14, (P<0.05)]) each day after the first 48 h (A).
Mice maintained on 2% NaCl had higher pNa+ on Day 5 compared with Day 1 (B;
P<0.05). Hematocrit (C) and plasma proteins (D) were similar between groups.
Water n=12, 2% NaCl n=13.
81
Figure 4-2. Individual fluid and food intake values. Circles represent the individual water (blue)
and 2% NaCl (red) daily fluid intakes values normalized to body weight for each 24
hr period (A). Bars represent the daily average of all mice. Individual daily chow
intake values normalized to body weight for mice with access to water (blue circles)
or 2% NaCl (red circles) (B). Bars represent the daily average of all mice.
82
Figure 4-3. Correlation graphs for food and fluid intake. Individual values plotted for daily food
and fluid intake for mice drinking water (black squares) or 2% NaCl (red circles).
Significant correlations were determined for Water Day 1 (p=0.01; R2=0.5393) and
2% NaCl Day 2-5 [Day 2 (p=0.04; R2=0.3283); Day 3 (p=0.04; R2=0.3356); Day 4
(p=0.02; R2=0.4477); Day 5 (p=0.01; R2=0. 4876)].
83
Figure 4-4. Daily food intake and body mass measurements. The consumption of chow was
similar between salt-loaded mice and water-drinking controls as there were no
significant differences in daily intakes (A), however, there was a main effect of 2%
NaCl [F(1, 115) = 9.53, (P<0.05)]. Body weights did not differ significantly (B). Water
n=12, 2% NaCl n=13.
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Figure 4-5. Stress induced neuronal activation. (top left) Coronal sections through the PVN
depicting neurons labeled with vasopressin-NP (green cell bodies) and Fos (red
nuclei). Fos expression was similar for restrained water drinking (A) and saline-
drinking mice (B). (bottom left) Coronal sections through the SON depicting Fos
labeling. Fos expression was similar for restrained water drinking (C) and saline-
drinking mice (D) in the SON. (top right) Overall Fos activation in the PVN was
increased with restraint in both the “neurosecretory” region (Bregma -0.70 to -0.82)
[F(1,24) = 58.13, P <0.05; Fig. 3-5E] and “preautonomic” region (Bregma -0.94 to -
1.06) [F(1,24) = 44.09, P <0.05; Fig. 3-5E]. Unrestrained salt-loaded mice had
increased Fos compared with unrestrained controls in the “preautonomic” region of
the PVN [Main effect of 2% NaCl: F(1,24) = 4.53, P <0.05; Fig. 3-5E]. (bottom right)
Fos activation in tdTomato neurons was increased in the PVN with restraint at both
PVN regions (P<0.05). Salt-loading did not affect Fos expression in tdTomato
neurons (F). Water n=12, 2% NaCl n=12. Error bars = SEM. Scale bars = 100 µm.
3v, 3rd ventricle. ot, optic tract.
85
Figure 4-6. Chronic salt-loading reduces the CORT response to restraint during the recovery
period. The integrated CORT response was not significantly reduced. *p<0.05.
AUC=Area Under the Curve. Error bars indicate SEM.
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CHAPTER 5
CHRONIC SALT-LOADING-ASSOCIATED NEUROPEPTIDE PLASTICITY
Background
The PVN contains peptidergic neurons that respond to dehydration through endocrine
and neural compensatory mechanisms that maintain and restore hydromineral balance.
Specifically, the intracellular dehydration that occurs with acute elevations in the pNa+
influences the activation of PVN neurons to prevent diuresis but promote natriuresis and the
maintenance of blood pressure by controlling the systemic and central release of vasopressin,
oxytocin and CRH. Elevations in the pNa+ or hypernatremia activates magnocellular
vasopressin and oxytocin neurons in the PVN to elicit neurohypophyseal secretion of these
neuropeptides which act on the kidney to promote water retention and the excretion of sodium in
urine (Verbalis et al., 1991; Ludwig et al., 1994; Pirnik et al., 2004). In contrast, acute
hypernatremia inhibits neurosecretory CRH neurons and plasma renin activity resulting in
blunted stress-induced HPA axis activation and lowered levels of corticosterone (Krause et al.,
2011a), thereby attenuating renal sodium retention (Husain et al., 1987; Papanek et al., 1997).
The PVN contains preautonomic neurons that also produce vasopressin, oxytocin and CRH that
project to the hindbrain and spinal cord to control sympathoexcitation and cardiovascular
function (Lee et al., 2013). In contrast to their neurosecretory counter-parts, peptidergic
preautonomic neurons are thought to influence sympathetic nervous system activity and
cardiovascular responses to hydromineral imbalance by affecting neurotransmission through the
release of vasopressin, oxytocin and CRH from axons terminating on postsynaptic neurons.
Thus, acute elevations in body sodium levels trigger coordinated excitation and inhibition of
peptidergic neurons in the PVN to initiate endocrine and neural signals that buffer against further
dehydration while maintaining blood pressure.
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Chronic salt-loading, as a consequence of drinking hypertonic saline instead of water,
also is associated with attenuated HPA axis activity and increased sympathetic outflow and
blood pressure (Sapirstein et al., 1950; Amaya et al., 2001), and these changes occur
concomitantly with altered expression of neuropeptides in the PVN and amygdala (Watts et al.,
1995). For example, studies conducted in rats found that magnocellular neurons of the PVN and
SON adapt to chronic salt-loading by upregulating CRH (Lightman and Young, 1987; Kovacs
and Sawchenko, 1993), which may facilitate the systemic release of oxytocin and natriuresis
(Verbalis et al., 1991); however, parvocellular neurons in the PVN down-regulate CRH
expression (Amaya et al., 2001) while neurons in the amygdala increase CRH (Watts et al.,
1995). As mentioned, the neuropeptide plasticity that follows chronic salt-loading is associated
with increased osmoregulatory capacity, decreased responsiveness of the HPA axis and
sympathetically-mediated hypertension (Lightman and Young, 1987; Watts, 1992a; 1996;
Amaya et al., 2001; Hartner et al., 2003). Thus, chronic salt-loading stimulates neuropeptide
plasticity in the PVN that may serve as a compensatory mechanism that permits the altered
endocrine axis activity and enhanced sympathetic outflow that is required to cope with chronic
osmotic stress. Whether chronic salt-loading elicits neuropeptide plasticity in preautonomic
neurons of the PVN or amygdala has not been investigated.
The primary goal of this study was to characterize the effect of chronic salt-loading on
CRH plasticity within vasopressin, oxytocin, and preautonomic neurons in the PVN. For
comparative purposes, preautonomic neurons in the amygdala were analyzed. Here, we utilized
a CRH-reporter mouse to specifically focus on the neural subtypes that respond to dehydration
by upregulating CRH. Additionally, relatively few chronic salt-loading experiments have been
performed in mice and the organization of neurons in the mouse PVN has only recently been
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thoroughly characterized (Biag et al., 2012). Consequently, we also sought to analyze CRH
expression in the PVN and SON for comparative purposes with similar studies using rats.
Collectively, our results serve to solidify the structure and functional neuroanatomy of the mouse
PVN and provide evidence that a prolonged hyperosmotic stimulus can influence CRH
expression in neuroendocrine and preautonomic neurons.
Methods
Animals
CRH reporter mice were generated as detailed in Chapter 2. A group of mice (n=23)
were used to quantify the effect of chronic salt-loading on CRH colocalization with oxytocin or
vasopressin and another group of CRH reporter mice (n=33) were used to determine the extent to
which these colocalizations occurred in PVN neurons projecting to the hindbrain. Mice either
continued ad libitum access to water (control group; Water) or had water replaced with a 2%
NaCl solution for five days (experimental group; 2% NaCl). All procedures were approved by
the Institutional Animal Care and Use Committee at the University of Florida.
In situ Hybridization
Localization of CRH mRNA through fluorescent detection was performed as described in
Chapter 2. Localization of CRH mRNA without fluorescence was accomplished with an alkaline
phosphatase reaction. Following the overnight hybridization, coverslips were removed and
slides were washed 3 X 5 min in maleic acid buffer containing Tween 20 at room temperature
and 2 X 30 min in 50% formamide, 1x SSC, 0.1% Tween-20. Sections were then blocked for 1
h at room temperature with 1x Blocking Reagent (Roche) in maleic acid buffer containing
Tween 20 with 10% heat-inactivated sheep serum before coverslipping and incubation with
primary antibody (Sheep anti-digoxigen alkaline phosphatase Fab Fragment, 1:1500; Roche)
overnight at 4° C. The following day, coverslips were removed and slides were rinsed in maleic
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acid buffer containing Tween 20 3 X 10 min and predeveloping buffer (100 mM Tris pH 9.8,
100 mM NaCl, 50 mM magnesium chloride) for 2 X 2 min at room temperature. Slides were
then transferred to developing solution (100 mM Tris pH 9.8, 100 mM NaCl, 50 mM magnesium
chloride, 5% polyvinyl alcohol, 0.11 mM nitroblue tetrazolium salt in 70% dimethylformamide,
5-bromo-4-chloro-3-indolyl-phosphate in 100% dimethylformamide) in a foil-wrapped coplin jar
at 37° C for 36 h. Slides were then briefly rinsed in tap water to stop the color reaction. Sections
were then dehydrated in 2 min rinses of increasing concentrations of ethanol, defatted 2 X 2 min
in xylene, and then coverslipped with DPX mounting media (VWR).
Image Capture and Analysis
Brightfield images of CRH digoxigen in situ hybridization labeled sections were taken
with a Plan-Apochromat 10x/0.45 M27 objective using light filters to obtain maximum contrast.
Densitometry of CRH mRNA labeling was performed using Image J (NIH) by first outlining a
region of interest based on boundaries established in The Mouse Brain in Stereotaxic
Coordinates 3rd Ed (Franklin and Paxinos, 2008) for separate rostral-caudal levels of the PVN,
and then using the “measure” function to determine the raw integrated density of the confined
area. Bi-lateral densities for each PVN atlas-matched level were averaged for brain sections
taken from control and salt-loaded mice.
Brightfield images of thionin-stained were taken with a Plan-Apochromat 20x/0.8 M27
objective using light filters to obtain maximum contrast. PVN boundaries were established using
figures in The Mouse Brain in Stereotaxic Coordinates 3rd Ed (Franklin and Paxinos, 2008) for
each rostral-caudal levels of the PVN.
Fluorescent images were captured using an AxioImager M.2 fluorescent microscope
(Carl Zeiss, Thornwood, New York) equipped with an Apotome.2 and connected to a PC
running Axiovision 4.8. Using a Plan-Apochromat 20x/0.8 M27 objective, z-stacks of
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tdTomato, fluorescently-labeled CRH mRNA, and vasopressin/oxytocin-NP were captured from
sections corresponding to various rostral-caudal levels through the PVN (from Bregma -0.46 mm
to -1.22 mm). The same sections that included the three most caudal regions of the PVN were
also used to image the amygdala. Each image was assigned to a specific atlas figure by an
individual experimenter that analyzed the shape of the PVN and amygdala proper and matched it
with the shapes of adjacent anatomical landmarks found in The Mouse Brain in Stereotaxic
Coordinates 3rd Ed (Franklin and Paxinos, 2008). Image size was approximately 1388 X 1040
pixels and each z-step was 1 µm with an average of 20 optical sections per PVN image. When
capturing images from sections processed for immunohistochemistry, exposure time was
determined based on an overexposure reporting function automatically set by the software.
Exposure time during the imaging process varied from 30-40 ms for the channel capturing
tdTomato images and 125-175 ms for vasopressin/oxytocin neurophysin images. When
capturing images from sections processed for in situ hybridization, the longest exposure time that
still resulted in a lack of signal when imaging sections hybridized with the negative control probe
was used. Images were adjusted using the brightness and contrast settings available in
Axiovision to increase clarity.
Quantification of colocalized tdTomato with either vasopressin or oxytocin neurophysin
neurons took place using a method similar to the “direct three-dimensional counting” process
(Williams and Rakic, 1988). Each z-stack was opened in Axiovision 4.8 or Zen lite 2012 (Carl
Zeiss) and then each image was inspected while alternately turning the respective channels on
and off to identify potential colocalizations. Candidate cells were then further analyzed as
necessary by removing the area of the image containing the cell into a separate region of interest
(ROI) image to generate a 3D reconstruction. If, as a 3D reconstruction, the cell contained
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fluorescent labeling on each individual channel throughout the z-plane of the image, then the cell
was counted. The number of bi-lateral colocalizations were determined for each atlas-matched
section containing the PVN. An identical procedure was repeated for image capture and analysis
of the SON. Statistical analyses were performed by grouping according to atlas region and in a
separate analysis by grouping all images obtained from each subject.
Sections from CRH-reporter mice were processed for double immunofluorescent labeling
of vasopressin-NP and FG and imaged for quantification of preautonomic neurons expressing
vasopressin-NP or tdTomato. FG injection site analysis was accomplished by imaging untreated
hindbrain sections with a DAPI filter set (E/A, 329/461 nm) to observe FG fluorescence and
determine injection sites that were comparable for PVN analysis. Specifically, each image was
opened in PowerPoint (Microsoft) and manipulated to make the image transparent. The images
corresponding to a particular atlas region were then superimposed over a tracing of the relevant
atlas figure. After correcting for the boundaries and matching anatomical landmarks, a circle
was drawn over the FG-labeled injection site and included in a composite figure comprised of all
injections for either control or salt-loaded mice for each atlas figure containing the RVLM.
The total number of FG positive cells in the PVN were determined from mice with
injection sites that were observed to be similar in location and area. Tissue from mice with
injection sites that did not show FG coverage in the area of the RVLM were excluded from the
study. Analysis of the forebrain from successful injections included three atlas-matched PVN
images per mouse (from Bregma -0.94, -1.06, and -1.22mm) that also contained the amygdala.
These areas were determined to be rich in hindbrain-projecting neurons based on the distribution
of FG positive cells. Images were acquired as described above and z-stacks were scrolled
through to identify FG positive cells based on a clear cellular profile and uniform labeling. Cells
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that met these criteria were marked with an event marker function and totals were recorded. This
process was repeated for tdTomato and vasopressin-NP. Finally, single-channel images and
superimpositions were used to determine the total number of FG positive cells, FG+vasopressin-
NP positive cells as a percentage of all FG cells, and FG+tdTomato positive cells as a percentage
of all FG cells.
Statistics
All grouped data presented as mean +/- SEM. Atlas-matched colocalization data were
assessed with a two-factor analysis of variance. Main effects or interactions (P<0.05) were
assessed post hoc with the Bonferroni method. Total neuropeptide/tdTomato colocalization data
obtained from grouping all PVN atlas regions from each subject were assessed with an unpaired
2-tailed t-test. Significance for all analyses was set at P<0.05. Statistical analyses were
performed and graphs were created using GraphPad Prism 5.
Results
Salt-Loading Reduces CRH mRNA Expression in the PVN
Figure 5-1 shows representative images of sequential (A and C, B and D) coronal brain
sections taken from a series through the PVN and processed for in situ hybridization and
digoxigen immunohistochemistry. At Bregma -0.70 mm, the CRH mRNA labeling represents a
dense triangular region (Fig. 5-1, A) that is markedly reduced in the ventral portion of the PVN
with salt-loading (Fig. 5-1, B). At Bregma -0.82 mm, CRH expression following salt-loading
appears localized to a cluster in the dorsal PVN (Fig.5-1, C) that is lessened in area, but not in
density with salt-loading (Fig. 5-1, D). Quantification of the average density for each PVN
region revealed a significant (P<0.05) decrease in CRH mRNA density with salt-loading at
Bregma -0.70 mm, but no overall difference at Bregma -0.82 mm (Fig. 5-1, E).
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Fluorescent Tracking of CRH mRNA by tdTomato
Assessment of the specificity of Cre/loxP-mediated reporting for CRH transcription was
performed using in situ hybridization. CRH transcript was labeled in PVN sections of mice
drinking water, 2% NaCl, water + restraint, or 2% NaCl + restraint. Adjacent sections were
processed for thionin staining for determination of PVN subregions at Bregma -0.82 (Fig. 5-2,
A). Subregions were outlined and used to compare DAPI-labeled nuclei, CRH mRNA+DAPI
nuclei, and CRH mRNA+tdTomato expressing cells. Semi-quantitative analysis revealed that
the area of each PVN subregion contained comparable numbers of neurons in each group as the
number of DAPI-positive nuclei were similar (Fig.5-2; D, E). Furthermore, while restraint
increased the number of DAPI+CRH mRNA counts and DAPI+tdTomato counts, salt-loading
alone decreased the number of DAPI+CRH mRNA counts in each subregion, suggesting a trend
toward inhibition of CRH mRNA with salt-loading; however, this inhibitory trend was not
reflected in the number of DAPI+tdTomato counts.
Fluorescently-labeled CRH mRNA and tdTomato-filled neurons of coronal sections
through the PVN were quantified in three atlas–matched brain sections representing the majority
of the rostral-caudal extent of the PVN from an euhydrated unrestrained mouse (Fig 5-2, F1).
CRH mRNA was colocalized with tdTomato 94.36 +/- 4.63% of the time while CRH mRNA that
was not colocalized with tdTomato (arrows) represented 9.63 +/-5.74% of total CRH mRNA
positive cells (Fig 5-2, F2).
Total tdTomato Unaffected by Salt-Loading or Restraint
The total number of tdTomato positive neurons was similar between groups (No main
effects or interaction; Fig. 5-3) although the mean count tended to be higher for salt-loaded
restrained mice vs water-drinking restrained mice at Bregma -0.82mm (Water+Restraint:
176.60+/-16.56, 2% NaCl+Restraint: 196.00+/-4.97) and Bregma -0.94mm (Water+Restraint:
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72.00+/-9.85, 2% NaCl+Restraint: 109.33+/-18.35). In contrast, the mean counts for
unrestrained mice were nearly the same: Bregma -0.82mm (Water No Restraint: 183.00+/-8.22,
2% NaCl No Restraint: 183.500+/-5.41) and Bregma -0.94mm (Water No Restraint: 87.60+/-
15.19, 2% NaCl No Restraint: 84.00+/-12.29).
Salt-Loading is not Associated with Alterations in the Extent of Oxytocin-NP/tdTomato
Colocalizations in the PVN
Figures 5-4 and 5-5 show representative images of coronal sections through the PVN of
mice drinking water or 2% NaCl. Comparable atlas-matched sections from both groups were
immunofluorescently labeled for oxytocin-NP. As indicated by the large number of tdTomato
positive neurons in Fig. 5-4, this region contains a large number of putative neurosecretory
neurons, while Fig. 5-5 contains primarily preautonomic neurons (confirmed in Fig. 5-11).
Sections corresponding to Bregma -0.70 mm or Bregma -0.82 mm had only scant colocalizations
in both the Water (Fig. 5-4 A, C) and 2% NaCl (Fig. 5-4 B, D) groups. The distribution of
tdTomato and oxytocin-NP in these two particular regions was clearly distinct from other regions
of the PVN. At Bregma -0.70 mm, tdTomato cells were organized as a dense cluster of neurons
surrounded by a comparatively much smaller number of oxytocin-NP cells. The tdTomato and
oxytocin-NP expression at Bregma -0.70 mm was very similar to that seen at Bregma -0.82 mm
except for a reduction in the amount of tdTomato expression in the ventral portion of the PVN.
At Bregma -0.94 mm and Bregma -1.06 mm, colocalizations were observed more frequently in
both control and salt-loaded mice compared with the other regions analyzed. tdTomato
expression in these two caudal regions decreased compared with the more rostral areas and
furthermore oxytocin-NP expression was more laterally distributed (Fig. 5-5 A-D).
Two-way ANOVA revealed a main effect of atlas region [F(4, 40) =17.30 (P<0.05)], but
not treatment and no interactions were found. The average number of tdTomato and oxytocin-
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NP colocalizations increased in the two most caudal sections through the PVN but this increase
was similar for both groups (summarized by atlas region in Fig. 5-4 E and Fig. 5-5 E, and
averaged for all subjects in Fig. 5-8 A).
Salt-Loading is Associated with an Increase in Vasopressin-NP/tdTomato Colocalizations
in the PVN
Figures 5-6 and 5-7 shows representative unilateral coronal sections
immunofluorescently labeled for vasopressin-NP through the PVN of CRH-reporter mice
drinking either water or 2% NaCl. The overall distribution of vasopressin-NP neurons appeared
much closer to and interspersed with tdTomato cells compared with the distribution of oxytocin-
NP cells. This was especially true in the ventral portion of the PVN (Fig. 5-6 A-D).
Furthermore, vasopressin-NP-positive soma were found more frequently in the neurosecretory
region, with the majority of vasopressin-NP immunoreactivity occurring at Bregma -0.70 mm
and Bregma -0.82 mm. The high density of tdTomato and vasopressin-NP cells observed in
these two atlas regions were also associated with a significant increase in colocalizations.
Specifically, an atlas-region x treatment interaction [F(4,81)=4.59 (P<0.05)] occurred with main
effects of both atlas region [F(4,81)=25.01 (P<0.05)] and 2% NaCl treatment [F(1,81)=21.68
(P<0.05)]. Post hoc analysis revealed sections related to Bregma -0.70 mm and Bregma -0.82
mm having significantly more (P<0.05) tdTomato/vasopressin-NP colocalizations with salt-
loading (Fig. 5-6 E). This effect contributed to an overall increase in the average number of
tdTomato/vasopressin-NP colocalizations throughout the PVN which more than doubled in
response to salt-loading (P<0.05) (Fig. 5-8 B).
Salt-Loading does not Affect CRH Expression in Vasopressin-NP or Oxytocin-NP
Expressing Neurons of the SON
Figure 5-9 depicts representative images of sections through the SON from Water and
2% NaCl mice. Analysis of tdTomato/vasopressin-NP colocalizations (Fig. 5-9 C) and
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tdTomato/oxytocin-NP colocalizations (Fig. 5-9 D) indicates no apparent effects of salt-loading.
Furthermore, both groups exhibited only sparse expression of tdTomato in the SON that
amounted to only one or two cells (data not shown).
Fluorogold Injections were Localized to the Nucleus Ambiguous, Pre-Bötzinger Complex,
and Rostroventrolateral Medulla
The distribution patterns of FG injection sites were consistent between Water and 2%
NaCl mice, indicating similar delivery of retrograde tracer to the hindbrain of both groups (Fig.
5-10). Of 33 injected mice, 7 were included from the Water group and 8 from the 2% NaCl
group. Overall, the injections delivered similar amounts of tracer to regions including and also
adjacent to the RVLM, specifically the nucleus ambiguous and pre-Bötzinger complex.
Only injection sites determined to have tracer injected into the RVLM were included for
subsequent FG quantification in the PVN. The most rostral atlas region (Bregma -6.48 mm) that
shows the RVLM (Fig. 5-10, A1 and B1) is also characterized by the lack of a nucleus
ambiguous, and at this level one section from the Water group was counted as a hit (Fig. 5-10
A1). While five sections from the 2% NaCl exhibited FG at this same level, only one of these
overlapped with the RVLM area (Fig. 5-10 B1, excluded injection sites shown in red). The next
region moving caudally (Bregma -6.64 mm; Fig. 5-10, A2 and B2) shows four RVLM hits in the
Water group (Fig. 5-10 A2) and four out of five hits in the 2% NaCl group (Fig. 5-10 B2). The
region at Bregma -6.72 mm included five hits in the Water (Fig. 5-10 A3), and four out of five
from 2% NaCl groups, respectively (Fig. 5-10 B3). At Bregma -6.84 mm, five out of six hits
were determined in the Water group (Fig. 5-10 A4) and five out of seven in the 2% NaCl group
(Fig. 5-10 B4).
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Retrogradely-Labeled Neurons Increase Expression of tdTomato and Vasopressin-NP in
PVN
Injection site analysis resulted in the inclusion of 7 mice from the Water group and 8
mice from the 2% NaCl group for quantification of soma in the PVN. Sections labeled for FG
and vasopressin-NP were analyzed first for the number of FG-labeled cells in each section.
Representative images are of the three atlas-matched regions containing the majority of
preautonomic cells (Fig. 5-11 A-F). Quantification of FG-labeled cells did not reveal a
significant interaction or a main effect of 2% NaCl but there was a main effect of atlas-region
[F(2, 36)=3.90 (P<0.05)] (Fig. 5-11 G). Average FG-expressing cells counted for all sections were
not significantly different (Fig. 5-11 H).
Vasopressin-NP cells in the caudal PVN were not labeled with the same intensity as more
anterior regions (Fig. 5-12 A). Labeling was often faint and granular (Fig. 5-12 B). There were
significant effects of both atlas-region [F(2,36)=4.91 (P<0.05)] and 2% NaCl [F(1,36)=4.40
(P<0.05)]. Post hoc analysis did not reveal significant differences in any particular atlas region
(Fig. 5-12 E); however, the average percentage of vasopressin-NP/FG colocalizations were
increased in response to salt-loading (P<0.05) (Fig. 5-12 F).
The tdTomato expression in all three atlas regions comprising the caudal PVN was much
less abundant and scattered than in more anterior PVN regions. Figure 5-13 shows
representative images of coronal atlas-matched sections through the PVN. Compared with the
controls (Fig. 5-13 A), salt-loading increased tdTomato colocalizations with FG (Fig. 5-13 B).
Two-way ANOVA detected a main effect of 2% NaCl [F(1,36)=17.02 (P<0.05)]. Post hoc
analysis revealed a significant increase in tdTomato/FG colocalizations at Bregma -0.94 mm
(P<0.05) (Fig. 5-13 C). The average percentage of tdTomato/FG colocalizations within the PVN
was increased in response to salt-loading (P<0.05) (Fig. 5-13 D).
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Retrogradely-Labeled Neurons in the Amygdala are Distinct from tdTomato-Expressing
Neurons
Analysis of brain sections including the amygdala was performed from three salt-loaded
mice and three water-drinking controls for the same atlas-matched regions described above for
the PVN. Figure 5-14 shows the distribution of tdTomato-expressing neurons and neurons
labeled with FG at Bregma -1.06. The densest expression of tdTomato was found in the lateral
portion of the amygdala which was almost entirely populated with neurons reporting for CRH.
FG expression was found almost exclusively in neurons of the medial amygdala, and there were
no colocalizations with very sparsely distributed tdTomato-expressing neurons. Other regions
with scant expression of tdTomato include the basolateral amygdala, the extended amygdala, the
anterior part of the basomedial amygdaloid nucleus, and the central nucleus of the amygdala;
regions which also had no retrogradely-labeled neurons.
Discussion
We used fluorescent indication of CRH transcription in conjunction with
immunohistochemistry and retrograde neuronal tract tracing to evaluate how salt-loading caused
by consumption of 2% NaCl changes neuropeptide expression within distinct populations of
PVN neurons. Salt-loading significantly decreased and redistributed CRH mRNA within
subregions of the PVN. Subsequent experiments utilized tdTomato reporting of CRH
transcription to decipher which PVN neurons exhibited changes in neuropeptide expression as a
consequence of salt-loading. Consumption of 2% NaCl did not affect tdTomato expression in
the SON or tdTomato colocalization with oxytocin-NP in the PVN. However, salt-loading did
significantly increase the colocalization of vasopressin and tdTomato in putative magnocellular
PVN neurons. To evaluate changes in neuropeptide expression within preautonomic neurons,
CRH-reporter mice were injected with FG targeting the RVLM. Interestingly, salt-loading
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significantly increased the colocalization of vasopressin and FG as well as tdTomato and FG
within preautonomic neurons in the PVN. This effect seemed to be specific to the PVN, as no
tdTomato colocalizations with FG were observed in the amygdala. Our results demonstrate that
chronic osmotic stress increases expression of CRH in neuronal circuits that maintain plasma
tonicity and blood pressure. The implication is that such neuropeptide plasticity alleviates the
deleterious consequences of chronic dehydration but may progressively become a
pathophysiological mechanism that contributes to the etiology of stress-related disease.
The parvocellular and magnocellular subdivisions of the mouse PVN are not well defined
which impedes the localization of CRH mRNA to these subdivisions (Biag et al., 2012). To
circumvent this impediment, we used the Cre-loxP system to report the transcription of CRH
mRNA with the red fluorescent protein, tdTomato, which allowed evaluation of CRH production
within specific neuronal phenotypes. Using this approach, we determined that salt-loading did
not affect the colocalization of tdTomato with oxytocin-NP, suggesting that salt-loading does not
increase CRH and oxytocin colocalization within the PVN of mice. In addition, we observed
few tdTomato expressing cells in the SON under basal conditions and did not observe an effect
of salt-loading on tdTomato expression within this nucleus. Studies conducted using rats have
found that CRH mRNA is expressed in magnocellular neurons of the PVN and SON that
produce oxytocin, and that oxytocin mRNA and oxytocin/CRH colocalizations increase in
response to chronic salt-loading (Majzoub et al., 1983; Lightman and Young, 1987; Kovacs and
Sawchenko, 1993; Imaki et al., 2003). This plasticity may influence pituitary physiology
(reviewed in (Bondy et al., 1989)) because CRH derived from magnocellular neurons acts in the
intermediate lobe of the pituitary to augment systemic neuropeptide release through a mechanism
dependent, in part, on melanocyte-stimulating hormone signaling. Our results, consistent with
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those of others (Imaki et al., 2003), suggest that the osmotically-induced CRH plasticity that
occurs in magnocellular oxytocin neurons within the PVN and SON of rats does not occur in
mice. The implication is that the effects of salt-loading on CRH expression and systemic
neuropeptide release may also differ between rats and mice.
In contrast to what was observed with tdTomato and oxytocin-NP, salt-loading increased
the colocalization of tdTomato with vasopressin-NP in the PVN. These colocalizations occurred
most frequently in a region of the PVN known to contain a dense cluster of CRH-positive
parvocellular neurons. In this area, vasopressin-positive neurons closely surround and are
interspersed with CRH neurons that project to the median eminence (Biag et al., 2012).
Colocalization of CRH and vasopressin in neurosecretory parvocellular neurons allows their
corelease, which facilitates the secretion of ACTH into the systemic circulation (Gillies et al.,
1982; Vale et al., 1983). It is unlikely that the increase in tdTomato/vasopressin-NP
colocalizations that we observed occurred in neurosecretory parvocellular neurons that initiate
HPA-axis activation because salt-loading attenuates CRH and does not increase vasopressin
mRNA in the parvocellular subdivision of the PVN (Scott Young, 1986; Kovacs and
Sawchenko, 1993; Aguilera and Rabadan-Diehl, 2000; Grinevich et al., 2001). It is possible that
chronic osmotic stress allows the corelease of CRH and vasopressin to act at intermediate
pituitary CRH receptors to facilitate vasopressin secretion, similar to the corelease of oxytocin
and CRH that occurs in magnocellular neurons of rats (Bondy and Gainer, 1989). This
interpretation is consistent with previous reports demonstrating that chronic salt-loading elevates
vasopressin (Morita et al., 2001). Furthermore, osmotically-induced release of vasopressin into
the systemic circulation is inhibited by corticosterone (Papanek et al., 1997) and suppressing
HPA axis activity could permit heightened release of vasopressin to alleviate the hypertonicity
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that occurs with chronic salt-loading. Here, we propose that, within the PVN, salt-loading
increases CRH expression in magnocellular neurons to elicit water reabsorption through
vasopressin secretion.
The expression of CRH in the PVN is not limited to neurons that project to the median
eminence, but is also found in neurons making extrahypothalamic synapses where CRH acts as a
neurotransmitter or modulator (Dunn and Berridge, 1990). In particular, neurons within the PVN
that project to hindbrain nuclei, deemed preautonomic neurons, are known to express CRH and
are implicated in the control of sympathetic outflow and arterial blood pressure (Sawchenko,
1987; Milner et al., 1993; Yang and Coote, 1998). In the present study, we identified
preautonomic neurons as those labeled with retrograde tracer delivered into the RVLM and its
adjacent nuclei. Importantly, we established similar tracer distribution and number of
retrogradely labeled preautonomic neurons in the PVN of control and salt-loaded mice, thereby
allowing quantitative comparisons between these groups. A portion of retrogradely-labeled PVN
neurons were also positive for tdTomato and the distribution of these colocalizations was similar
to that of RVLM-projecting CRH neurons previously reported in rats (Milner et al., 1993). In
contrast, none of the FG-positive neurons in the amygdala were positive for tdTomato as neurons
labeled for FG were confined to the medial portion of the amygdala while tdTomato-positive
neurons were found densely expressed in the lateral portion. Interestingly, mice subjected to
salt-loading had a greater percentage of retrogradely labeled neurons in the PVN that expressed
tdTomato, suggesting chronic osmotic stress initiates the transcription of CRH in PVN neurons
with efferent projections to the RVLM. The RVLM is a hub for many converging and diverging
signals relating to sympathetic tone (reviewed (Guyenet, 2006)) and injection of CRH into the
RVLM increases arterial pressure in rats (Milner et al., 1993). Thus, the increased expression of
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CRH that occurs in PVN preautonomic neurons subsequent to salt-loading may allow these
neurons to utilize CRH receptors in the RVLM to promote sympathetic outflow and
cardiovascular adaptations to chronic osmotic stress.
The increased expression of CRH in preautonomic neurons that occurs with salt-loading
may work in concert with vasopressin to influence autonomic outflow subsequent to chronic
osmotic stress. Consistent with the results of Pretel & Piekut (Pretel and Piekut, 1989), we found
that PVN neurons retrogradely identified as projecting to the RVLM and its adjacent nuclei were
immunoreactive for vasopressin-NP. These results are consistent with prior reports that PVN
vasopressinergic neurons project to the RVLM in both rats and mice (Kc et al., 2002; Kc et al.,
2010; Rood et al., 2013) but conflict with other studies indicating that very few if any
vasopressin neurons project to the RVLM (Gomez et al., 1993; Stocker et al., 2006). This
discrepancy can be explained by the induction of vasopressin in preautonomic neurons by salt-
loading or could also be due to inadvertent administration of retrograde tracer to the ambiguous
nucleus, which receives vasopressinergic projections from the PVN (Wang et al., 2002; Rood et
al., 2013). In the latter case, there is evidence that vasopressin promotes parasympathetic
withdrawal in the ambiguous nucleus (Wang et al., 2002) that may complement the action of
vasopressin by increasing sympathetic output via the RVLM (Kc et al., 2010).
The increase in colocalization of tdTomato with FG and AVP does not agree well with
our data that did not find any significant changes in tdTomato expression. The average number
of tdTomato positive neurons in the PVN of salt-loaded restrained mice was higher than the
average number determined for restrained water-drinking controls (Fig. 5-3), suggesting a trend
towards increased reporting for CRH. The statistical methods employed to determine any
differences between groups rely on comparing mean counts relative to the variability observed to
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make the claim that the increase was significant. It is highly likely that the variability
contributing to total tdTomato came from additional sources beyond those influencing the
variability observed in the determination of colocalization of tdTomato with AVP and FG. If
this is the case, then the true effect size of salt-loading on total tdTomato may be small or
medium, while the true effect size of salt-loading on tdTomato in discrete populations of PVN
neurons may be large. In support of this, the experiments performed by Imaki et al. suggest that
in mice, but not rats, total CRH expression in the PVN is only subtly altered during salt-loading,
promoting the idea that significant increases in CRH expression are likely only observed in a
narrowly defined population (Imaki et al., 2003).
Taken together, our results illustrate potential mechanisms by which CRH and
vasopressin may be differentially expressed in the PVN to counter chronic saline consumption.
Both of these neuropeptides become dysregulated in widespread diseases and disorders including
those with psychiatric and cardiovascular components (discussed in more detail in Chapter 6).
The increased colocalization of CRH and vasopressin may work to increase systemic
vasopressin, but the consequences of this plasticity on the rest of the network of which the PVN
is a hub has yet to be determined. Similarly, the increase in preautonomic neuropeptide
expression suggests either an adaptation to maintain normal cardiovascular function during an
extreme overload of salt or is a marker for a maladaptive consequence of some other mechanism.
Given our data supporting a lack of dehydration-induced anorexia or hypovolemia, it is likely the
former case; however, it may be that the duration of salt-loading in the present experiments did
not allow for sufficient development of other mechanisms to surface. These mechanisms might
include the involvement of SON neurons or oxytocin neurons and future experiments would
indeed shed additional light on the overall plasticity of PVN neurons during chronic salt-loading.
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Figure 5-1. Representative images of PVN sections processed for in situ hybridization of
digoxigenin-conjugated probe and CRH mRNA. Comparable sections from mice in
the Water (A, C) and 2% NaCl (B, D) groups. Salt-loading was associated with a
decrease in density at Bregma -0.70 mm, but did not differ at Bregma -0.82 (E,
P<0.05). Water n=4-6, 2% NaCl n=4. Scale bars = 100 µm. 3v, 3rd ventricle.
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Figure 5-2. Analysis of tdTomato reporting for CRH mRNA expression in the PVN. Thionin
staining for determination of PVN subregions at Bregma -0.82 (A). Coronal section
through the PVN depicting DAPI (blue nuclei) and CRH mRNA in situ hybridization
(magenta dots) (B). Superimposed image of CRH mRNA in situ hybridization
(magenta dots) and tdTomato-filled cells (green) (C). Total counts of DAPI,
DAPI+CRH mRNA, and DAPI+tdTomato in the lateral magnocellular (PaLM) (D)
and dorsal cap (PaDC) (E). Coronal section through the PVN depicting CRH mRNA
and tdTomato positive neurons as well as neurons with CRH mRNA expression
without tdTomato expression (F1). Graph depicting reporter fidelity (F2). Inset:
closeup of CRH mRNA expression colocalized with DAPI and tdTomato, or DAPI
only. Arrows indicate false negatives. Lateral Magnocellular, PaLM. Dorsal Cap,
PaDC. Error bars = SEM. Scale bars = 50 µm (A-C, F), 10 µm (F1, inset). 3v, 3rd
ventricle.
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Figure 5-3. Analysis of total tdTomato in the PVN. Neither restraint or salt-loading significantly
increased the number of tdTomato positive neurons in the PVN.
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Figure 5-4. Representative unilateral images and quantification of oxytocin-neurophysin (OT-
NP)/tdTomato colocalizations in the “neurosecretory” region of the PVN. Total
bilateral colocalizations were determined for mice maintained on water (left side of
each outline; A and C) and mice maintained on 2% NaCl (right side of each outline;
B and D). Maintenance on 2% NaCl did not affect the number of OT-NP/tdTomato
colocalizations although there was a main effect of atlas-region [F(1, 16)=29.18
(P<0.05)] with more colocalizations occurring at Bregma -0.82 (E). Water n=5, 2%
NaCl n=5. Error bars = SEM. Scale bars = 100 µm. 3v, 3rd ventricle.
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Figure 5-5. Representative unilateral images and quantification of oxytocin-neurophysin (OT-
NP)/tdTomato colocalizations in the “preautonomic” region of the PVN. Total
bilateral colocalizations were determined for mice maintained on water (left side of
each outline; A and C) and mice maintained on 2% NaCl (right side of each outline;
B and D). Maintenance on 2% NaCl did not affect the number of OT-NP/tdTomato
colocalizations (P>0.05E). Water n=5, 2% NaCl n=5. Error bars = SEM. Scale bars
= 100 µm. 3v, 3rd ventricle.
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Figure 5-6. Representative unilateral images and quantification of vasopressin-neurophysin
(AVP-NP)/tdTomato colocalizations in the “neurosecretory” region of the PVN.
Total bilateral colocalizations were determined for mice maintained on water (left
side of each outline; A and C) and mice maintained on 2% NaCl (right side of each
outline; B and D). Maintenance on 2% NaCl revealed a significant effect of 2% NaCl
[F(1,34)=19.64 (P<0.05)] with sections related to Bregma -0.70 mm and Bregma -0.82
mm having significantly more (P<0.05) AVP-NP/tdTomato colocalizations with salt-
loading (E). Water n=8, 2% NaCl n=9-10. Error bars = SEM. Scale bars = 100 µm.
3v, 3rd ventricle.
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Figure 5-7. Representative unilateral images and quantification of vasopressin-neurophysin
(AVP-NP)/tdTomato colocalizations in the “preautonomic” region of the PVN. Total
bilateral colocalizations were determined for mice maintained on water (left side of
each outline; A and C) and mice maintained on 2% NaCl (right side of each outline;
B and D). Maintenance on 2% NaCl had no effect on AVP-NP/tdTomato
colocalizations (P>0.05, E). Water n=8, 2% NaCl n=9-10. Error bars = SEM. Scale
bars = 100 µm. 3v, 3rd ventricle.
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Figure 5-8. Average total colocalizations in the PVN. The average total number of oxytocin-
neurophysin (OT-NP)/tdTomato colocalizations for all PVN regions from each
subject was not significantly different between groups (P>0.05, A). Salt-loading was
associated with significantly increased (P<0.05, B) total vasopressin-neurophysin
(AVP-NP)/tdTomato colocalizations. Water (open bars) n=5, 2% NaCl (grey bars)
n=5. Error bars = SEM.
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Figure 5-9. Representative unilateral images used for quantification of colocalizations in the
supraoptic nucleus. There was no obvious qualitative change in tdTomato expression
due to salt-loading (A, B). The tdTomato expression that did occur was not
significantly affected in terms of colocalization with either vasopressin-NP (C) or
oxytocin-NP (D). Water (open bars) n=3, 2% NaCl (grey bars) n=3. Error bars =
SEM. Scale bars = 50 µm. ot, optic tract.
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Figure 5-10. A summary of FG injections targeting the RVLM. Injections from the control
group are shown in the left column (A1-4) and injections from salt-loaded mice are
shown to the right (B1-4). The atlas figures are tracings from individual plates
published in The Mouse Brain in Stereotaxic Coordinates 3rd Ed containing the four
regions (from top to bottom: Bregma -6.48 mm, -6.64 mm, -6.72 mm, -6.84 mm)
outlining the boundaries of the RVLM. The images to the left of each atlas drawing
(a, b, c, etc) are the actual images from which the location of FG-labeled area was
determined. Each image is labeled with a lower case letter designating its
corresponding injection site on the figure tracing to the right. Any image with FG
labeling was included for analysis regardless of whether or not it was determined to
be close enough to the RVLM to be acceptable for PVN analysis, thus hits are
designated with a green outline while misses are designated with a red outline.
Images are ordered with misses first (if any) followed by hits. Scale bars = 500µm
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Figure 5-11. Representative unilateral images of z-stack 3D reconstructions used for
quantification of PVN neurons labeled with Fluorogold (FG). Images shown are
sequential from a series of sections taken from a single control subject (A, C, E) and a
single subject from the salt-loaded group (B, D, F). Total bilateral counts of FG-
positive cells were determined. Quantification revealed a similar number of FG-
positive cells for each PVN atlas region in each group (G). The average total number
of FG cells for all PVN regions from each subject was also not significantly different
(H). Water n=6-7, 2% NaCl n=7-8. Error bars = SEM. Scale bars = 50 µm. 3v, 3rd
ventricle.
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Figure 5-12. Representative images and quantification of Fluorogold (FG)/vasopressin-NP
(AVP-NP) colocalization. In contrast to more anterior sections, AVP-NP labeling
appeared granular (B). Quantification revealed a non-significant increase in the
number of FG/AVP-NP colocalizations (D) with salt-loading for each PVN atlas
region (E). The average total number of FG/AVP-NP colocalizations for all PVN
regions from each subject was higher (F) in salt-loaded mice compared with controls
(P<0.05). Error bars = SEM. Scale bars: A = 50 µm, B-D = 10 µm. 3v, 3rd ventricle.
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Figure 5-13. Representative images and quantification of Fluorogold (FG)/tdTomato
colocalization (A, B). Quantification revealed a significant increase in the number of
FG/tdTomato colocalizations (C) with salt-loading at Bregma -0.82 mm. The average
total number of FG/tdTomato colocalizations for all PVN regions from each subject
(D) was higher in salt-loaded mice compared with controls (P<0.05). Error bars =
SEM. Scale bars = 50 µm; 3v, 3rd ventricle.
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Figure 5-14. Representative image of Fluorogold (FG) and tdTomato in the amygdala. Typical
FG expression was located in the medial portion of the amygdala (CeM) while the
densest expression of tdTomato was in the lateral division (CeL). Only scant
expression of tdTomato occurred in the CeM, and in no case did tdTomato colocalize
with FG. Central nucleus of the amygdala (CeC), basolateral amygdala (BLA),
internal capsule (ic). Scale bar = 200 µm.
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CHAPTER 6
GENERAL DISCUSSION AND FUTURE DIRECTIONS
The data presented here serves two important general functions that allow for more
thorough future investigation into neuroplastic changes in the PVN that accompany salt-loading.
First, the extension of reported findings in the acute salt-loading paradigm from the rat model to
the mouse model was a necessary step towards gaining valuable tools to investigate the effects of
salt-loading on a genetic level. Similarly, the comparison of the general effects of chronic salt-
loading on ingestive behavior and measures of plasma constituency to results obtained using rats
is valuable to the researcher considering utilizing mice in place of or in conjunction with
experiments using rats. Second, the use of a transgenic reporter mouse to visualize CRH-
positive neurons represents a significant step forward in overcoming the technical limitations
inherent in visualizing CRH-positive neurons with high spatial precision. Granted this
improvement, like any method, has its limitations that must be acknowledged upfront, and
associated data must be interpreted in light of these limitations. These two general points will be
discussed together with the more specific points that might be better understood in the context of
species differences and methodological concerns.
The results of Chapter 3 confirmed that the effects of acute salt-loading involving
suppression of the HPA axis using a rat model could be replicated in a mouse model and further
implicated a centrally-mediated inhibitory mechanism. The results obtained by both Krause et
al. and Frazier et al. in rats included slight increases in pNa+ levels, without associated increases
in hematocrit as well as a blunted restraint-induced CORT (Krause et al., 2011a; Frazier et al.,
2013). All of these effects were found and reported here in Chapter 3 suggesting that the murine
response to restraint experienced in the context of acute hypernatremia involves suppression of
the HPA axis. However, Krause et al. also reported that restraint-induced ACTH was attenuated
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during restraint which was a measure not investigated with mice (Krause et al., 2011a). The
significance of this finding in rats was that the decrease in ACTH with CORT suggested that the
inhibitory effect on the HPA axis associated with acute hypernatremia was centrally mediated.
However, because only CORT and not ACTH were measured in our experiments in mice, we did
not have sufficient evidence to implicate a centrally-mediated effect based solely on endocrine
measures. To more directly test this hypothesis, and to do so in a direct way that is not possible
in rats, we utilized a CRH-reporter mouse to visualize Fos activation in PVN CRH neurons from
mice rendered acutely hypernatremic. In these mice, Fos-positive nuclei were significantly
reduced in transgenically-identified CRH neurons compared with controls providing evidence for
a source of inhibition to these cells. Regarding this inhibition, the study in rats reported by
Frazier et al. similarly lacked data showing a decrease in restraint-induced ACTH, but provided
electrophysiological data supporting an inhibitory tone on putative CRH neurons in the PVN that
was dependent on oxytocin-receptors (Frazier et al., 2013). The results from the current study
using mice support the activity of oxytocin as an inhibitory factor in attenuation of CRH neurons
and the HPA axis as mice rendered acutely hypernatremic had more frequent Fos-positive nuclei
in PVN neurons expressing oxytocin. However, additional experimentation is necessary to
confirm an action of oxytocin on CRH neurons or on neurons that innervate CRH neurons in the
PVN. These additional experiments benefit from the translation of findings from rat to mouse as
well as the initial investigation into the validity of the CRH reporter mouse used here. The
validation of the CRH reporter mouse included not only that fluorescent reporting for CRH
transcription colocalized well with fluorescent labeling of CRH mRNA (Figs. 2-1 and 5-2), but
for the first time showed that an osmotic stimulus via 2.0 M NaCl injection had an effect on
activation of genetically modified neurons altered to report for CRH in the PVN. These steps
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provide for future experiments to be performed that require the in vitro or post hoc identification
of PVN neurons that are known to be CRH-producing and which are also sensitive to inputs
which convey information regarding elevation of pNa+ concentration.
Chapter 3 included experiments to assess whether the anxiolytic effects of acute
hypernatremia produced in rats extended to the mouse model. Krause et al. found that slightly
elevating the pNa+ concentration resulted in pro-social effects as hypernatremic rats spent more
time interacting a novel conspecific (Krause et al., 2011a). Furthermore, Frazier et al. found that
the same treatment reduced anxiety-like behavior as assessed on the elevated plus maze (Frazier
et al., 2013). The increased activation of oxytocin neurons in the PVN noted in both of these
studies as well as our neuroanatomical data here imply that oxytocin is being released in the
brain, as oxytocin acting in the brain has been shown to have pro-social and anxiolytic effects on
behavior (Landgraf and Neumann, 2004; Neumann and Landgraf, 2012b; Peters et al., 2014).
Anecdotally speaking, rats generally appear to be less anxious and struggle less when confronted
with human handling which indicates that basal anxiety or reactionary behavior is pronounced in
mice in comparison to rats, thus it was unclear whether this behavior could be overcome with the
physiological alterations associated with hypertonic saline treatment. Our results here in mice
found that exploration of the open arms of the elevated plus maze was increased following
hypertonic saline injections indicating that the mechanism influencing anxiety in rats likely is
occurring in mice as well, and that innate behavioral differences between these two species do
not seem to influence the behavioral alterations caused by the hypernatremic state. This could be
because of the well-conserved mechanisms that balance hydromineral homeostasis in both
species which may be very similar despite other neuropeptide or circuit-level differences.
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The well-conserved mechanisms responsible for differences in innate behavior, but
similar anxiolytic responses during hypernatremia are likely mediated through the actions of
oxytocin and vasopressin, which seem to have similar centrally- and systemically-mediated
effects in both rats and mice (Insel et al., 1998), but which also display marked gender
differences (Francis et al., 2002). It has been postulated that gender differences exist in
neuropeptide expression for two possible reasons, 1) to promote behavioral differences and 2) to
prevent them (De Vries, 2004) i.e. in the first case, males and females must have very different
social and sexual behavior mediated by oxytocin and vasopressin, but in the latter case both
genders must be able to maintain adequate hydration, blood pressure, body temperature, and
other critical functions which are in part mediated by the same neuropeptides. It may be that this
explanation for differences on a sex basis also may be applicable on a species level as there are
many differences between rats and mice as far as oxytocin and vasopressin expression are
concerned. As previously discussed, perhaps the most obvious is the lack of segregated groups
of magnocellular neurons in the PVN of the mouse (Biag et al., 2012). This anatomical
difference may have functional consequences as magnocellular neurons that are more
interspersed with other neurons in the PVN may have more opportunity for cross-talk with other
neuronal phenotypes. The potential for cross-talk is an interesting and ongoing area of research
(Potapenko et al., 2013; Stern, 2015; Biancardi et al., 2016) involving the study of molecular and
glial-mediated signaling between neurons of the PVN to locally coordinate the output of specific
types of neurons. It is possible that the difference in the “scattered” murine expression of
magnocellular oxytocin and vasopressin in the PVN represents an anatomical basis for
differential control of basal anxiety levels through differential cross-talk with other PVN neurons
or differential projection patterns to limbic brain regions mediating fear and anxiety-like
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behavior. However, when confronted with an osmotic challenge, the differences in oxytocin and
vasopressin expression may either be inhibited or become secondary to similarly expressed
oxytocin and vasopressin neurons leading to a reduction in anxiety in both species in a similar
way. If true, the reason for a species difference in basal anxiety and anatomical PVN
magnocellular distribution, but a species similarity in the anxiolytic response to acute
hypernatremia might be found in consideration for the natural habitats of rats and mice. Both
rats and mice evolved in a commensalistic relationship with humans (Atkinson, 1985), but mice
are generally considered to be much more dependent on this association than rats are, thus the
common name house mouse (the house mouse or Mus musculus is the most widely-used
laboratory mouse species). Because of this close association, it may be that in order to survive,
mice developed a heightened sense of awareness due to the greater likelihood of encountering
natural predators that also associate with humans such as dogs, cats, foxes, rats, etc. which would
make them seem more anxious when handled. Rats on the other hand, being a predator of mice
and often exhibiting non-commensal behavior (Aplin et al., 2011; Varudkar and Ramakrishnan,
2015), may have developed a comparatively more “calm” demeanor. However, regardless of the
environmental obligations, each species would need to face exposure in seeking out water which
is analogous to the physiological state hypertonic saline injections induce before behavioral
testing in an apparatus such as the elevated plus maze. Thus it may be that while basal anxiety
levels are different between rats and mice, the activation of anxiolytic pathways related to
hyperosmotic conditions similarly induce changes in behavior.
There are several effects of acute salt-loading in rats that have yet to be confirmed in
mice. Krause et al. found that plasma vasopressin levels were not elevated at the time of
behavioral testing, restraint, and brain sample collection (Krause et al., 2011a). Furthermore,
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this study found that plasma renin activity was suppressed during restraint, but that plasma
oxytocin levels were increased, which are also effects not determined by the study performed
here in mice. These measures are important controls for several reasons. While hematocrit and
plasma protein levels are important indicators of plasma volume and therefore activation of the
RAAS, they do not provide a comprehensive view of the humoral consistency. The
determination of plasma vasopressin concentration and renin activity are important controls to
evaluate the endocrine response to hypertonic saline injections. These measures would indicate
whether the dehydration induced by the injections caused only intracellular dehydration or in
addition, caused extracellular dehydration and volume loss through increased urinary output.
High pNa+ generally suppresses the RAAS. Furthermore, vasopressin release is known to
quickly and transiently be released before returning to normal when the behavioral tests and
samples were collected in the acute salt-loading paradigm employed (Stricker and Verbalis,
1986). These points suggest that vasopressin and plasma renin activity would similarly be
affected in our experiments as previous reports using rats. However, in both cases water bottles
were removed following saline injections and the water deprivation period may have impacted
mice differently than rats. Regarding motivated behavior, the additional measures are important
because of the distinct mechanisms involved with hypovolemic thirst compared with hypertonic
thirst. Each would have specific consequences in terms of the circuits activated and the degree
to which various neurotransmitter and neuropeptides were involved in behavior and stress-
responsiveness (Stricker and Verbalis, 1986; Stricker and Sved, 2000). However, given that the
behavioral and CORT responses we observed in our experiments here with mice were consistent
with previous reports in rats it is likely that plasma vasopressin and renin activity were similarly
suppressed. Moreover, we found that oxytocin neurons were significantly activated following
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acute hypernatremia which implies that plasma oxytocin levels would be higher than controls
(Krause et al., 2011a), but this cannot be concluded without the proper experiments being
performed. Evidence exists that oxytocin is released systemically in response to elevated pNa+,
but that this is specific to rodents and does not occur in humans (Goodwin et al., 1970; Williams
et al., 1985; Seckl et al., 1989; Rasmussen et al., 2004). It would therefore be beneficial to know
if the increased activation of oxytocin neurons we observed was specific to neurons dedicated to
central projections or included pituitary projections.
As the primary purpose of these experiments was to better understand the capacity for
neuroendocrine and behavioral alterations associated with mediators of hydromineral
homeostasis in humans, it is important to relate these data to possible mechanisms of human
physiology. Clinical correlates to the presented experimental findings using animal models are
abundant, and the following discussion highlights these studies. They show that increased
activity of the RAAS is predictive of depression and anxiety, and when medication that blocks or
inhibits a specific component of the RAAS is prescribed for hypertension, there has also been
improvement in mood and affect. Large scale studies have provided evidence that aldosterone
levels may be a possible solution to the long-sought after biomarker for affective disorders.
Furthermore, direct evidence for the anxiolytic and stress-dampening effects of oxytocin is
available although there have been conflicting reports.
Interest in the relationship between anxiety or depression and RAAS activity found
significant correlations. In a two-year long study involving 1805 participants, researchers found
that depressed hypertensive individuals had elevated aldosterone levels compared with
individuals that were not depressed or those that were not hypertensive (Hafner et al., 2013).
Participants that were either only depressed or, surprisingly, only hypertensive did not present
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with heighted renin or aldosterone. In a smaller, but much more specific study carried out while
participants were asleep, Murck et al. monitored HPA and RAAS hormones of unmedicated
depressed and age-matched control subjects finding that aldosterone was significantly higher in
depressed individuals (Murck et al., 2003a; Murck et al., 2012). Furthermore, more than half of
patients with primary aldosteronism surveyed also suffered from an anxiety disorder and these
individuals had higher levels of self-reported stress, psychological distress, and a lower level of
well-being (Sonino et al., 2006; Sonino et al., 2011). As previously discussed, aldosterone
affects specific areas of the brain in rodent models, including the PVN, nucleus of the solitary
tract, locus coeruleus, and amygdala. The clinical data showing a correlation between elevated
aldosterone with anxiety and depression suggests that these areas are perhaps viable targets for
intervention if the primary cause of hyperaldosteronism cannot be directly addressed.
MacDonald and Feifel recently reviewed progress in developing oxytocin-based
therapeutics, citing over 30 clinical studies using intranasal administration of oxytocin in normal
individuals as well as single and multi-dose trials in those suffering from a brain-based illness
(Macdonald and Feifel, 2014). Results in terms of efficacy in reducing anxiety were conflicting;
however, some promising anatomical data was identified. For instance, females with a common
oxytocin receptor gene variant present with a psychological phenotype that includes excessive
worrying, cautiousness, pessimism, shyness, and fearfulness (Wang et al., 2013a). This variant is
associated with reduced resting state PFC to amygdala connectivity as assessed by fMRI and this
circuit’s functional connectivity is improved with oxytocin administration (Dodhia et al., 2014).
It may be that more specific routes of altering the central oxytocin system will be necessary in
order to tailor oxytocin-based therapies to individual disorders.
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A better understanding of the oxytocinergic circuitry may lead to novel therapeutic
strategies for treating core symptoms of autism spectrum disorders (ASD). Anecdotal reports and
clinical case studies have found that illness accompanied by fever markedly alleviates symptoms
of ASD (Sullivan et al., 1980; Cotterill, 1985; Curran et al., 2007). A hallmark of fever is
insensible water loss that causes acute hypernatremia or increases in the pNa+ concentration.
Thus, it is plausible that hypernatremia driven increases in central levels of endogenous oxytocin
might alleviate symptoms of ASD. This might be an especially attractive mechanism to exploit
if systemic levels of oxytocin are not affected, which as previously discussed, seems to be the
case in humans. Accumulating preclinical and clinical evidence suggests that brain
oxytocinergic circuits are valid candidate therapeutic targets for relief of ASD symptoms (Andari
et al., 2010; Guastella et al., 2010; Domes et al., 2013; Taurines et al., 2014).
The ability of oxytocin to suppress the activity of PVN CRH neurons seems to be an
effect seen in both humans and animal models. As discussed previously, an acute hypernatremic
state is associated with increased central release of oxytocin that potentiates an inhibitory
oxytocin-dependent tone on CRH neurons of the PVN. In support of this, human males were
given intranasal oxytocin in a randomized, placebo-controlled, double-blind experiment to test
the effect on exercise-induced salivary cortisol levels finding 24 IU, but not 48 IU oxytocin
significantly attenuated cortisol levels compared with placebo (Cardoso et al., 2013). Further
research of this caliber is necessary to determine if similar results are obtained with the induction
of a psychogenic stressor.
These clinical findings and experimental results using human subjects illustrate a
relationship between factors that mediate salt and water handling in the body, and neural circuits
involving the PVN which are often intimately involved in a variety of mental health disorders.
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The possibility exists that the reduced anxiety, HPA axis responsiveness, and increased
activation of oxytocin-producing neurons in the PVN presented here in association with acute
hypernatremia might be in some way involved with these disorders. It is possible that repeated
instances of recruiting these mechanisms may lead to conscious or unconscious learning of the
association between the hypernatremic state and perhaps a state of well-being - if it is true that a
human can induce a similar acute hypernatremic state. The injection of hypertonic saline
subcutaneously is an introduction of sodium that is for practical purposes never a mode of
increasing pNa+ levels in humans; rather, this method is meant to efficiently raise pNa+ in a
controlled experimental setting. Any translational potential of these experiments must therefore
ask the question of what a similar stimulus might be in humans in their normal environment or
simply, is it possible for one to raise his/her own pNa+ level to an extent that causes anxiolytic
and HPA axis-dampening effects. These are important questions to answer in order to make the
case that raising one’s pNa+ has reinforcing effects due to a “calming” or positive effect on a
subjective feeling of well-being. One of the major differences between our experimental design
and human behavior in terms of raising the pNa+ is that in our experiments there is no sensation
of taste while obviously humans mediate salt intake largely through gustatory processing. In this
sense, it may be that taste-related central processing might augment the effects of acute salt-
loading in some way and additional experiments that stimulate these pathways might test this
hypothesis. It is also possible that the effects of acute salt-loading might reinforce raising the
pNa+ through unconscious mechanisms as well. Another possibility is that an individual might
not increase the pNa+ solely by increasing salt-intake, but by altering fluid intake and urination
frequency to experience brief periods of hypertonic plasma without an associated reduction in
plasma volume. Finally, there may be no direct translation from rodent model to human
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physiology and behavior, but only an indirect summation of findings that, taken together with the
collective findings of others, adds to the general understanding of how mechanisms that control
hydromineral homeostasis also influences behavior, stress-responsiveness, and the activity of
PVN neurons.
To begin to understand the similarities and differences between acute and chronic salt-
loading and to add the element of ingestive behavior to our experimental paradigm, we
performed chronic salt-loading experiments to assess changes in the PVN and behavior.
Importantly, these experiments relied heavily on the identification of neurons in the PVN as
CRH-positive using a CRH-reporter mouse. Direct quantification of CRH protein or transcript
allows for the determination of CRH expression levels at the time brain tissue is sampled. In
contrast, the CRH reporter mouse utilized in the present study allows visualization of cells that
are stimulated to produce CRH during development or in response to an experimental treatment,
but is not indicative of the quantity of CRH. Regarding the possibility of ectopic expression, the
reliable colocalization of tdTomato with CRH mRNA has been previously validated using
various methods (Wamsteeker Cusulin et al., 2013; Chen et al., 2015; Pleil et al., 2015),
suggesting that increases in the number of cells that transcribe CRH in response to a stimulus can
be observed, quantified, and compared to controls. Reporting for CRH transcription following
Cre/lox-mediated recombination is accomplished through ROSA-26 promoter-driven tdTomato
expression, and consequently, the fluorescent indicator is understood to be persistent, meaning
that downregulation of CRH expression will not be accompanied by a downregulation of
tdTomato. While downregulation of CRH mRNA in response to salt-loading is a known and
interesting phenomenon (Watts, 1992a; Watts et al., 1995; Amaya et al., 2001), we designed our
experiments to focus on potential upregulation of CRH in vasopressin, oxytocin, and
130
preautonomic neurons. Thus, while further characterizing the rearrangement of CRH in the PVN
during salt-loading was the impetus for the current study, the primary goals were to use the
improved spatial resolution of the CRH reporter mouse to isolate specific PVN cell-types that
increased CRH expression. It is true that in terms of CRH expression, spatial precision was
prioritized over temporal precision in that a tdTomato-positive neuron may have expressed CRH
at any point and to any extent; however, comparison of the total number of colocalizations
relative to water-drinking controls is indicative of CRH upregulation in specific cellular
phenotypes.
Another technical consideration that is important to the interpretation of the
colocalization data presented in Chapter 5 is the potential for changing numbers of vasopressin-
and oxytocin-positive neurons which might contribute to the colocalization counts presented in
Figures 5-3 to 5-7. The overall purpose of counting colocalizations in the PVN and SON was to
test the hypothesis that chronic salt-loading causes CRH expression to increase in magnocellular
oxytocin neurons, a hypothesis that was later extended to include magnocellular vasopressin
neurons after it was determined that the data did not support our hypothesis. Qualitative
assessment of the images and quantification of colocalized neurons that were labeled for
oxytocin and tdTomato indicate that salt-loading did not increase the number of
tdTomato/oxytocin colocalizations. However, as discussed elsewhere, the mouse PVN is
characterized by less segregation between magnocellular and parvocellular neurons and the
fluorescent indication for CRH is not known to extinguish when CRH transcription rates go
down or stop. Thus, interpretation of any colocalization becomes more complicated. If there
were changes, however small, in the total number of oxytocin neurons or the total number of
tdTomato neurons in salt-loaded mice compared with controls, then it may be that a change in
131
the number of colocalizations would reflect this difference, but this need not be the case. No
differences in colocalization of oxytocin and tdTomato was detected through repeated random
sampling of several specific areas of the PVN in either group during a statistical test of
significance. We similarly assessed vasopressin- and tdTomato-expressing neurons and found
significant increases in colocalization which, in contrast to the investigation regarding oxytocin,
brought up an important question of whether it was CRH neurons making vasopressin or
vasopressin neurons making CRH. However, from a functional perspective the real question is
whether or not salt-loading is increasing CRH expression in neurons projecting to the posterior
pituitary or increasing vasopressin expression in neurons projecting to the median eminence
which might be answered through future experiments utilizing tract tracing of those projections.
As discussed in Chapter 5, it is likely that increased CRH in magnocellular projections to the
pituitary may potentiate vasopressin or oxytocin release (Bondy and Gainer, 1989) while
increased vasopressin in median eminence projecting neurons may potentiate ACTH release
(Abousamra et al., 1987). Because exposure to stress has likely already caused the reporting for
CRH in most if not all of the parvocellular median eminence-projecting neurons which cannot
subsequently decrease, tdTomato is likely going up or staying the same in the population of
magnocellular vasopressin neurons. Regarding the possibility of vasopressin increasing in
parvocellular median eminence-projecting neurons, total vasopressin counts might provide
additional information as to the nature of the colocalized neurons, but would not be a good
substitute for isolating neurons based on their projection targets, as salt-loading increased
vasopressin in another population of hindbrain-projecting neurons in this study. Essentially, the
colocalization data is best interpreted as an indication of possible functional reconfigurations
132
occurring in the PVN, and these indications require additional experiments for conclusive
evidence concerning functionality.
The fluorescent indication of CRH-positive neurons was first used in the chronic salt-
loading study to identify PVN neurons that were also activated by salt-loading or restraint. In
contrast to our acute salt-loading experiment, we found that chronic salt-loading was not
associated with a decrease in restraint-induced Fos-positive nuclei specifically within neurons
that reported for CRH transcription. The results of Krause et al. and Frazier et al. found that in
acute salt-loaded rats, restraint-induced CORT was attenuated (Krause et al., 2011a; Frazier et
al., 2013), which was also a finding here in acutely salt-loaded mice. As discussed above, there
were several indications that this effect was centrally-mediated, including reduced ACTH, an
inhibitory tone on putative CRH neurons, and reduced Fos activation in CRH neurons.
However, similar restraint-induced Fos between chronic salt-loaded mice and controls is not
sufficient evidence to conclude that restraint-induced HPA axis activation is not inhibited during
chronic salt-loading. On the contrary, the results of Chapter 5 suggested that the recovery to
baseline levels of CORT after restraint stress occurs faster in chronic salt-loaded mice.
Additional mechanisms for HPA axis inhibition exist both upstream and downstream of CRH
neurons that are likely influencing levels of ACTH and CORT during the course of the extended
osmotic challenge. Previous reports in rats have shown that the rise and fall of CORT which
normally occurs in a circadian rhythm becomes dysregulated during chronic salt-loading by
eliminating the daily nadir (Watts, 1992a). Circadian rhythms are controlled by neurons in the
suprachiasmatic nucleus which changes transcriptional activity in response to salt-loading (Watts
et al., 1995), suggesting that information regarding external and internal cues that are sent from
the suprachiasmatic nucleus to the PVN (Watts et al., 1987; Vujovic et al., 2015) conveying the
133
appropriate timing for low activity of CRH neurons may also be dysregulated. It is possible that
this dysregulation in a circadian regulatory circuit generates heightened activity of CRH neurons
at all times, and this effect supersedes an inhibitory input generated by the osmosensitive circuits
converging on the PVN CRH neurons during chronic salt-loading. Apart from central
mechanisms, it is also possible that the HPA axis may be attenuated while restraint-induced Fos
was unaffected through negative feedback involving actions in the pituitary. The incongruence
of the activity of CRH neurons with our results showing an attenuated HPA axis with chronic
salt-loading warrants further investigation.
Chronic salt-loading without restraint was associated with a significantly increased
number of Fos-positive nuclei in the caudal portion of the PVN. This region contains a large
population of neurons projecting to hindbrain and spinal cord autonomic centers (Biag et al.,
2012). The method of phenotyping PVN neurons based on position in the rostral-caudal extent is
a very general tool useful for a broad indication of the types of neurons involved with the
response to a particular stimulus. However, immunohistochemical and tract tracing molecules
directly provide evidence related to the function and destination of neuronal projections. Our
hypothesis that restraint-induced Fos in CRH neurons would be inhibited with chronic salt-
loading was not supported by the data we collected. However, the pattern of Fos activation
without respect to phenotypical marker provided some insight as to which populations of
neurons might be activated. These neurons were then specifically isolated in the experiments of
Chapter 5, which found that there was an increase in markers for vasopressin and CRH in
neurons projecting to the RVLM. Future experiments are necessary to determine the
consequences of this increase in expression.
134
One of the most striking differences between what we observed in the PVN and SON of
salt-loaded mice and previously published studies using rats was the lack of a robust alteration in
CRH expression. Our findings did include significant changes in digoxigen-labeled hybridized
probe as well as increases in colocalization of tdTomato reporting for CRH with vasopressin and
FG. However, based on the majority of the findings in the literature, we hypothesized that the
increase in CRH expression in magnocellular PVN and SON would be dramatic in a visually-
noticeable way. It may be that the other differences between what we found in mice and
previously published findings in rats (Watts et al., 1995) may provide insight into this disparity.
Most chronic salt-loading experiments included results that indicated dehydration-induced
anorexia, that is, coexistent markers for hypovolemia such as increased hematocrit along with
reduced body weight. Chronic salt-loading is associated with increased CRH mRNA expression
in the lateral hypothalamus, a region of the brain that is part of a feeding behavior network
(Watts, 1999), receives afferent projections from osmosensitive neurons located in the SFO
(Miselis, 1981), and sends efferent projections to the PVN (Watts, 1999). It is therefore possible
that without an increase in angiotensin-II caused by hypovolemia, and associated stimulation of
SFO neurons projecting to the lateral hypothalamus, the stimulation and alteration of anorexic
signals in the lateral hypothalamus may not occur. In the same way, a lack of stimulation in the
lateral hypothalamus could result in a lack of input to PVN neurons which may be involved with
a dramatic increase in CRH expression with salt-loading. Furthermore, knife cuts dissociating
the OVLT from the PVN prevented chronic salt-loading induced rearrangement of CRH
suggesting that signals in the plasma are necessary for causing the change (Kovacs and
Sawchenko, 1993). Together it seems that without adequate stimulation by hypovolemia-related
plasma-born substances acting in the circumventricular organs, neuropeptide rearrangement may
135
not occur or at least not occur in a dramatic way. Alternatively, there may be a species-related
difference that would prevent any such rearrangement from occurring under any circumstances,
and future studies would be necessary to confirm this.
Our results show that in mice CRH mRNA expression following five days of drinking
2% NaCl differs from the expression previously reported in rats and illustrates that CRH is
involved in both the neuroendocrine and autonomic reconfiguration caused by chronic salt-
loading. Our results together with previous reports (Amaya et al., 2001) suggest that salt-loading
reduces parvocellular CRH mRNA expression and increases expression of CRH and vasopressin
in neurons that project to the RVLM. The major implication is that this shift in expression is
coupled to increased sympathetic drive that may either maintain or elevate blood pressure during
the salt-loading period, and may contribute to long-lasting cardiovascular pathology. Sustained
exposure to either a stressful environment or chronic elevation of the pNa+ results in significant
risk for the development of psychiatric disorders or cardiovascular disease, respectively
(Chrousos and Gold, 1992; Strazzullo et al., 2009). Thus, the mechanisms that support the
changes in expression detailed here along with their respective components may be viable
therapeutic targets for intervention in certain disease states. The mechanisms that contribute to
the plasticity likely include changes on the transcriptional level. Increased transcription for CRH
may be induced via steroid signaling, as chronic salt-loading increases ACTH and CORT after
the first day before becoming suppressed (Elias et al., 2002). Activation of glucocorticoid
receptors in PVN parvocellular CRH neurons generally represents negative feedback of the HPA
axis (Herman et al., 2012); however, in other PVN neurons and areas of the brain, glucocorticoid
receptor activation increases CRH expression (Swanson and Simmons, 1989) in a similar way to
the increase caused by water deprivation (Watts and Sanchezwatts, 1995). It may be that the
136
increased number of RVLM-projecting neurons reporting for CRH in our study was also
glucocorticoid-mediated early in the salt-loading period. As discussed previously, additional
influences from osmosensitive regions of the brain also contribute to altered CRH expression and
the relationship between these influences and steroid signaling should be included in future
endeavors.
There are interesting similarities between the neuropeptide plasticity found in the current
study and that observed in postmortem tissue obtained from patients with cardiovascular or
affective disorders. For example, the increased colocalization of CRH and vasopressin that
occurred with salt-loading is also observed in patients that have died from complications arising
from chronic heart failure. Specifically, patients that died from chronic heart failure have twice
as many neurons colocalizing CRH and vasopressin in the PVN when compared to that of
controls (Sivukhina et al., 2009). Relative to normotensive individuals, patients that have died
from complications due to essential hypertension exhibit a profound increase in CRH mRNA
within discrete regions of the PVN (Goncharuk et al., 2007); alterations that are evocative of the
CRH plasticity that we found within PVN preautonomic neurons as a consequence of salt-
loading. Cardiovascular disease and affective disorders are highly comorbid (Vogelzangs et al.,
2010), and intriguingly, affective disorders are also accompanied by neuroendocrine and
autonomic adaptations predictive of neuropeptide plasticity in the PVN. That is, affective
disorders are associated with increased CRH and vasopressin expression in the PVN and SON as
well as sympatho-vagal imbalance (Raadsheer et al., 1994; Meynen et al., 2006; Kemp et al.,
2012). Here, we propose that chronic challenges to body fluid homeostasis lead to neuropeptide
plasticity in hypothalamic nuclei that promote endocrine and autonomic alterations that mitigate
137
the challenge, but progressively contribute to the etiology of cardiovascular and affective
disorders.
Our results show that in mice CRH mRNA expression following five days of drinking
2% NaCl differs from the expression previously reported in rats and further illustrates that CRH
is involved in both the neuroendocrine and autonomic reconfiguration caused by chronic salt-
loading. However, more detailed cellular investigation revealed that salt-loading increased CRH
expression in putative magnocellular vasopressinergic neurons of the PVN and increased
expression of CRH and vasopressin-NP in neurons that project to the RVLM. The major
implication is that this shift in expression is coupled to increased vasopressin release and
sympathetic drive that not only maintains blood tonicity and pressure during the salt-loading, but
also may contribute to the etiology of cardiovascular and affective disorders.
Closing Remarks
Relatively free, widespread access to potable water and an excess of salt are
contemporary additions to most economically advanced societies, representing only a small
fraction of human evolution. This suggests that the hydration-related instincts that for millennia
guided behavior, may be as limited in their usefulness to modern man as many misappropriated
aspects of the stress response. However, for many non-human species the centrally-mediated
mechanisms connecting hydromineral homeostasis with anxiety levels may have developed to
improve the likelihood of survival. Activation of the RAAS is associated with physiological
states such as sodium and volume depletion, situations which would also have the potential to
produce an anxious state. For example, species that do not consume meat have a dietary
imperative to locate and consume salt. Freely accessible salt may represent isolated areas that
may also be known to predators as a successful hunting area. Thus, the low-sodium state and
locating of a salt source may be hazardous and necessitate the increased alertness of heightened
138
anxiety. Furthermore, a mild hypernatremic state is relieved primarily by approaching a water
source that may also be frequented by a variety of species implying that the increased sociability
and decreased anxiety that accompanies central oxytocin release may be of survival value as
well. It may be that these evolutionarily conserved systems as well as the circuits that interact
with them may become dysregulated by a variety of genetic and environmental influences.
The understanding of a pathological process obviously begins with a clear understanding
of normal physiological function. Indeed, the development of our understanding of the RAAS
occupies a large segment of modern medical history which has revolutionized pharmacological
treatment for hypertension. Pushing the boundaries of this knowledge has uncovered local
tissue-specific systems with RAAS components as well as a counter-regulatory limb that will
likely result in novel therapeutic targets. Furthermore, it is clear that increased activation of the
RAAS promotes anxiety and potentiates the response to a psychogenic stressor. While existing
drugs have been shown to counteract these effects, it is still unclear exactly how these off-target
effects are accomplished. Similarly, oxytocin and vasopressin have well-characterized endocrine
effects in the body, but are just beginning to be thought of as centrally acting mediators of
behavior. Together, the mechanisms of salt and water homeostasis represent a toolbox for
uncovering what may be fundamental central processes with unrealized potential.
The primary advantages of studying the central effects of both the RAAS and the
neuropeptides oxytocin and vasopressin is that their systemic roles in normal physiological
function have been extensively studied and the routes by which they activate brain circuits are
well-understood. With these rigorously reviewed research findings at hand, it is possible to
manipulate the normal homeostatic mechanisms governing hydromineral balance for the purpose
of studying the effect on the brain circuits involved. Often this can be accomplished without the
139
potential confounds of introducing a foreign substance into the brain or facing the many
drawbacks of injecting a receptor blocker or agonist. Moreover, hydromineral balance is
primarily regulated by a complex series of behavioral adjustments involving motivation, reward,
social function, problem solving, and risk assessment. Given the diversity and far-reaching
implications of these areas of research, there exists a translational potential for basic science
understanding targeted at further revealing the mechanistic relationships between homeostatic
and stress-responsive systems.
The unpleasant anxious state associated with water deprivation is an obvious example of
how mood can be governed by hydromineral imbalance; however, the idea that the neurological
basis of the cravings for water, or salt for that matter, is somehow also the basis for the
pathogenesis of a clinical psychiatric disorder is beyond the scope of the present work.
Furthermore, to contend that the treatment of anxiety disorders, or psychiatric disorders in
general, can be trivialized to simply a monitoring and responding to the hydration state of tissues
is also not being suggested. In the extreme, such a connection does exist however, in the case of
the very serious condition of polydipsic schizophrenia in which patients are susceptible to water
intoxication (Goldman, Gnerlich, & Hussain, 2007). Less severe and more common are the
anecdotal cases of poor eating and drinking habits that result in transient states of subclinical
malaise due to mild dehydration or dysnatremias, as well as the relief that comes from satiating
thirst or satisfying a craving for salty food. Again, it would be easy to make the connection that
individuals use water or salt intake to self-medicate, consciously or unconsciously, in an effort to
alleviate the subjective effects of stress. While these are interesting and plausible ideas, none of
them are directly addressed here; rather, experimental evidence for mechanisms and circuits that
are reliably influenced by treatments involving water or salt are analyzed for the purpose of
140
understanding the parameters in which normal and dysfunctional stress-responsiveness occurs
and associated measures of anxiety are altered.
Future Directions
A major finding of these experiments was that the robust alteration in chronic salt-
loading induced CRH expression reported previously using a rat model did not occur in a
dramatic way using a mouse model. Therefore, repeating the experiments with increased
concentrations of saline to determine if the osmoregulatory capacity of mice prevented the
dynamic changes in CRH expression reported in rats is an important experiment.
We found that there was an increase in tdTomato/vasopressin colocalizations with salt-
loading. Future experiments will determine conclusively whether it is vasopressin increasing in
CRH parvocellular neurons or CRH increasing in vasopressin magnocellular neurons. One such
set of experiments would systematically measure soma diameter to classify neurons as
magnocellular or parvocellular. These experiments might include a method or methods that
could detect decreases in CRH such as standard IHC and in situ hybridization.
The time course of Cre expression is related to the expression of tdTomato reporting for
CRH which is increased with restraint. It may be possible to begin to determine this time course
experimentally in vitro by stimulating PVN neurons to induce tdTomato expression. For
example, this could be accomplished with time-lapse microscopy in an in vitro preparation of a
hypothalamic section bathed in artificial cerebrospinal fluid. While it has been determined that
CRH mRNA increases in response to restraint, it has not been determined experimentally how
long after a restraint-induced increase in CRH mRNA it takes for a rise in Cre and tdTomato. It
would be best to perform these experiments in young mice that had been protected from
exposure to stress as much as possible to limit the number of tdTomato neurons in the PVN.
While not a specific contribution to furthering the understanding of PVN plasticity that this
141
dissertation embodied, the technical advancement would include a better understanding of the
capabilities of a relatively new transgenic mouse model. Future experiments utilizing reporter
mice would benefit from knowing how long after stimulation it takes to induce Cre-mediated
excision of the STOP codon and subsequent expression of tdTomato.
Further experimentation will be aimed at exploring the mechanistic relationship between
increased glutamatergic innervation to the PVN with salt-loading (Fig. 6-1) and altered
neuropeptide expression. Qualitative images of astrocytic coverage suggest that retraction of
astrocytic processes during dehydration may influence synaptic physiology related to the
induction of plasticity in the PVN (Fig. 6-2).
142
Figure 6-1. Increased vesicular glutamate transporter 2 (vGlut-2) appositions on tdTomato
neurons in the PVN with salt-loading. The dorsal-medial parvocellular region of the
PVN (A) labeled for vGlut-2 (magenta) in CRH reporter mouse tissue (B). High
magnification images in the hydrated state (C1) and salt-loaded state (C2) were
analyzed and quantified. A significant increase in vGlut-2 appositions were found on
tdTomato neurons in the PVN (D).
A B
C1
C2
D
143
Figure 6-2. Decreased labeling for the astrocytic marker glial fibrillary acidic protein (GFAP)
around tdTomato reporting for CRH in the PVN with salt-loading.
B
A
144
LIST OF REFERENCES
Abousamra AB, Harwood JP, Catt KJ, Aguilera G. 1987. Mechanisms of Action of Crf and
Other Regulators of Acth Release in Pituitary Corticotrophs. Annals of the New York
Academy of Sciences 512:67-84.
Adachi T, Hisano S, Daikoku S. 1985. Intragranular Colocalization of Immunoreactive
Methionine Enkephalin and Oxytocin within the Nerve-Terminals in the Posterior
Pituitary. Journal of Histochemistry & Cytochemistry 33(9):891-899.
Aguilera G, Kiss A, Luo X. 1995a. Increased Expression of Type 1 Angiotensin II Receptors in
the Hypothalamic Paraventricular Nucleus following Stress and Glucocorticoid
Administration. Journal of Neuroendocrinology 7(10):775-783.
Aguilera G, Rabadan-Diehl C. 2000. Vasopressinergic regulation of the hypothalamic-pituitary-
adrenal axis: implications for stress adaptation. Regul Pept 96(1-2):23-29.
Aguilera G, Young WS, Kiss A, Bathia A. 1995b. Direct regulation of hypothalamic
corticotropin-releasing-hormone neurons by angiotensin II. Neuroendocrinology
61(4):437-444.
Akmayev IG. 1971. Morphological aspects of the hypothalamic-hypophyseal system. II.
Functional morphology of pituitary microcirculation. Z Zellforsch Mikrosk Anat
116(2):178-194.
Akmayev IG, Popov AP. 1977. Morphological Aspects of Hypothalamo-Hypophyseal System .7.
Tanycytes - Their Relation to Hypophyseal Adrenocorticotropic Function -
Ultrastructural-Study. Cell and Tissue Research 180(2):263-282.
Albrecht D. 2010. Physiological and pathophysiological functions of different angiotensins in the
brain. British Journal of Pharmacology 159(7):1392-1401.
Amaya F, Tanaka M, Hayashi S, Tanaka Y, Ibata Y. 2001. Hypothalamo-pituitary-adrenal axis
sensitization after chronic salt loading. Neuroendocrinology 73(3):185-193.
Amaya F, Tanaka M, Tamada Y, Tanaka Y, Nilaver G, Ibata Y. 1999. The influence of salt
loading on vasopressin gene expression in magno- and parvocellular hypothalamic
neurons: an immunocytochemical and in situ hybridization analysis. Neuroscience
89(2):515-523.
Andari E, Duhamel JR, Zalla T, Herbrecht E, Leboyer M, Sirigu A. 2010. Promoting social
behavior with oxytocin in high-functioning autism spectrum disorders. Proceedings of the
National Academy of Sciences of the United States of America 107(9):4389-4394.
Anil Kumar K, Nagwar S, Thyloor R, Satyanarayana S. 2014. Anti-stress and nootropic activity
of drugs affecting the renin-angiotensin system in rats based on indirect biochemical
evidence. Journal of the renin-angiotensin-aldosterone system : JRAAS.
145
Antunes-Rodrigues J, Ruginsk SG, Mecawi AS, Margatho LO, Cruz JC, Vilhena-Franco T, Reis
WL, Ventura RR, Reis LC, Vivas LM, Elias LL. 2013. Mapping and signaling of neural
pathways involved in the regulation of hydromineral homeostasis. Brazilian journal of
medical and biological research. 46(4):327-338.
Aoyagi T, Koshimizu TA, Tanoue A. 2009. Vasopressin regulation of blood pressure and
volume: findings from V1a receptor-deficient mice. Kidney Int 76(10):1035-1039.
Aplin KP, Suzuki H, Chinen AA, Chesser RT, Ten Have J, Donnellan SC, Austin J, Frost A,
Gonzalez JP, Herbreteau V, Catzeflis F, Soubrier J, Fang YP, Robins J, Matisoo-Smith E,
Bastos AD, Maryanto I, Sinaga MH, Denys C, Van Den Bussche RA, Conroy C, Rowe
K, Cooper A. 2011. Multiple geographic origins of commensalism and complex dispersal
history of Black Rats. Plos One 6(11):e26357.
Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB. 1998. The role of corticotropin-releasing
factor in depression and anxiety disorders. The Journal of endocrinology 160(1):1-12.
Armario A. 2006. The Hypothalamic-Pituitary-Adrenal Axis: What can it Tell us About
Stressors? CNS & Neurological Disorders - Drug Targets 5(5):485-501.
Atkinson IAE. 1985. The spread of commensal species of Rattus to oceanic islands and their
effects on island avifaunas. International Council for Bird Preservation Technical
Publication:35-81.
Badaue-Passos D, Jr., Godino A, Johnson AK, Vivas L, Antunes-Rodrigues J. 2007. Dorsal
raphe nuclei integrate allostatic information evoked by depletion-induced sodium
ingestion. Exp Neurol 206(1):86-94.
Baertschi AJ, Vallet PG. 1981. Osmosensitivity of the hepatic portal vein area and vasopressin
release in rats. The Journal of physiology 315:217-230.
Bains JS, Ferguson AV. 1993. Paraventricular nucleus neurons projecting to the spinal cord in
the rat are influenced by subfornical organ stimulation. Society for Neuroscience
Abstracts 19(1-3):956-956.
Bancila M, Giuliano F, Rampin O, Mailly P, Brisorgueil MJ, Calas A, Verge D. 2002. Evidence
for a direct projection from the paraventricular nucleus of the hypothalamus to putative
serotoninergic neurons of the nucleus paragigantocellularis involved in the control of
erection in rats. The European journal of neuroscience 16(7):1240-1248.
Bardgett ME, Chen QH, Guo Q, Calderon AS, Andrade MA, Toney GM. 2014. Coping with
dehydration: sympathetic activation and regulation of glutamatergic transmission in the
hypothalamic PVN. Am J Physiol Regul Integr Comp Physiol 306(11):R804-813.
146
Barnes V, Schneider R, Alexander C, Staggers F. 1997. Stress, stress reduction, and hypertension
in African Americans: an updated review. Journal of the National Medical Association
89(7):464-476.
Beinfeld MC, Meyer DK, Brownstein MJ. 1980. Cholecystokinin Octapeptide in the Rat
Hypothalamo-Neurohypophyseal System. Nature 288(5789):376-378.
Ben-Barak Y, Russell JT, Whitnall MH, Ozato K, Gainer H. 1985. Neurophysin in the
hypothalamo-neurohypophysial system. I. Production and characterization of monoclonal
antibodies. J Neurosci 5(1):81-97.
Benarroch EE. 2005. Paraventricular nucleus, stress response, and cardiovascular disease.
Clinical autonomic research : official journal of the Clinical Autonomic Research Society
15(4):254-263.
Benarroch EE. 2013a. Oxytocin and vasopressin Social neuropeptides with complex
neuromodulatory functions. Neurology 80(16):1521-1528.
Bernard C. 1949. An introduction to the study of experimental medicine. New York : Henry
Schuman, Inc.
Biag J, Huang Y, Gou L, Hintiryan H, Askarinam A, Hahn JD, Toga AW, Dong HW. 2012.
Cyto- and chemoarchitecture of the hypothalamic paraventricular nucleus in the
C57BL/6J male mouse: a study of immunostaining and multiple fluorescent tract tracing.
J Comp Neurol 520(1):6-33.
Biancardi VC, Stranahan AM, Krause EG, de Kloet AD, Stern JE. 2016. Cross talk between AT1
receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS
production in the hypothalamic paraventricular nucleus. Am J Physiol Heart Circ Physiol
310(3):H404-415.
Biller KJ, Unwin RJ, Shirley DG. 2000. Distal tubular electrolyte transport during inhibition of
renal 11beta-hydroxysteroid dehydrogenase. American journal of physiology Renal
physiology 280(1):9.
Bledsoe RK, Madauss KP, Holt JA, Apolito CJ, Lambert MH, Pearce KH, Stanley TB, Stewart
EL, Trump RP, Willson TM, Williams SP. 2005. A Ligand-mediated Hydrogen Bond
Network Required for the Activation of the Mineralocorticoid Receptor. Journal of
Biological Chemistry 280(35):31283-31293.
Boler J, Enzmann F, Folkers K, Bowers CY, Schally AV. 1969. The identity of chemical and
hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl-
proline amide. Biochem Biophys Res Commun 37(4):705-710.
147
Bondy CA, Gainer H. 1989. Corticotrophin-Releasing Hormone Stimulates Neurohypophyslal
Hormone Release through an Interaction with the Intermediate Lobe of the Pituitary. J
Neuroendocrinol 1(1):5-8.
Bondy CA, Whitnall MH, Brady LS, Gainer H. 1989. Coexisting Peptides in Hypothalamic
Neuroendocrine Systems - Some Functional Implications. Cell Mol Neurobiol 9(4):427-
446.
Bosch OJ, Dabrowska J, Modi ME, Johnson ZV, Keebaugh AC, Barrett CE, Ahern TH, Guo J,
Grinevich V, Rainnie DG, Neumann ID, Young LJ. 2016. Oxytocin in the nucleus
accumbens shell reverses CRFR2-evoked passive stress-coping after partner loss in
monogamous male prairie voles. Psychoneuroendocrinology 64:66-78.
Bota M, Sporns O, Swanson LW. 2012. Neuroinformatics analysis of molecular expression
patterns and neuron populations in gray matter regions: the rat BST as a rich exemplar.
Brain Res 1450:174-193.
Bourque CW. 2008. Central mechanisms of osmosensation and systemic osmoregulation. Nat
Rev Neurosci 9(7):519-531.
Boyle CN, Lorenzen SM, Compton D, Watts AG. 2012. Dehydration-anorexia derives from a
reduction in meal size, but not meal number. Physiol Behav 105(2):305-314.
Brazeau P, Ling N, Bohlen P, Esch F, Ying SY, Guillemin R. 1982. Growth-Hormone
Releasing-Factor, Somatocrinin, Releases Pituitary Growth-Hormone Invitro.
Proceedings of the National Academy of Sciences of the United States of America-
Biological Sciences 79(24):7909-7913.
Burgus R, Dunn TF, Desiderio D, Ward DN, Vale W, Guillemin R. 1970. Characterization of
ovine hypothalamic hypophysiotropic TSH-releasing factor. Nature 226(5243):321-325.
Burgus R, Ling N, Butcher M, Guillemi.R. 1973. Primary Structure of Somatostatin -
Hypothalamic Peptide That Inhibits Secretion of Pituitary Growth-Hormone. Proceedings
of the National Academy of Sciences of the United States of America 70(3):684-688.
Busnardo C, Alves FHF, Crestani CC, Scopinho AA, Resstel LBM, Correa FMA. 2013.
Paraventricular nucleus of the hypothalamus glutamate neurotransmission modulates
autonomic, neuroendocrine and behavioral responses to acute restraint stress in rats.
European Neuropsychopharmacology 23(11):1611-1622.
Byrum CE, Guyenet PG. 1987. Afferent and efferent connections of the A5 noradrenergic cell
group in the rat. J Comp Neurol 261(4):529-542.
Caeiro X, Vivas L. 2008. beta-Endorphin in the median preoptic nucleus modulates the pressor
response induced by subcutaneous hypertonic sodium chloride. Exp Neurol 210(1):59-66.
148
Calhoun DA. 1992. Hypertension in blacks: socioeconomic stress and sympathetic nervous
system activity. Am J Med Sci 304(5):306-311.
Calhoun DA, Oparil S. 1995. Racial differences in the pathogenesis of hypertension. Am J Med
Sci 310 Suppl 1:S86-90.
Cardoso C, Ellenbogen MA, Orlando MA, Bacon SL, Joober R. 2013. Intranasal oxytocin
attenuates the cortisol response to physical stress: a dose-response study.
Psychoneuroendocrinology 38(3):399-407.
Castel M, Morris JF. 1988. The neurophysin-containing innervation of the forebrain of the
mouse. Neuroscience 24(3):937-966.
Chan-Palay V, Zaborszky L, Kohler C, Goldstein M, Palay SL. 1984. Distribution of tyrosine-
hydroxylase-immunoreactive neurons in the hypothalamus of rats. J Comp Neurol
227(4):467-496.
Chen J, Gomez-Sanchez CE, Penman A, May PJ, Gomez-Sanchez E. 2014. Expression of
mineralocorticoid and glucocorticoid receptors in preautonomic neurons of the rat
paraventricular nucleus. Am J Physiol Regul Integr Comp Physiol 306(5):R328-340.
Chen Y, Molet J, Gunn BG, Ressler K, Baram TZ. 2015. Diversity of Reporter Expression
Patterns in Transgenic Mouse Lines Targeting Corticotropin-Releasing Hormone-
Expressing Neurons. Endocrinology 156(12):4769-4780.
Christensen EI, Grann B, Kristoffersen IB, Skriver E, Thomsen JS, Andreasen A. 2014a. Three-
dimensional reconstruction of the rat nephron. Am J Physiol Renal Physiol 306(6):F664-
671.
Chrousos GP, Gold PW. 1992. The concepts of stress and stress system disorders. Overview of
physical and behavioral homeostasis. Jama 267(9):1244-1252.
Clarke IJ, Cummins JT. 1982. The Temporal Relationship between Gonadotropin-Releasing
Hormone (Gnrh) and Luteinizing-Hormone (Lh) Secretion in Ovariectomized Ewes.
Endocrinology 111(5):1737-1739.
Coote JH. 1995. Cardiovascular function of the paraventricular nucleus of the hypothalamus.
Biological signals 4(3):142-149.
Coote JH. 2005. A role for the paraventricular nucleus of the hypothalamus in the autonomic
control of heart and kidney. Exp Physiol 90(2):169-173.
Cotterill RM. 1985. Fever in autistics. Nature 313(6002):426.
149
Cowley AW. 1997. Role of the renal medulla in volume and arterial pressure regulation.
American Journal of Physiology-Regulatory Integrative and Comparative Physiology
273(1):R1-R15.
Curran LK, Newschaffer CJ, Lee LC, Crawford SO, Johnston MV, Zimmerman AW. 2007.
Behaviors associated with fever in children with autism spectrum disorders. Pediatrics
120(6):e1386-1392.
Dabrowska J, Hazra R, Ahern TH, Guo JD, McDonald AJ, Mascagni F, Muller JF, Young LJ,
Rainnie DG. 2011. Neuroanatomical evidence for reciprocal regulation of the
corticotrophin-releasing factor and oxytocin systems in the hypothalamus and the bed
nucleus of the stria terminalis of the rat: Implications for balancing stress and affect.
Psychoneuroendocrinology 36(9):1312-1326.
Daniels D, Mietlicki EG, Nowak EL, Fluharty SJ. 2009. Angiotensin II stimulates water and
NaCl intake through separate cell signalling pathways in rats. Exp Physiol 94(1):130-137.
Davis M, Rainnie D, Cassell M. 1994. Neurotransmission in the rat amygdala related to fear and
anxiety. Trends Neurosci 17(5):208-214.
de Kloet ER, Joels M, Holsboer F. 2005. Stress and the brain: from adaptation to disease. Nat
Rev Neurosci 6(6):463-475.
De Luca LA, Menani JV, Johnson AK. 2013. Neurobiology of Body Fluid Homeostasis:
Transduction and Integration: Taylor & Francis Group.
De Vries GJ. 2004. Minireview: Sex differences in adult and developing brains: compensation,
compensation, compensation. Endocrinology 145(3):1063-1068.
de Wardener HE, He FJ, MacGregor GA. 2004. Plasma sodium and hypertension. Kidney Int
66(6):2454-2466.
Dimitrov EL, Yanagawa Y, Usdin TB. 2013. Forebrain GABAergic projections to locus
coeruleus in mouse. J Comp Neurol 521(10):2373-2397.
Dodhia S, Hosanagar A, Fitzgerald DA, Labuschagne I, Wood AG, Nathan PJ, Phan KL. 2014.
Modulation of resting-state amygdala-frontal functional connectivity by oxytocin in
generalized social anxiety disorder. Neuropsychopharmacology 39(9):2061-2069.
Domes G, Heinrichs M, Kumbier E, Grossmann A, Hauenstein K, Herpertz SC. 2013. Effects of
intranasal oxytocin on the neural basis of face processing in autism spectrum disorder.
Biological psychiatry 74(3):164-171.
Dong HW, Petrovich GD, Watts AG, Swanson LW. 2001. Basic organization of projections
from the oval and fusiform nuclei of the bed nuclei of the stria terminalis in adult rat
brain. J Comp Neurol 436(4):430-455.
150
Dong HW, Swanson LW. 2006. Projections from bed nuclei of the stria terminalis, dorsomedial
nucleus: implications for cerebral hemisphere integration of neuroendocrine, autonomic,
and drinking responses. J Comp Neurol 494(1):75-107.
Dooley CP, Valenzuela JE. 1984. Duodenal volume and osmoreceptors in the stimulation of
human pancreatic secretion. Gastroenterology 86(1):23-27.
Duchen LW. 1962. Effects of Ingestion of Hypertonic Saline on Pituitary Gland in Rat -
Morphological Study of Pars Intermedia and Posterior Lobe. Journal of Endocrinology
25(2):161-&.
Dunn AJ, Berridge CW. 1990. Physiological and behavioral responses to corticotropin-releasing
factor administration: is CRF a mediator of anxiety or stress responses? Brain research
reviews 15(2):71-100.
Duvernoy HM, Risold PY. 2007. The circumventricular organs: an atlas of comparative anatomy
and vascularization. Brain Res Rev 56(1):119-147.
Elias LL, Dorival Campos A, Moreira AC. 2002. The opposite effects of short- and long-term
salt loading on pituitary adrenal axis activity in rats. Horm Metab Res 34(4):207-211.
Englemann M, Wotjak CT, Ebner K, Landgraf R. 2000. Behavioural impact of intraseptally
released vasopressin and oxytocin in rats. Experimental Physiology 85(s1):125s-130s.
Epstein DE, Sherwood A, Smith PJ, Craighead L, Caccia C, Lin PH, Babyak MA, Johnson JJ,
Hinderliter A, Blumenthal JA. 2012. Determinants and consequences of adherence to the
dietary approaches to stop hypertension diet in African-American and white adults with
high blood pressure: results from the ENCORE trial. J Acad Nutr Diet 112(11):1763-
1773.
Fang Z, Carlson SH, Peng N, Wyss JM. 2000. Circadian rhythm of plasma sodium is disrupted
in spontaneously hypertensive rats fed a high-NaCl diet. Am J Physiol Regul Integr
Comp Physiol 278(6):R1490-1495.
Ferguson AV, Latchford KJ, Samson WK. 2008. The paraventricular nucleus of the
hypothalamus - a potential target for integrative treatment of autonomic dysfunction.
Expert Opin Ther Targets 12(6):717-727.
Fisher JP, Paton JF. 2012. The sympathetic nervous system and blood pressure in humans:
implications for hypertension. J Hum Hypertens 26(8):463-475.
Fitzsimons JT. 1998. Angiotensin, thirst, and sodium appetite. Physiol Rev 78(3):583-686.
151
Francis DD, Young LJ, Meaney MJ, Insel TR. 2002. Naturally occurring differences in maternal
care are associated with the expression of oxytocin and vasopressin (V1a) receptors:
gender differences. J Neuroendocrinol 14(5):349-353.
Franklin KBJ, Paxinos G. 2008. The Mouse Brain in Stereotaxic Coordinates, Compact Third
Edition. Amsterdam: Boston.
Frazier CJ, Pati D, Hiller H, Nguyen D, Wang L, Smith JA, MacFadyen K, de Kloet AD, Krause
EG. 2013. Acute hypernatremia exerts an inhibitory oxytocinergic tone that is associated
with anxiolytic mood in male rats. Endocrinology 154(7):2457-2467.
Freeman ME, Kanyicska B, Lerant A, Nagy G. 2000. Prolactin: structure, function, and
regulation of secretion. Physiol Rev 80(4):1523-1631.
Fuxe K, Agnati LF, Ganten D, Lang RE, Calza L, Poulsen K, Infantellina F. 1982.
Morphometric evaluation of the coexistence of renin-like and oxytocin-like
immunoreactivity in nerve cells of the paraventricular hypothalamic nucleus of the rat.
Neuroscience letters 33(1):19-24.
Gabor A, Leenen FH. 2012. Central neuromodulatory pathways regulating sympathetic activity
in hypertension. J Appl Physiol (1985) 113(8):1294-1303.
Gaillard RC, Grossman A, Gillies G, Rees LH, Besser GM. 1981. Angiotensin II stimulates the
release of ACTH from dispersed rat anterior pituitary cells. Clin Endocrinol (Oxf)
15(6):573-578.
Ganesan R, Sumners C. 1989. Glucocorticoids potentiate the dipsogenic action of angiotensin II.
Brain Res 499(1):121-130.
Gaymann W, Martin R. 1989. Immunoreactive Galanin-Like Material in Magnocellular
Hypothalamoneurohypophysial Neurons of the Rat. Cell and Tissue Research
255(1):139-147.
Geerling JC, Loewy AD. 2006. Aldosterone-sensitive neurons in the nucleus of the solitary tract:
bidirectional connections with the central nucleus of the amygdala. J Comp Neurol
497(4):646-657.
Geerling JC, Loewy AD. 2009. Aldosterone in the brain. Am J Physiol Renal Physiol
297(3):F559-576.
Geerling JC, Shin JW, Chimenti PC, Loewy AD. 2010. Paraventricular hypothalamic nucleus:
axonal projections to the brainstem. J Comp Neurol 518(9):1460-1499.
Gillies GE, Linton EA, Lowry PJ. 1982. Corticotropin Releasing Activity of the New Crf Is
Potentiated Several Times by Vasopressin. Nature 299(5881):355-357.
152
Gomez-Sanchez EP, Ahmad N, Romero DG, Gomez-Sanchez CE. 2005. Is aldosterone
synthesized within the rat brain? American journal of physiology Endocrinology and
metabolism 288(2):E342-346.
Gomez RE, Cannata MA, Milner TA, Anwar M, Reis DJ, Ruggiero DA. 1993. Vasopressinergic
mechanisms in the nucleus reticularis lateralis in blood pressure control. Brain Res
604(1-2):90-105.
Goncharuk VD, Buijs RM, Swaab DF. 2007. Corticotropin-releasing hormone neurons in
hypertensive patients are activated in the hypothalamus but not in the brainstem. J Comp
Neurol 503(1):148-168.
Goodwin FJ, Ledingham JG, Laragh JH. 1970. The effects of prolonged administration of
vasopressin and oxytocin on renin, aldosterone and sodium balance in normal man. Clin
Sci 39(5):641-651.
Gouzenes L, Desarmenien MG, Hussy N, Richard P, Moos FC. 1998. Vasopressin regularizes
the phasic firing pattern of rat hypothalamic magnocellular vasopressin neurons. J
Neurosci 18(5):1879-1885.
Grinevich V, Ma XM, Verbalis J, Aguilera G. 2001. Hypothalamic pituitary adrenal axis and
hypothalamic-neurohypophyseal responsiveness in water-deprived rats. Exp Neurol
171(2):329-341.
Grippo AJ, Francis J, Beltz TG, Felder RB, Johnson AK. 2005. Neuroendocrine and cytokine
profile of chronic mild stress-induced anhedonia. Physiol Behav 84(5):697-706.
Guastella AJ, Einfeld SL, Gray KM, Rinehart NJ, Tonge BJ, Lambert TJ, Hickie IB. 2010.
Intranasal oxytocin improves emotion recognition for youth with autism spectrum
disorders. Biological psychiatry 67(7):692-694.
Guo J, Dabrowska J, Hazra R, Rainnie DG. 2013. CRF neurons in the paraventricular nucleus
and the bed nucleus of the stria terminalis: distinct physiological properties and genetic
phenotypes. Society for Neuroscience Abstract Viewer and Itinerary Planner 43.
Guyenet PG. 2006. The sympathetic control of blood pressure. Nat Rev Neurosci 7(5):335-346.
Hafner S, Baumert J, Emeny RT, Lacruz ME, Bidlingmaier M, Reincke M, Ladwig KH. 2013.
Hypertension and depressed symptomatology: a cluster related to the activation of the
renin-angiotensin-aldosterone system (RAAS). Findings from population based KORA
F4 study. Psychoneuroendocrinology 38(10):2065-2074.
Hainsworth R. 2014. Cardiovascular control from cardiac and pulmonary vascular receptors. Exp
Physiol 99(2):312-319.
153
Haque M, Wilson R, Sharma K, Mills NJ, Teruyama R. 2015. Localisation of 11beta-
Hydroxysteroid Dehydrogenase Type 2 in Mineralocorticoid Receptor Expressing
Magnocellular Neurosecretory Neurones of the Rat Supraoptic and Paraventricular
Nuclei. J Neuroendocrinol 27(11):835-849.
Hartner A, Cordasic N, Klanke B, Veelken R, Hilgers KF. 2003. Strain differences in the
development of hypertension and glomerular lesions induced by deoxycorticosterone
acetate salt in mice. Nephrology, dialysis, transplantation : official publication of the
European Dialysis and Transplant Association - European Renal Association
18(10):1999-2004.
Hattangady NG, Olala LO, Bollag WB, Rainey WE. 2012. Acute and chronic regulation of
aldosterone production. Mol Cell Endocrinol 350(2):151-162.
Hattori T, Morris M, Alexander N, Sundberg DK. 1990. Extracellular oxytocin in the
paraventricular nucleus: hyperosmotic stimulation by in vivo microdialysis. Brain
Research 506(1):169-171.
He FJ, Markandu ND, MacGregor GA. 2005. Modest salt reduction lowers blood pressure in
isolated systolic hypertension and combined hypertension. Hypertension 46(1):66-70.
Heilig CW, Stromski ME, Blumenfeld JD, Lee JP, Gullans SR. 1989. Characterization of the
major brain osmolytes that accumulate in salt-loaded rats. Am J Physiol 257(6 Pt
2):F1108-1116.
Herman JP, Cullinan WE. 1997. Neurocircuitry of stress: Central control of the hypothalamo-
pituitary-adrenocortical axis. Trends in Neurosciences 20(2):78-84.
Herman JP, McKlveen JM, Solomon MB, Carvalho-Netto E, Myers B. 2012. Neural regulation
of the stress response: glucocorticoid feedback mechanisms. Brazilian journal of medical
and biological research = Revista brasileira de pesquisas medicas e biologicas /
Sociedade Brasileira de Biofisica [et al] 45(4):292-298.
Hinks GL, Poat JA, Hughes J. 1995. Changes in Hypothalamic Cholecystokinin(a) and
Cholecystokinin(B) Receptor Subtypes and Associated Neuropeptide Expression in
Response to Salt-Stress in the Rat and Mouse. Neuroscience 68(3):765-781.
Hiroyuki K, Hirohito M, Tsutomu M, Akira N. 2013. Angiotensin II blockade and renal
protection. Current pharmaceutical design 19(17):3033-3042.
Hoffman GE, Smith MS, Verbalis JG. 1993. c-Fos and related immediate early gene products as
markers of activity in neuroendocrine systems. Front Neuroendocrinol 14(3):173-213.
154
Hollis JH, McKinley MJ, D'Souza M, Kampe J, Oldfield BJ. 2008. The trajectory of sensory
pathways from the lamina terminalis to the insular and cingulate cortex: a
neuroanatomical framework for the generation of thirst. Am J Physiol Regul Integr Comp
Physiol 294(4):R1390-1401.
Huang CC, Chu CY, Yeh CM, Hsu KS. 2014. Acute hypernatremia dampens stress-induced
enhancement of long-term potentiation in the dentate gyrus of rat hippocampus.
Psychoneuroendocrinology 46:129-140.
Husain A, DeSilva P, Speth RC, Bumpus FM. 1987. Regulation of angiotensin II in rat adrenal
gland. Circ Res 60(5):640-648.
Hwang BH, Wu JY, Severs WB. 1986. Effects of chronic dehydration on angiotensin II receptor
binding in the subfornical organ, paraventricular hypothalamic nucleus and adrenal
medulla of Long-Evans rats. Neuroscience letters 65(1):35-40.
Imaki T, Katsumata H, Konishi SI, Kasagi Y, Minami S. 2003. Corticotropin-releasing factor
type-1 receptor mRNA is not induced in mouse hypothalamus by either stress or osmotic
stimulation. Journal of Neuroendocrinology 15(10):916-924.
Imaki T, Katsumata H, Miyata M, Naruse M, Imaki J, Minami S. 2001. Expression of
corticotropin releasing factor (CRF), urocortin and CRF type 1 receptors in
hypothalamic-hypophyseal systems under osmotic stimulation. Journal of
Neuroendocrinology 13(4):328-338.
Insel TR, Winslow JT, Wang ZX, Young LJ. 1998. Oxytocin, vasopressin, and the
neuroendocrine basis of pair bond formation. In: Zingg HH, Bourque CW, Bichet DG,
eds. Vasopressin and Oxytocin: Molecular, Cellular, and Clinical Advances. Vol 449.
Advances in Experimental Medicine and Biology. p 215-224.
Jacobson L. 2014. Hypothalamic-pituitary-adrenocortical axis: neuropsychiatric aspects. Compr
Physiol 4(2):715-738.
Jankord R, Herman JP. 2008. Limbic regulation of hypothalamo-pituitary-adrenocortical
function during acute and chronic stress. Ann N Y Acad Sci 1148(1):64-73.
Johnson AK, Gross PM. 1993. Sensory circumventricular organs and brain homeostatic
pathways. FASEB journal : official publication of the Federation of American Societies
for Experimental Biology 7(8):678-686.
Kawabe T, Chitravanshi VC, Kawabe K, Sapru HN. 2008. Cardiovascular function of a
glutamatergic projection from the hypothalamic paraventricular nucleus to the nucleus
tractus solitarius in the rat. Neuroscience 153(3):605-617.
155
Kc P, Balan KV, Tjoe SS, Martin RJ, Lamanna JC, Haxhiu MA, Dick TE. 2010. Increased
vasopressin transmission from the paraventricular nucleus to the rostral medulla
augments cardiorespiratory outflow in chronic intermittent hypoxia-conditioned rats. The
Journal of physiology 588(Pt 4):725-740.
Kc P, Haxhiu MA, Tolentino-Silva FP, Wu M, Trouth CO, Mack SO. 2002. Paraventricular
vasopressin-containing neurons project to brain stem and spinal cord respiratory-related
sites. Respir Physiol Neurobiol 133(1-2):75-88.
Keller-Wood ME, Dallman MF. 1984. Corticosteroid inhibition of ACTH secretion. Endocrine
reviews 5(1):1-24.
Kemp AH, Quintana DS, Felmingham KL, Matthews S, Jelinek HF. 2012. Depression, comorbid
anxiety disorders, and heart rate variability in physically healthy, unmedicated patients:
implications for cardiovascular risk. Plos One 7(2):e30777.
Ken'ichi Y, Hitoshi H. 2010. Changes in vasopressin release and autonomic function induced by
manipulating forebrain GABAergic signaling under euvolemia and hypovolemia in
conscious rats. Endocrine journal 58(7):559-573.
Kilcoyne MM, Hoffman DL, Zimmerman EA. 1980. Immuno-Cytochemical Localization of
Angiotensin-Ii and Vasopressin in Rat Hypothalamus - Evidence for Production in the
Same Neuron. Clinical science 59:S57-S60.
Kim SY, Adhikari A, Lee SY, Marshel JH, Kim CK, Mallory CS, Lo M, Pak S, Mattis J, Lim
BK, Malenka RC, Warden MR, Neve R, Tye KM, Deisseroth K. 2013. Diverging neural
pathways assemble a behavioural state from separable features in anxiety. Nature
496(7444):219-223.
Kirchgessner AL, Sclafani A, Nilaver G. 1988. Histochemical identification of a PVN-hindbrain
feeding pathway. Physiol Behav 42(6):529-543.
Knobloch HS, Charlet A, Hoffmann LC, Eliava M, Khrulev S, Cetin AH, Osten P, Schwarz MK,
Seeburg PH, Stoop R, Grinevich V. 2012. Evoked axonal oxytocin release in the central
amygdala attenuates fear response. Neuron 73(3):553-566.
Kolaj M, Bai D, Renaud LP. 2004. GABAB receptor modulation of rapid inhibitory and
excitatory neurotransmission from subfornical organ and other afferents to median
preoptic nucleus neurons. J Neurophysiol 92(1):111-122.
Kolaj M, Renaud LP. 2007. Presynaptic alpha-adrenoceptors in median preoptic nucleus
modulate inhibitory neurotransmission from subfornical organ and organum vasculosum
lamina terminalis. Am J Physiol Regul Integr Comp Physiol 292(5):R1907-1915.
Koletsky S. 1958. Hypertensive vascular disease produced by salt. Laboratory investigation; a
journal of technical methods and pathology 7(4):377-386.
156
Koletsky S. 1959. Role of salt and renal mass in experimental hypertension. AMA archives of
pathology 68(1):11-22.
Koletsky S. 1961. Pathogenesis of experimental hypertension induced by salt. Am J Cardiol
8(4):576-581.
Kovacs KJ, Sawchenko PE. 1993. Mediation of osmoregulatory influences on neuroendocrine
corticotropin-releasing factor expression by the ventral lamina terminalis. Proceedings of
the National Academy of Sciences of the United States of America 90(16):7681-7685.
Krause EG, de Kloet AD, Flak JN, Smeltzer MD, Solomon MB, Evanson NK, Woods SC, Sakai
RR, Herman JP. 2011a. Hydration state controls stress responsiveness and social
behavior. J Neurosci 31(14):5470-5476.
Krause EG, de Kloet AD, Scott KA, Flak JN, Jones K, Smeltzer MD, Ulrich-Lai YM, Woods
SC, Wilson SP, Reagan LP, Herman JP, Sakai RR. 2011b. Blood-borne angiotensin II
acts in the brain to influence behavioral and endocrine responses to psychogenic stress. J
Neurosci 31(42):15009-15015.
Krause EG, Melhorn SJ, Davis JF, Scott KA, Ma LY, de Kloet AD, Benoit SC, Woods SC,
Sakai RR. 2008. Angiotensin type 1 receptors in the subfornical organ mediate the
drinking and hypothalamic-pituitary-adrenal response to systemic isoproterenol.
Endocrinology 149(12):6416-6424.
Kriz W. 1967. Der Architektonische Und Funktionelle Aufbau Der Rattenniere. Zeitschrift Fur
Zellforschung Und Mikroskopische Anatomie 82(4):495-&.
Kuramochi G, Kobayashi I. 2000. Regulation of the urine concentration mechanism by the
oropharyngeal afferent pathway in man. Am J Nephrol 20(1):42-47.
Labrie F, Giguere V, Proulx L, Lefevre G. 1984. Interactions between Crf, Epinephrine,
Vasopressin and Glucocorticoids in the Control of Acth-Secretion. Journal of Steroid
Biochemistry and Molecular Biology 20(1):153-160.
Landgraf R, Neumann ID. 2004. Vasopressin and oxytocin release within the brain: a dynamic
concept of multiple and variable modes of neuropeptide communication. Front
Neuroendocrinol 25(3-4):150-176.
Laragh JH, Angers M, Kelly WG, Lieberman S. 1960. Hypotensive agents and pressor
substances. The effect of epinephrine, norepinephrine, angiotensin II, and others on the
secretory rate of aldosterone in man. Jama 174(3):234-240.
Lee SK, Ryu PD, Lee SY. 2013. Differential distributions of neuropeptides in hypothalamic
paraventricular nucleus neurons projecting to the rostral ventrolateral medulla in the rat.
Neuroscience letters 556:160-165.
157
Leong DS, Terron JA, Falcon-Neri A, Armando I, Ito T, Johren O, Tonelli LH, Hoe KL,
Saavedra JM. 2002. Restraint stress modulates brain, pituitary and adrenal expression of
angiotensin II AT(1A), AT(1B) and AT(2) receptors. Neuroendocrinology 75(4):227-
240.
Leshem M. 2011. Low dietary sodium is anxiogenic in rats. Physiology & Behavior 103(5):453-
458.
Li YF, Jackson KL, Stern JE, Rabeler B, Patel KP. 2006. Interaction between glutamate and
GABA systems in the integration of sympathetic outflow by the paraventricular nucleus
of the hypothalamus. Am J Physiol Heart Circ Physiol 291(6):H2847-2856.
Li ZH, Ferguson AV. 1993. Subfornical Organ Efferents to Paraventricular Nucleus Utilize
Angiotensin as a Neurotransmitter. American Journal of Physiology 265(2):R302-R309.
Lightman SL. 2008. The neuroendocrinology of stress: a never ending story. J Neuroendocrinol
20(6):880-884.
Lightman SL, Young WS. 1987. Vasopressin, Oxytocin, Dynorphin, Enkephalin and
Corticotropin-Releasing Factor Messenger-Rna Stimulation in the Rat. Journal of
Physiology-London 394:23-39.
Llewellyn T, Zheng H, Liu X, Xu B, Patel KP. 2012. Median preoptic nucleus and subfornical
organ drive renal sympathetic nerve activity via a glutamatergic mechanism within the
paraventricular nucleus. Am J Physiol Regul Integr Comp Physiol 302(4):R424-432.
Loewy AD, McKellar S. 1980. The neuroanatomical basis of central cardiovascular control. Fed
Proc 39(8):2495-2503.
Lucas M, Chen A, Richter-Levin G. 2013. Hypothalamic corticotropin-releasing factor is
centrally involved in learning under moderate stress. Neuropsychopharmacology
38(9):1825-1832.
Ludwig M, Callahan MF, Neumann I, Landgraf R, Morris M. 1994. Systemic Osmotic
Stimulation Increases Vasopressin and Oxytocin Release Within the Supraoptic Nucleus.
Journal of Neuroendocrinology 6(4):369-373.
Ludwig M, Leng G. 1997. Autoinhibition of supraoptic nucleus vasopressin neurons in vivo: a
combined retrodialysis/electrophysiological study in rats. The European journal of
neuroscience 9(12):2532-2540.
Ludwig M, Leng G. 2006. Dendritic peptide release and peptide-dependent behaviours. Nat Rev
Neurosci 7(2):126-136.
158
Mabrouk OS, Kennedy RT. 2012. Simultaneous oxytocin and arg-vasopressin measurements in
microdialysates using capillary liquid chromatography-mass spectrometry. J Neurosci
Methods 209(1):127-133.
Macdonald K, Feifel D. 2014. Oxytocin’s role in anxiety: A critical appraisal. Brain Res
1580:22-56
Madden CJ, Morrison SF. 2009. Neurons in the paraventricular nucleus of the hypothalamus
inhibit sympathetic outflow to brown adipose tissue. Am J Physiol Regul Integr Comp
Physiol 296(3):R831-843.
Majzoub JA, Rich A, Vanboom J, Habener JF. 1983. Vasopressin and Oxytocin Messenger-Rna
Regulation in the Rat Assessed by Hybridization with Synthetic Oligonucleotides.
Journal of Biological Chemistry 258(23):4061-4064.
Mak P, Broussard C, Vacy K, Broadbear JH. 2012. Modulation of anxiety behavior in the
elevated plus maze using peptidic oxytocin and vasopressin receptor ligands in the rat.
Journal of psychopharmacology (Oxford, England) 26(4):532-542.
Makara GB, Kiss A, Lolait SJ, Aguilera G. 1996. Hypothalamic-pituitary corticotroph function
after shunting of magnocellular vasopressin and oxytocin to the hypophyseal portal
circulation. Endocrinology 137(2):580-586.
Martin R, Geis R, Holl R, Schafer M, Voigt KH. 1983. Co-Existence of Unrelated Peptides in
Oxytocin and Vasopressin Terminals of Rat Neurohypophyses - Immunoreactive
Methionines-Enkephalin-Like,Leucine5-Enkephalin-Like and Cholecystokinin-Like
Substances. Neuroscience 8(2):213-227.
Martin R, Voigt KH. 1981. Enkephalins co-exist with oxytocin and vasopressin in nerve
terminals of rat neurohypophysis. Nature 289(5797):502-504.
Masaharu N, Hiraki S. 2013. Central regulation of body-fluid homeostasis. Trends Neurosci
36(11):661-673.
McKinley MJ, Gerstberger R, Mathai ML, Oldfield BJ, Schmid H. 1999. The lamina terminalis
and its role in fluid and electrolyte homeostasis. J Clin Neurosci 6(4):289-301.
McKinley MJ, Mathai ML, McAllen RM, McClear RC, Miselis RR, Pennington GL, Vivas L,
Wade JD, Oldfield BJ. 2004. Vasopressin secretion: osmotic and hormonal regulation by
the lamina terminalis. J Neuroendocrinol 16(4):340-347.
McKinley MJ, McAllen RM, Davern P, Giles ME, Penschow J, Sunn N, Uschakov A, Oldfield
BJ. 2002. The sensory circumventricular organs of the mammalian brain: subfornical
organ, OVLT and area postrema. Berlin: Springer-Verlag.
159
Mecawi Ade S, Ruginsk SG, Elias LL, Varanda WA, Antunes-Rodrigues J. 2015.
Neuroendocrine Regulation of Hydromineral Homeostasis. Compr Physiol 5(3):1465-
1516.
Melander T, Hokfelt T, Rokaeus A. 1986a. Distribution of galaninlike immunoreactivity in the
rat central nervous system. J Comp Neurol 248(4):475-517.
Melander T, Hokfelt T, Rokaeus A, Cuello AC, Oertel WH, Verhofstad A, Goldstein M. 1986b.
Coexistence of galanin-like immunoreactivity with catecholamines, 5-
hydroxytryptamine, GABA and neuropeptides in the rat CNS. J Neurosci 6(12):3640-
3654.
Meneely GR, Ball CO. 1958. Experimental epidemiology of chronic sodium chloride toxicity
and the protective effect of potassium chloride. The American journal of medicine
25(5):713-725.
Menendez JA, Mcgregor IS, Healey PA, Atrens DM, Leibowitz SF. 1990. Metabolic Effects of
Neuropeptide-Y Injections into the Paraventricular Nucleus of the Hypothalamus. Brain
research 516(1):8-14.
Meynen G, Unmehopa UA, van Heerikhuize JJ, Hofman MA, Swaab DF, Hoogendijk WJ. 2006.
Increased arginine vasopressin mRNA expression in the human hypothalamus in
depression: A preliminary report. Biol Psychiatry 60(8):892-895.
Milner TA, Reis DJ, Pickel VM, Aicher SA, Giuliano R. 1993. Ultrastructural localization and
afferent sources of corticotropin-releasing factor in the rat rostral ventrolateral medulla:
implications for central cardiovascular regulation. J Comp Neurol 333(2):151-167.
Miselis RR. 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.
Morita M, Kita Y, Notsu Y. 2001. Mechanism of AVP release and synthesis in chronic salt-
loaded rats. The Journal of pharmacy and pharmacology 53(12):1703-1709.
Murck H, Held K, Ziegenbein M, Kunzel H, Koch K, Steiger A. 2003a. The renin-angiotensin-
aldosterone system in patients with depression compared to controls--a sleep endocrine
study. BMC psychiatry 3:15.
Murck H, Schussler P, Steiger A. 2012. Renin-angiotensin-aldosterone system: the forgotten
stress hormone system: relationship to depression and sleep. Pharmacopsychiatry
45(3):83-95.
Neumann ID, Landgraf R. 2012b. Balance of brain oxytocin and vasopressin: implications for
anxiety, depression, and social behaviors. Trends in Neurosciences 35(11):649-659.
160
Niebylski A, Boccolini A, Bensi N, Binotti S, Hansen C, Yaciuk R, Gauna H. 2012.
Neuroendocrine Changes and Natriuresis in Response to Social Stress in Rats. Stress and
Health 28(3):179-185.
O'Donaughy TL, Brooks VL. 2006. Deoxycorticosterone acetate-salt rats: hypertension and
sympathoexcitation driven by increased NaCl levels. Hypertension 47(4):680-685.
O'Donaughy TL, Qi Y, Brooks VL. 2006. Central action of increased osmolality to support
blood pressure in deoxycorticosterone acetate-salt rats. Hypertension 48(4):658-663.
Oka Y, Ye M, Zuker CS. 2015. Thirst driving and suppressing signals encoded by distinct neural
populations in the brain. Nature 520(7547):349-352.
Ozaki Y, Nomura M, Saito J, Luedke CE, Muglia LJ, Matsumoto T, Ogawa S, Ueta Y, Pfaff
DW. 2004. Expression of the arginine vasopressin gene in response to salt loading in
oxytocin gene knockout mice. J Neuroendocrinol 16(1):39-44.
Papanek PE, Sladek CD, Raff H. 1997. Corticosterone inhibition of osmotically stimulated
vasopressin from hypothalamic-neurohypophysial explants. Am J Physiol 272(1 Pt
2):R158-162.
Peter K. 1927. Studies on construction and development of the kidney. Jena: G. Fischer.
Peters S, Slattery DA, Uschold-Schmidt N, Reber SO, Neumann ID. 2014. Dose-dependent
effects of chronic central infusion of oxytocin on anxiety, oxytocin receptor binding and
stress-related parameters in mice. Psychoneuroendocrinology 42:225-236.
Phillips MI, Sumners C. 1998. Angiotensin II in central nervous system physiology. Regul Pept
78(1-3):1-11.
Pirnik Z, Mravec B, Kiss A. 2004. Fos protein expression in mouse hypothalamic paraventricular
(PVN) and supraoptic (SON) nuclei upon osmotic stimulus: colocalization with
vasopressin, oxytocin, and tyrosine hydroxylase. Neurochem Int 45(5):597-607.
Pleil KE, Rinker JA, Lowery-Gionta EG, Mazzone CM, McCall NM, Kendra AM, Olson DP,
Lowell BB, Grant KA, Thiele TE, Kash TL. 2015. NPY signaling inhibits extended
amygdala CRF neurons to suppress binge alcohol drinking. Nat Neurosci 18(4):545-552.
Polito AB, 3rd, Goldstein DL, Sanchez L, Cool DR, Morris M. 2006. Urinary oxytocin as a non-
invasive biomarker for neurohypophyseal hormone secretion. Peptides 27(11):2877-
2884.
Potapenko ES, Biancardi VC, Zhou Y, Stern JE. 2013. Astrocytes modulate a postsynaptic
NMDA-GABAA-receptor crosstalk in hypothalamic neurosecretory neurons. J Neurosci
33(2):631-640.
161
Poulain DA, Wakerley JB. 1982. Electrophysiology of Hypothalamic Magnocellular Neurons
Secreting Oxytocin and Vasopressin. Neuroscience 7(4):773-808.
Pretel S, Piekut DT. 1989. Mediation of changes in paraventricular vasopressin and oxytocin
mRNA content to the medullary vagal complex and spinal cord of the rat. J Chem
Neuroanat 2(6):327-334.
Pyner S. 2009. Neurochemistry of the paraventricular nucleus of the hypothalamus: implications
for cardiovascular regulation. J Chem Neuroanat 38(3):197-208.
Raadsheer FC, Hoogendijk WJ, Stam FC, Tilders FJ, Swaab DF. 1994. Increased numbers of
corticotropin-releasing hormone expressing neurons in the hypothalamic paraventricular
nucleus of depressed patients. Neuroendocrinology 60(4):436-444.
Rasmussen MS, Simonsen JA, Sandgaard NC, Hoilund-Carlsen PF, Bie P. 2004. Effects of
oxytocin in normal man during low and high sodium diets. Acta Physiol Scand
181(2):247-257.
Rivalland ET, Tilbrook AJ, Turner AI, Iqbal J, Pompolo S, Clarke IJ. 2006. Projections to the
preoptic area from the paraventricular nucleus, arcuate nucleus and the bed nucleus of the
stria terminalis are unlikely to be involved in stress-induced suppression of GnRH
secretion in sheep. Neuroendocrinology 84(1):1-13.
Rivest S, Laflamme N, Nappi RE. 1995. Immune challenge and immobilization stress induce
transcription of the gene encoding the CRF receptor in selective nuclei of the rat
hypothalamus. J Neurosci 15(4):2680-2695.
Rivier C, Vale W. 1983. Modulation of stress-induced ACTH release by corticotropin-releasing
factor, catecholamines and vasopressin. Nature 305(5932):325-327.
Rivier J, Spiess J, Thorner M, Vale W. 1982. Characterization of a Growth Hormone-Releasing
Factor from a Human Pancreatic-Islet Tumor. Nature 300(5889):276-278.
Roberts EM, Pope GR, Newson MJ, Lolait SJ, O'Carroll AM. 2011. The vasopressin V1b
receptor modulates plasma corticosterone responses to dehydration-induced stress. J
Neuroendocrinol 23(1):12-19.
Robson AC, Leckie CM, Seckl JR, Holmes MC. 1998. 11 Beta-hydroxysteroid dehydrogenase
type 2 in the postnatal and adult rat brain. Brain Res Mol Brain Res 61(1-2):1-10.
Rodaros D, Caruana DA, Amir S, Stewart J. 2007. Corticotropin-releasing factor projections
from limbic forebrain and paraventricular nucleus of the hypothalamus to the region of
the ventral tegmental area. Neuroscience 150(1):8-13.
162
Rokaeus A, Young WS, Mezey E. 1988. Galanin Coexists with Vasopressin in the Normal Rat
Hypothalamus and Galanins Synthesis Is Increased in the Brattleboro (Diabetes-
Insipidus) Rat. Neuroscience letters 90(1-2):45-50.
Rood BD, Stott RT, You S, Smith CJ, Woodbury ME, De Vries GJ. 2013. Site of origin of and
sex differences in the vasopressin innervation of the mouse (Mus musculus) brain. J
Comp Neurol 521(10):2321-2358.
Saavedra JM. 2012. Angiotensin II AT(1) receptor blockers ameliorate inflammatory stress: a
beneficial effect for the treatment of brain disorders. Cell Mol Neurobiol 32(5):667-681.
Saavedra JM, Pavel J. 2005. Angiotensin II AT1 receptor antagonists inhibit the angiotensin-
CRF-AVP axis and are potentially useful for the treatment of stress-related and mood
disorders. Drug Development Research 65(4):237-269.
Sampey DB, Burrell LM, Widdop RE. 1999. Vasopressin V2 receptor enhances gain of
baroreflex in conscious spontaneously hypertensive rats. Am J Physiol 276(3 Pt 2):R872-
879.
Sanders PW. 2009. Vascular consequences of dietary salt intake. Am J Physiol Renal Physiol
297(2):F237-243.
Sanders PW, Gibbs CL, Akhi KM, MacMillan-Crow LA, Zinn KR, Chen YF, Young CJ,
Thompson JA. 2001. Increased dietary salt accelerates chronic allograft nephropathy in
rats. Kidney international 59(3):1149-1157.
Sapirstein LA, Brandt WL, Drury DR. 1950. Production of arterial hypertension in the rat by
substitution of hypertonic sodium chloride solutions for drinking water. The American
journal of medicine 8(4):525-526.
Sawchenko PE. 1987. Evidence for differential regulation of corticotropin-releasing factor and
vasopressin immunoreactivities in parvocellular neurosecretory and autonomic-related
projections of the paraventricular nucleus. Brain Res 437(2):253-263.
Schally AV. 1978. Aspects of hypothalamic regulation of the pituitary gland. Science
202(4363):18-28.
Schally AV, Arimura A, Bowers CY, Kastin AJ, Sawano S, Reeding TW. 1968. Hypothalamic
neurohormones regulating anterior pituitary function. Recent Prog Horm Res 24:497-588.
Schally AV, Arimura A, Kastin AJ. 1973. Hypothalamic regulatory hormones. Science
179(4071):341-350.
Schweda F, Kurtz A. 2010. Regulation of renin release by local and systemic factors. Reviews of
physiology, biochemistry and pharmacology 161:1-44.
163
Scott Young W. 1986. Corticotropin-releasing factor mRNA in the hypothalamus is affected
differently by drinking saline and by dehydration. FEBS Letters 208(1):158-162.
Seckl JR, Johnson MR, Lightman SL. 1989. Vasopressin and oxytocin responses to hypertonic
saline infusion: effect of the opioid antagonist naloxone. Clin Endocrinol (Oxf)
30(5):513-518.
Selye H. 1943. Production of nephrosclerosis in the fowl by sodium chloride. Jour Amer Vet
Med Assoc 103((798)):140-143.
Selye H, Hall CE, Rowley EM. 1943. Malignant hypertension produced by treatment with
desoxycorticosterone acetate and sodium chloride. Canadian Medical Association Journal
49:88-92.
Selye H, Stone H. 1943. Role of sodium chloride in production of nephrosclerosis by steroids.
Proceedings of the Society for Experimental Biology and Medicine 52(3):190-193.
Sharp FR, Sagar SM, Hicks K, Lowenstein D, Hisanaga K. 1991. C-Fos Messenger-Rna, Fos,
and Fos-Related Antigen Induction by Hypertonic Saline and Stress. Journal of
Neuroscience 11(8):2321-2331.
Shelat SG, Flanagan-Cato LM, Fluharty SJ. 1999. Glucocorticoid and mineralocorticoid
regulation of angiotensin II type 1 receptor binding and inositol triphosphate formation in
WB cells. J Endocrinol 162(3):381-391.
Sivukhina EV, Poskrebysheva AS, Smurova Iu V, Dolzhikov AA, Morozov Iu E, Jirikowski GF,
Grinevich V. 2009. Altered hypothalamic-pituitary-adrenal axis activity in patients with
chronic heart failure. Horm Metab Res 41(10):778-784.
Sjoquist M, Huang W, Jacobsson E, Skott O, Stricker EM, Sved AF. 1999. Sodium excretion and
renin secretion after continuous versus pulsatile infusion of oxytocin in rats.
Endocrinology 140(6):2814-2818.
Sofroniew MV. 1980. Projections from vasopressin, oxytocin, and neurophysin neurons to neural
targets in the rat and human. The journal of histochemistry and cytochemistry : official
journal of the Histochemistry Society 28(5):475-478.
Son SJ, Filosa JA, Potapenko ES, Biancardi VC, Zheng H, Patel KP, Tobin VA, Ludwig M,
Stern JE. 2013. Dendritic peptide release mediates interpopulation crosstalk between
neurosecretory and preautonomic networks. Neuron 78(6):1036-1049.
Sonino N, Fallo F, Fava GA. 2006. Psychological aspects of primary aldosteronism. Psychother
Psychosom 75(5):327-330.
164
Sonino N, Tomba E, Genesia ML, Bertello C, Mulatero P, Veglio F, Fava GA, Fallo F. 2011.
Psychological assessment of primary aldosteronism: a controlled study. J Clin Endocrinol
Metab 96(6):E878-883.
Spencer S, Saper CB, Joh T, Reis DJ, Goldstein M, Raese JD. 1985. Distribution of
catecholamine-containing neurons in the normal human hypothalamus. Brain Res
328(1):73-80.
St-Louis R, Parmentier C, Raison D, Grange-Messent V, Hardin-Pouzet H. 2012. Reactive
oxygen species are required for the hypothalamic osmoregulatory response.
Endocrinology 153(3):1317-1329.
Stachniak TJ, Trudel E, Bourque CW. 2014. Cell-specific retrograde signals mediate antiparallel
effects of angiotensin II on osmoreceptor afferents to vasopressin and oxytocin neurons.
Cell Rep 8(2):355-362.
Stellar E, Hyman R, Samet S. 1954. Gastric Factors Controlling Water-Solution-Drinking and
Salt-Solution-Drinking. Journal of Comparative and Physiological Psychology 47(3):220-
226.
Stern JE. 2015. Neuroendocrine-autonomic integration in the paraventricular nucleus: novel roles
for dendritically released neuropeptides. J Neuroendocrinol 27(6):487-497.
Stocker SD, Cunningham JT, Toney GM. 2004. Water deprivation increases Fos
immunoreactivity in PVN autonomic neurons with projections to the spinal cord and
rostral ventrolateral medulla. Am J Physiol Regul Integr Comp Physiol 287(5):R1172-
1183.
Stocker SD, Hunwick KJ, Toney GM. 2005. Hypothalamic paraventricular nucleus differentially
supports lumbar and renal sympathetic outflow in water-deprived rats. Journal of
Physiology-London 563(1):249-263.
Stocker SD, Osborn JL, Carmichael SP. 2008. Forebrain osmotic regulation of the sympathetic
nervous system. Clin Exp Pharmacol Physiol 35(5-6):695-700.
Stocker SD, Simmons JR, Stornetta RL, Toney GM, Guyenet PG. 2006. Water deprivation
activates a glutamatergic projection from the hypothalamic paraventricular nucleus to the
rostral ventrolateral medulla. J Comp Neurol 494(4):673-685.
Stocker SD, Sved AF, Stricker EM. 2000. Role of renin-angiotensin system in hypotension-
evoked thirst: studies with hydralazine. Am J Physiol Regul Integr Comp Physiol
279(2):R576-585.
Strazzullo P, D'Elia L, Kandala NB, Cappuccio FP. 2009. Salt intake, stroke, and cardiovascular
disease: meta-analysis of prospective studies. BMJ 339:b4567.
165
Stricker EM, Sved AF. 2000. Thirst. Nutrition 16(10):821-826.
Stricker EM, Verbalis JG. 1986. Interaction of osmotic and volume stimuli in regulation of
neurohypophyseal secretion in rats. Am J Physiol 250(2 Pt 2):R267-275.
Sullivan RC, Fay WH, Schuler AL, Campbell M, Coleman M, Caparulo B, Cohen DJ. 1980.
Why Do Autistic-Children. J Autism Dev Disord 10(2):231-241.
Sun N, Roberts L, Cassell MD. 1991. Rat Central Amygdaloid Nucleus Projections to the Bed
Nucleus of Thestria Terminalis. Brain research bulletin 27(5):651-662.
Sunn N, McKinley MJ, Oldfield BJ. 2003. Circulating angiotensin II activates neurones in
circumventricular organs of the lamina terminalis that project to the bed nucleus of the
stria terminalis. Journal of Neuroendocrinology 15(8):725-731.
Swanson LW, Kuypers HG. 1980. The paraventricular nucleus of the hypothalamus:
cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal
vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-
labeling methods. J Comp Neurol 194(3):555-570.
Swanson LW, Sawchenko PE. 1980. Paraventricular nucleus: a site for the integration of
neuroendocrine and autonomic mechanisms. Neuroendocrinology 31(6):410-417.
Swanson LW, Sawchenko PE. 1983. Hypothalamic integration: organization of the
paraventricular and supraoptic nuclei. Annu Rev Neurosci 6:269-324.
Swanson LW, Sawchenko PE, Berod A, Hartman BK, Helle KB, Vanorden DE. 1981. An
immunohistochemical study of the organization of catecholaminergic cells and terminal
fields in the paraventricular and supraoptic nuclei of the hypothalamus. J Comp Neurol
196(2):271-285.
Swanson LW, Simmons DM. 1989. Differential steroid hormone and neural influences on
peptide mRNA levels in CRH cells of the paraventricular nucleus: a hybridization
histochemical study in the rat. J Comp Neurol 285(4):413-435.
Tang Y, McKenna KE. 2001. Paraventricular nucleus projections (PVN) to spinal and brainstem
nuclei involved in the control of sexual function. Society for Neuroscience Abstracts
27(2):2227-2227.
Taniguchi H, He M, Wu P, Kim S, Paik R, Sugino K, Kvitsiani D, Fu Y, Lu J, Lin Y, Miyoshi
G, Shima Y, Fishell G, Nelson SB, Huang ZJ. 2011. A resource of Cre driver lines for
genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71(6):995-1013.
166
Taurines R, Schwenck C, Lyttwin B, Schecklmann M, Jans T, Reefschlager L, Geissler J,
Gerlach M, Romanos M. 2014. Oxytocin plasma concentrations in children and
adolescents with autism spectrum disorder: correlation with autistic symptomatology.
Atten Defic Hyperact Disord 6(3):231-239.
Teitelbaum P, Stellar E. 1954. Recovery from the failure to eat produced by hypothalamic
lesions. Science 120(3126):894-895.
Tilbrook AJ, Rivalland ETA, Turner AI, Pompolo S, Clarke IJ. 2006. Isolation/restraint stress in
sheep activates projections to the preoptic area arising from the paraventricular nucleus
but not those arising from the bed nucleus of the stria terminalis or the arcuate nucleus.
Frontiers in neuroendocrinology 27(1):40-41.
Tobian L, Hanlon S. 1990. High Sodium-Chloride Diets Injure Arteries and Raise Mortality
without Changing Blood-Pressure. Hypertension 15(6):900-903.
Tsuruo Y, Ceccatelli S, Villar MJ, Hokfelt T, Visser TJ, Terenius L, Goldstein M, Brown JC,
Buchan A, Walsh J. 1988. Coexistence of TRH with other neuroactive substances in the
rat central nervous system. J Chem Neuroanat 1(5):235-253.
Turkkan JS. 1994. Biobehavioral effects of extended salt loading and conflict stress in intact
baboons. J Exp Anal Behav 61(2):263-272.
Ulrich-Lai YM, Herman JP. 2009. Neural regulation of endocrine and autonomic stress
responses. Nat Rev Neurosci 10(6):397-409.
Vale W, Rivier C, Brown M. 1977. Regulatory peptides of the hypothalamus. Annu Rev Physiol
39:473-527.
Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J. 1986.
Purification and characterization of an FSH releasing protein from porcine ovarian
follicular fluid. Nature 321(6072):776-779.
Vale W, Spiess J, Rivier C, Rivier J. 1981. Characterization of a 41-Residue Ovine
Hypothalamic Peptide That Stimulates Secretion of Corticotropin and Beta-Endorphin.
Science 213(4514):1394-1397.
Vale W, Vaughan J, Smith M, Yamamoto G, Rivier J, Rivier C. 1983. Effects of Synthetic Ovine
Corticotropin-Releasing Factor, Glucocorticoids, Catecholamines, Neurohypophyseal
Peptides, and Other Substances on Cultured Corticotropic Cells. Endocrinology
113(3):1121-1131.
Valentino RJ, Pavcovich LA, Hirata H. 1995. Evidence for corticotropin-releasing hormone
projections from Barrington's nucleus to the periaqueductal gray and dorsal motor
nucleus of the vagus in the rat. J Comp Neurol 363(3):402-422.
167
Vanderhaeghen JJ, Lotstra F, Liston DR, Rossier J. 1983. Proenkephalin, [Met]enkephalin, and
oxytocin immunoreactivities are colocalized in bovine hypothalamic magnocellular
neurons. Proceedings of the National Academy of Sciences of the United States of
America 80(16):5139-5143.
Vanderhaeghen JJ, Lotstra F, Vandesande F, Dierickx K. 1981. Coexistence of Cholecystokinin
and Oxytocin-Neurophysin in Some Magnocellular Hypothalamo-Hypophyseal Neurons.
Cell and Tissue Research 221(1):227-231.
Vandesande F, Dierickx K. 1975. Identification of Vasopressin Producing and of Oxytocin
Producing Neurons in Hypothalamic Magnocellular Neurosecretory-System of Rat. Cell
and Tissue Research 164(2):153-162.
Varudkar A, Ramakrishnan U. 2015. Commensalism facilitates gene flow in mountains: a
comparison between two Rattus species. Heredity (Edinb) 115(3):253-261.
Verbalis JG, Mangione MP, Stricker EM. 1991. Oxytocin Produces Natriuresis in Rats at
Physiological Plasma-Concentrations. Endocrinology 128(3):1317-1322.
Vogelzangs N, Seldenrijk A, Beekman AT, van Hout HP, de Jonge P, Penninx BW. 2010.
Cardiovascular disease in persons with depressive and anxiety disorders. Journal of
affective disorders 125(1-3):241-248.
Voisin DL, Bourque CW. 2002. Integration of sodium and osmosensory signals in vasopressin
neurons. Trends Neurosci 25(4):199-205.
Vujovic N, Gooley JJ, Jhou TC, Saper CB. 2015. Projections from the subparaventricular zone
define four channels of output from the circadian timing system. J Comp Neurol
523(18):2714-2737.
Wamsteeker Cusulin JI, Fuzesi T, Watts AG, Bains JS. 2013. Characterization of corticotropin-
releasing hormone neurons in the paraventricular nucleus of the hypothalamus of Crh-
IRES-Cre mutant mice. Plos One 8(5):e64943.
Wang J, Irnaten M, Venkatesan P, Evans C, Mendelowitz D. 2002. Arginine vasopressin
enhances GABAergic inhibition of cardiac parasympathetic neurons in the nucleus
ambiguus. Neuroscience 111(3):699-705.
Wang J, Qin W, Liu B, Zhou Y, Wang D, Zhang Y, Jiang T, Yu C. 2013a. Neural mechanisms
of oxytocin receptor gene mediating anxiety-related temperament. Brain Struct Funct
219(5):1543-1554.
Wang YF, Sun MY, Hou Q, Hamilton KA. 2013b. GABAergic inhibition through synergistic
astrocytic neuronal interaction transiently decreases vasopressin neuronal activity during
hypoosmotic challenge. The European journal of neuroscience 37(8):1260-1269.
168
Watson SJ, Akil H, Fischli W, Goldstein A, Zimmerman E, Nilaver G, van wimersma Griedanus
TB. 1982. Dynorphin and vasopressin: common localization in magnocellular neurons.
Science 216(4541):85-87.
Watts AG. 1992a. Disturbance of Fluid Homeostasis Leads to Temporally and Anatomically
Distinct Responses in Neuropeptide and Tyrosine-Hydroxylase Messenger-Rna Levels in
the Paraventricular and Supraoptic Nuclei of the Rat. Neuroscience 46(4):859-879.
Watts AG. 1992b. Osmotic Stimulation Differentially Affects Cellular-Levels of Corticotropin-
Releasing Hormone and Neurotensin Neuromedin-N Messenger-Rnas in the Lateral
Hypothalamic Area and Central Nucleus of the Amygdala. Brain research 581(2):208-
216.
Watts AG. 1996. The impact of physiological stimuli on the expression of corticotropin-releasing
hormone (CRH) and other neuropeptide genes. Front Neuroendocrinol 17(3):281-326.
Watts AG. 1999. Dehydration-associated anorexia: Development and rapid reversal. Physiology
& Behavior 65(4-5):871-878.
Watts AG, Kelly AB, Sanchez-Watts G. 1995. Neuropeptides and thirst: the temporal response
of corticotropin-releasing hormone and neurotensin/neuromedin N gene expression in rat
limbic forebrain neurons to drinking hypertonic saline. Behav Neurosci 109(6):1146-
1157.
Watts AG, Sanchezwatts G. 1995. A Cell-Specific Role for the Adrenal-Gland in Regulating Crh
Messenger-Rna Levels in Rat Hypothalamic Neurosecretory Neurons after Cellular
Dehydration. Brain Research 687(1-2):63-70.
Watts AG, Swanson LW, Sanchez-Watts G. 1987. Efferent projections of the suprachiasmatic
nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucoagglutinin in
the rat. J Comp Neurol 258(2):204-229.
Whitnall MH, Gainer H, Cox BM, Molineaux CJ. 1983. Dynorphin-a-(1-8) Is Contained within
Vasopressin Neurosecretory Vesicles in Rat Pituitary. Science 222(4628):1137-1139.
Wiedenmayer CP, Magarinos AM, McEwen BS, Barr GA. 2005. Age-specific threats induce
CRF expression in the paraventricular nucleus of the hypothalamus and hippocampus of
young rats. Horm Behav 47(2):139-150.
Williams RW, Rakic P. 1988. Three-dimensional counting: an accurate and direct method to
estimate numbers of cells in sectioned material. J Comp Neurol 278(3):344-352.
Williams TD, Abel DC, King CM, Jelley RY, Lightman SL. 1985. Vasopressin and oxytocin
responses to acute and chronic osmotic stimuli in man. The Journal of endocrinology
108(1):163-168.
169
Windle RJ, Kershaw YM, Shanks N, Wood SA, Lightman SL, Ingram CD. 2004. Oxytocin
attenuates stress-induced c-fos mRNA expression in specific forebrain regions associated
with modulation of hypothalamo-pituitary-adrenal activity. J Neurosci 24(12):2974-2982.
Windle RJ, Shanks N, Lightman SL, Ingram CD. 1997. Central oxytocin administration reduces
stress-induced corticosterone release and anxiety behavior in rats. Endocrinology
138(7):2829-2834.
Wittmann G, Fuzesi T, Singru PS, Liposits Z, Lechan RM, Fekete C. 2009. Efferent projections
of thyrotropin-releasing hormone-synthesizing neurons residing in the anterior
parvocellular subdivision of the hypothalamic paraventricular nucleus. J Comp Neurol
515(3):313-330.
Yang G, Xi ZX, Wan Y, Wang H, Bi G. 1993. Changes in circulating and tissue angiotensin II
during acute and chronic stress. Biological signals 2(3):166-172.
Yang Z, Coote JH. 1998. Influence of the hypothalamic paraventricular nucleus on
cardiovascular neurones in the rostral ventrolateral medulla of the rat. The Journal of
physiology 513 ( Pt 2)(2):521-530.
Yao ST, Antunes VR, Bradley PM, Kasparov S, Ueta Y, Paton JF, Murphy D. 2011. Temporal
profile of arginine vasopressin release from the neurohypophysis in response to
hypertonic saline and hypotension measured using a fluorescent fusion protein. J
Neurosci Methods 201(1):191-195.
Yu HCM, Burrell LM, Black MJ, Wu LL, Dilley RJ, Cooper ME, Johnston CI. 1998. Salt
induces myocardial and renal fibrosis in normotensive and hypertensive rats. Circulation
98(23):2621-2628.
Yue C, Mutsuga N, Sugimura Y, Verbalis J, Gainer H. 2008. Differential kinetics of oxytocin
and vasopressin heteronuclear RNA expression in the rat supraoptic nucleus in response
to chronic salt loading in vivo. J Neuroendocrinol 20(2):227-232.
Zhai XY, Thomsen JS, Birn H, Kristoffersen IB, Andreasen A, Christensen EI. 2006. Three-
dimensional reconstruction of the mouse nephron. Journal of the American Society of
Nephrology : JASN 17(1):77-88.
Ziegler DR, Herman JP. 2002. Neurocircuitry of stress integration: anatomical pathways
regulating the hypothalamo-pituitary-adrenocortical axis of the rat. Integr Comp Biol
42(3):541-551.
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BIOGRAPHICAL SKETCH
Justin entered academia later in life, attending Clark State Community College in
Springfield, OH in January 2008 at the age of 32. After one year of coursework, he transferred
to the University of Cincinnati where he completed premedical coursework and explored
professions related to medicine and basic science research. In December of 2010, he accepted a
position working in the laboratory of Dr. Randall Sakai under the mentorship of Dr. Eric Krause.
This experience exposed him to the basics of neuroanatomical methods and research aims
exploring the central actions of neuropeptides and rodent models of behavior. Graduating with a
BS in neuroscience and a minor in mathematics in March of 2012, Justin then moved to the
University of Florida to work with Dr. Krause before beginning graduate studies in August of the
same year. During the course of his training, Justin was involved with several research projects
producing eight published peer-reviewed manuscripts with four more manuscripts either
submitted for review or in preparation at the time of this publication. Following graduation,
Justin plans to move to Boston College and Michigan State University to continue his training as
a postdoctoral fellow under the mentorship of Dr. Alexa Veenema.