The Effect of High Salt Intake on Osmoreceptor Gain in Salt

The Effect of High Salt Intake on Osmoreceptor Gain in

Salt-Sensitive Hypertension

David Levi Integrated Program in Neuroscience McGill University Montreal, QC August 2018

A thesis submitted to McGill University in partial fulfillment of the requirement of the degree of Master of Science.

Table of Contents

Abstract ...... i Resumé ...... ii Acknowledgements ...... iii Preface and Contribution of Authours ...... iv Symbols and Abbreviations ...... v 1. Introduction ...... 1 1.1 General Introduction to Hypertension ...... 1 1.1.1 Epidemiology of Hypertension ...... 1 1.1.2 Risk Factors of Hypertension ...... 2 1.2 Homeostasis of Blood Pressure ...... 3 1.2.1 Introduction to Blood Pressure Regulation ...... 3 1.2.2 The Nervous System as an Initial Regulator of Blood Pressure ...... 4 1.2.3 The Kidney: Bridging Blood Pressure Regulation and Fluid Balance ...... 5 1.3 Regulation of Fluid Balance ...... 7 1.3.1 Osmosis in the Context of a Single Cell ...... 7 1.3.2 Systemic Osmoregulation ...... 7 1.3.3 The OVLT as the Central Osmoreceptor ...... 8 1.3.4 Neuroendocrine Mechanisms Underlying Vasopressin Secretion ...... 9 1.3.5 Electrophysiological Characterization of VP-Containing MNCs ...... 10 1.3.6 Intrinsic and Extrinsic Modulation of Vasopressin MNCs ...... 11 1.3.6.1 Intrinsic Osmosensitivity of VP MNCs ...... 11 1.3.6.2 Glial-MNC Interactions ...... 13 1.3.6.3 Afferent Modulation of VP MNCs ...... 13 1.3.6.3a Osmotic Modulation: OVLT ...... 13 1.3.6.3b Cardiovascular Modulation: Baroreceptors ...... 14 1.3.7 Fluid Balance and Plasma Sodium ...... 14 1.4 High Dietary Sodium Intake as a Risk Factor for Hypertension ...... 16 1.4.1 Changes in the Perception and Intake of Salt Throughout Human History ...... 16 1.4.2 Forming the Relationship between High Dietary Salt Intake and Hypertension ...... 17 1.4.3 Sodium Reduction as a Strategy to Reduce Blood Pressure ...... 19 1.4.4 Projected Effect of Reducing Sodium Intake on Cardiovascular Health ...... 20 1.5 Mechanisms Underlying Salt Sensitive Hypertension ...... 22 1.5.1 Salt Sensitivity: An Overview ...... 22 1.5.2 Salt-Sensitive Hypertension as a Continuous, Multifactorial Disease ...... 24 1.5.3 Renal Dysfunction in Salt-Sensitive Hypertension ...... 26 1.5.3.1 Overview of Pressure Natriuresis ...... 26 1.5.3.2 Impaired Pressure-Natriuresis in Salt Sensitive Hypertension ...... 26 1.5.4 Role of the Autonomic Nervous System in Salt Sensitivity ...... 28 1.5.4.1 Sympathetic Control of Cardiovascular and Renal Function ...... 28 1.5.4.2 Abnormally Elevated SNA Following High Salt Intake ...... 29 1.5.4.3 Detection of Plasma Hypernatremia as the Mediator of Sympathoexcitation ...... 30 1.5.5 Role of Vasopressin in Rat Models of Salt-Sensitive Hypertension ...... 31

1.5.5.1 Exaggerated Release of Vasopressin in Models of Salt-Sensitive Hypertension . 31 1.5.5.2 GABA is Excitatory in Salt-Sensitive Hypertension ...... 32 1.5.5.3 Increased Osmoreceptor Drive in Salt Sensitive Hypertension? ...... 34 1.5.6 Specific Aim ...... 35 1.5.7 AngII-Salt Model of Salt-Sensitive Hypertension ...... 36 2. Methods ...... 38 2.1 Animal Care ...... 38 2.1.1 Salt Loading Model of Salt-Sensitive Hypertension ...... 38 2.1.2 AngII-Salt Model of Salt-Sensitive Hypertension ...... 38 2.2 Electrophysiology in Acute Hypothalamic Slices ...... 39 2.2.1 Acute Slice Preparation and Recording Conditions ...... 39 2.2.2 Chemicals and Drugs ...... 40 2.2.3 Spontaneous Current Clamp Recordings ...... 41 2.2.4 Spontaneous Voltage Clamp Recordings ...... 41 2.2.5 Evoked Voltage Clamp Recordings ...... 41 2.3 Statistics ...... 42 3. Results ...... 43 3.1 Results Overview ...... 43 3.2 Effects of Salt Loading (SL) ...... 43 3.2.1 SL Increases SONVP MNC Osmoresponsiveness ...... 43 3.2.2 SL Increases SONVP MNC Intrinsic Osmosensitivity ...... 44 3.2.3 Salt Loading Does Not Enhance Osmoreceptor Signalling ...... 45 3.2.4 SL Discussion ...... 46 3.2.4.1 Summary of SL Results ...... 46 3.2.4.2 Possible Mechanisms Underlying Increased Osmoresponsiveness in SL ...... 46 3.3 Effects of AngII-Salt Hypertension ...... 53 3.3.1 AngII-Salt Hypertension Increases SONVP MNC Overall Osmoresponsiveness ...... 53 3.3.2 AngII-Salt Does Not Increase SONVP MNC Intrinsic Osmoresponsiveness ...... 54 3.3.3 AngII-Salt Increases Synaptic Gain in the OVLT→SONVP Pathway ...... 55 3.3.4 AngII-Salt Increases Probability of Release of the OVLT→SONVP Connection ...... 56 3.3.5 AngII-Salt Increases Quantal Size of SONVP MNCs ...... 57 3.3.6 AngII-Salt Increases the Frequency and Amplitude of Asynchronous EPSCs ...... 58 3.3.6 AngII-Salt Discussion ...... 59 3.3.6.1 Summary of AngII-Salt Results ...... 59 3.3.6.2 Possible Mechanism Underlying Increased Synaptic Gain in AngII-Salt ...... 60 4. General Discussion and Conclusion ...... 70 4.1 Summary of Thesis Motivation and Results ...... 70 4.2 Comparison of the Salt-Sensitive Hypertension Models ...... 70 4.3 Perspectives of Thesis Results in Regards to Global Health ...... 72

This work is dedicated to the memories of Elie Sotto and Dr. Irving Levi

Abstract

High dietary salt intake (HDSI) is a significant risk factor for hypertension and is strongly correlated with the incidence of cardiovascular and renal diseases. Increases in osmotic pressure resulting from enhanced plasma sodium levels are detected by osmosensitive neurons in the hypothalamus called osmoreceptors. Osmoreceptors in the organum vasculosum laminae terminalis (OVLT) send an excitatory projection to the supraoptic nucleus (SON) and activate specialized magnocellular neurosecretory cells (MNCs), which are also intrinsically osmosensitive. These MNCs project to the neurohypophysis, from which they release vasopressin

(VP) into the circulation. Recent studies, including those from our lab, have demonstrated that chronic exposure of rats to HDSI results in excessive activation of MNCs, leading to VP-mediated increases in blood pressure. Although this effect is associated with a reduction in the efficacy of inhibitory synaptic signaling by baroreceptors, it remains possible that a facilitation of excitatory osmoreceptor signaling can also contribute to this process.

This proposed research program aims to elucidate whether the synaptic strength of the

OVLT→SONVP synaptic connection is enhanced in animal models of salt-sensitive hypertension.

This thesis aims to provides a better understanding of how high salt intake alters signalling pathways that control VP secretion, and may allow for the innovation of novel therapeutic approaches to treat hypertension by targeting this system.

Resumé

L'apport élevé de sel alimentaire est un facteur de risque majeur d'hypertension et fortement corrélé à l'incidence des maladies cardiovasculaires et rénales. Les augmentations de la pression osmotique dues à l'augmentation des niveaux de sodium dans le plasma sont détectées par les neurones osmosensoriels de l'hypothalamus, appelés osmorécepteurs. Les osmorécepteurs de l'organum vasculosum laminae terminalis (OVLT) communiquent avec le noyau supraoptique

(SON) et excitent des cellules magnoceullulaires neurosécrétoires (CMNs) pour liberer la vasopressine (VP) dans la circulation. Des études récentes ont démontré que l'exposition chronique des rats à l'apport élevé en sel alimentaire entraîne une activation excessive des CMNs, et augmente la tension artérielle induites par la VP. Bien que cet effet soit associé à une réduction de l'efficacité de la signalisation synaptique inhibitrice par les barorécepteurs, il reste possible qu'une facilitation de la signalisation excitatoire par les osmorécepteurs puisse également contribuer à ce processus.

Ce programme de recherche vise à déterminer si la force synaptique de la connexion synaptique OVLT → SONVP est améliorée dans les modèles animaux d'hypertension sensible au sel. L'objectif est de mieux comprendre comment l'apport élevé en sel modifie les voies de signalisation qui contrôlent la sécrétion de VP et peut conduire à de nouvelles approches pour le traitement de l'hypertension en ciblant ce système.

Acknowledgements

The work presented in this thesis was made possible by the Heart and Stroke Foundation of Canada and the Canadian Institutes of Health Research. Their financial support has given me a priceless perspective of working in health research, and has helped me perceive our world through a more curious and innovative lens.

I am indebted to my supervisor and mentor, Dr. Charles Bourque, for his unwavering support and confidence in my ability to succeed not only as a scientist but as a person. Thank you for giving me the privilege of working with you. The genuine appreciation, patience and support you continue to share is the framework of how I hope to mentor my future students. I thank my co-supervisor, Dr. Masha Prager-Khoutorsky, for the impacts she has made on me both personally and professionally. I wish to highlight the helpful insights into this project provided by Dr. David

Stellwagen and Dr. Arkady Khoutorsky of my advisory committee.

I am grateful to the Bourque lab for accepting me as one of their own. The personal and professional relationships I have forged with my colleagues have helped me mature as I continue to work alongside individuals with unique and fascinating lives. Eric, Cristian, Claire, Zahra,

Daniel, Josh, Xander, Nate, thank you for making my experience as a scientist a positive one.

I’d also like to mention Dr. David Hornstein and Dr. Ashvini Gursahaney of the Montreal

General Hospital’s Intensive Care Unit. While balancing graduate responsibilities, you both provided me with the opportunity to develop my interpersonal skills and foster my passion for medicine. I feel prepared to start the next chapter of my life in this exciting and rewarding career.

Finally, it goes without mention to acknowledge those who have persevered with me on this seemingly-endless path. Mom, Dad, Jonathan, Sarah and Skye, we finally made it!

iii

Preface and Contribution of Authours

The scholarly works presented in this thesis are the first to investigate alterations in osmoreceptor signaling pathways that regulate vasopressin secretion in animal models of salt- sensitive hypertension. Charles W. Bourque and Masha Prager-Khoutorsky conceptualized and designed the experiments. David Levi bred and maintained all colonies of transgenic animals, performed all surgical and electrophysiological procedures, accumulated and analyzed all collected data, and wrote the complete thesis.

Symbols and Abbreviations

ACE angiotensin converting enzyme ACSF artificial cerebrosprinal fluid aEPSC asynchronous EPSC AngII angiotensin II AngII- salt angiotensin II-salt AV3V anteroventral third ventricle BDNF brain-derived neurotrophic factor BIC bicuculline ECF extracellular fluid EPSC excitatory postsynaptic current EPSP excitatory postsynaptic potential EU euhydrated GABA γ-aminobutyric acid GFP green fluorescent protein GlyR glycine receptor Hz Hertz HDSI High dietary salt intake IML intermediolateral KCC2 K-Cl transporter 2 kg kilogram mEPSC miniature EPSC mmHg millimeters of mercury MNC magnocellular neurosecretory cell MnPO median preoptic nucleus OVLT organum vasculosum laminae terminalis PVN paraventricular nucleus RVLM rostroventolateral medulla SEM standard error of the mean sEPSC spontaneous excitatory postsynaptic current SFO subfornical organ SIC stretch inactivated cation channel SL salt loading SNA sympathetic nerve activity SON supraoptic nucleus TPR total peripheral resistance TRPV1 transient receptor potential vanilloid 1 receptor

TTX tetrodotoxin V1R vasopressin receptor type 1 V2R vasopressin receptor type 2 VRAC volume regulated anion channel Ω ohm

1. Introduction

1.1 General Introduction to Hypertension

The state of chronically elevated blood pressure, or hypertension, represents a major health problem in the developed world [1-8]. While abrupt rises in blood pressure can lead to hypertensive emergencies with life-threatening implications, high blood pressure is often asymptomatic for long periods of time throughout one’s life. Besides increasing the risk of a sudden cardiovascular accident such as a stroke or myocardial infarction, hypertension can lead to cardiac hypertrophy, heart failure, aortic dissection, and renal failure [3, 9, 10]. While the molecular pathways of blood pressure regulation are reasonably well understood, the mechanisms leading to hypertension in the vast majority of affected individuals remain unknown. Contemporary perspectives suggest hypertension to be a product of environmental factors interplayed with genetic polymorphisms, which together, lead to chronically increased blood volume and peripheral resistance [11].

1.1.1 Epidemiology of Hypertension

Hypertension is recognized as one of the leading risk factors for human mortality and morbidity. Elevated blood pressure is thought to account for 62% of deaths due to stroke and 49% of deaths due to heart disease [7, 12]. Complications associated with hypertension are responsible for 9.4 million global deaths annually [13], a number only expected to rise. Indeed, epidemiological reports suggest that one quarter of the global adult population was hypertensive in 2005, a year when over 30% of all deaths were accounted for by cardiovascular disease, and project the incidence of hypertension to be about one third of the adult population by 2025 [14].

In Canada, hypertension affects one of five citizens while representing the most common reason for hospital admission and medication prescription. Although numerous effective antihypertensive

drugs are now available, reports suggest 10-30% of patients with hypertension do not respond to any specific or combination of treatment regimens [15, 16]. Overall, the costs associated with treating hypertension in North America are staggering. In 2003, Canada spent over $2 billion in total costs towards the treatment of hypertension, while the United States spent $50.3 billion [4].

1.1.2 Risk Factors of Hypertension

One reason why hypertension remains such an important global health challenge is because there is no known specific cause. Importantly, blood pressure is a continuously distributed variable among the general population. Although there are no rigidly defined thresholds of blood pressures that reliably predict whether an individual will suffer from hypertension, the incidence of symptoms rises as blood pressure increases [11]. Indeed, the probability of developing hypertension increases with age [16], maintains some genetic component [17], and reflects one’s social determinants of health [12], yet these factors cannot reliably predict its onset. Environmental factors and behavioural choices can also increase blood pressure, and thus remain the only modifiable causes of hypertension. These include physical inactivity, obesity and stress, as well as the consumption of alcohol, tobacco and sodium [18, 19]. Although all of these modifiable risk factors deserve extensive discussion, this thesis will focus exclusively on the importance of sodium in the pathogenesis of hypertension.

To investigate the potential mechanisms by which the excessive consumption of dietary sodium can induce and maintain hypertension, it is critical to first review the physiological processes that mediate healthy cardiovascular function and fluid balance.

1.2 Homeostasis of Blood Pressure

1.2.1 Introduction to Blood Pressure Regulation

The circulatory system transports compounds, including gases, nutrients, electrolytes and waste to and from the body’s organs through its red blood cells and plasma. Effective and continued function of this system is a prerequisite for survival, and requires one’s blood pressure be sufficiently high to ensure adequate rates of perfusion. As such, changes in blood pressure are routinely made to direct appropriate amounts of oxygen and nutrients to specific parts of the body in different environmental contexts, including when exercising, and when changes in gravity cause fluids to redistribute within the body, including the use of elevators. Importantly, these changes in blood pressure rarely deviate from normal by more than 10-15% on a daily basis [20]. Blood pressure is measured in millimeters of mercury (mmHg) and is recorded as a ratio of two numbers.

The numerator represents the intra-arterial pressure when the heart is contracting and is defined as the systolic pressure. The denominator represents the intra-arterial pressure between heartbeats and is defined as the diastolic pressure. These two values represent the blood pressure range, where systolic represents the highest blood pressure and diastolic represents the lowest. In humans, healthy adult blood pressure is currently defined as 120 mmHg systolic 80 mmHg diastolic, while hypertension is currently defined as blood pressures greater than 140 mmHg systolic 90 mmHg diastolic [12].

Three primary factors physiologically adjust blood pressure: cardiac output, vascular resistance, and blood volume. Cardiac output, defined as the volume of blood pumped by the heart per minute, can be altered by changing the stroke volume, defined as the blood volume pumped per beat, as well as the heart rate. Vascular resistance is determined by the diameter and compliance of blood vessels and can be quickly altered by changing their diameter. Dilating vessels

(vasodilation) increases blood flow, while constricting vessels (vasoconstriction) decreases blood flow. Total blood volume can be changed by controlling the ratio of fluid intake and excretion.

Importantly, the body has evolved a variety of mechanisms that alter these three factors to maintain blood pressures within physiological working ranges and have been extensively characterized at the molecular and genetic level [10, 21-23]. Relevant blood pressure control systems for this thesis relate to the nervous system and kidney. The capacity to osmoregulate, or to control fluid balance, is a vital regulator of blood pressure and will also be separately reviewed in section 1.3.

1.2.2 The Nervous System as an Initial Regulator of Blood Pressure

The brain constantly receives frontline information of changes in blood pressure from sensory receptors, and can engage in feedback mechanisms to quickly regulate blood pressure via its autonomic division. This interplay of physiology helps maintain adequate cardiovascular function in the face of a dynamic environment (section 1.2.1). The medullary cardiovascular center within the brainstem houses sympathetic and parasympathetic autonomic neurons that can alter cardiac output. Sympathetic cardiac nerves increase the heart rate and its contractility, while parasympathetic fibers within the vagus nerve lower cardiac output by reducing the heart rate.

Additionally, neurons within the medullary vasomotor nucleus innervate arteriole smooth muscle cells throughout the body to maintain appropriate levels of vasoconstriction. The nervous system underlines this simple and rapid control of blood pressure through specialized mechanosensitive receptors embedded within blood vessels called baroreceptors. Located primarily in the carotid sinus in the walls of the aorta and internal carotid artery, these sensory neurons become activated when blood vessels become stretched as a result of increased pressure. Extensively innervating the brain [24] and circulatory system [25], baroreceptor activation dilates peripheral blood vessels and

reduces the heartbeat, effectively lowering blood pressure. Chemoreceptors are another type of sensory neuron that lie adjacent to baroreceptors in the carotid and aortic bodies. These neurons monitor the content and levels of gases and toxins within the blood and initiate feedback mechanisms in states of insufficient oxygen or excess carbon dioxide [26]. This system allows us to place high strain on muscles in conditions where they consume large amounts of oxygen and produce excess carbon dioxide, including the evasion of predators in ancient times and exercise in modern times [20].

1.2.3 The Kidney: Bridging Blood Pressure Regulation and Fluid Balance

The kidney plays a critical role in regulating the components and volume of blood through the production of urine. Specifically, urine represents the solution of excess solutes and fluids expelled by the body in order to maintain extracellular fluid homeostasis. Its composition therefore changes in response to environmental perturbations that indirectly disturb the composition of the millieu interiereur. The kidney plays a key role in regulating blood pressure through its ability to regulate blood volume [20, 25]. When arterial blood pressure rises above normal, the excess pressure causes the kidneys to reduce blood volume by excreting more water and salt than are entering the body. This has hypotensive effects through its ability to decrease cardiac output

(section 1.2). The opposite is true in conditions of low blood volume, where the reduced blood pressure causes the kidney to reduce urine excretion. The functional units of the kidney, or nephrons, receive blood flowing through the renal artery via afferent glomerular arterioles. The complex physiological subdivisions of the nephron highlight its ability to modulate the reabsorption of essential nutrients including amino acids, glucose and electrolytes, while expelling waste particles and excess water [27, 28]. Epithelial cells lining the basolateral membrane of the nephron’s distal portion allow for the controlled reabsorption of sodium and water. These cells

express hormone receptors that can modulate reabsorption, including those for vasopressin and aldosterone.

Vasopressin is a peptide hormone secreted from the posterior pituitary that increases the ability for water reabsorption via actions on the vasopressin-2 receptor (V2R). This reduces urine volume and urination frequency, explaining why vasopressin is also known as the antidiuretic hormone. The kidney also secretes hormones to modulate reabsorption. In response to low blood pressure, endothelial cells lining the glomerlus’ afferent arteriole secrete the enzyme renin into the circulation to cleave the plasma protein angiotensinogen into angiotensin I, which is then converted to angiotensin II (AngII) by the angiotensin-converting-enzyme (ACE) in the lungs and plasma [29]. AngII helps counter low blood pressure via direct and indirect mechanisms. First, it is a potent vasoconstrictor and can act directly on the nephron’s proximal tubule to increase sodium reabsorption. In higher doses, AngII stimulates the secretion of the steroid hormone aldosterone from the adrenal cortex. Aldosterone amplifies AngII’s sodium retaining capacities by increasing the ability of the nephron’s distal epithelial cells to reabsorb sodium into the circulation.

Physiological mechanisms that modulate vasopressin function or activity of the renin-angiotensin- aldosterone system (RAAS) will effectively alter renal function and directly affect blood pressure.

For example, analogues of vasopressin are commonly administered in critical care medicine to increase fluid retention in the management of hemorrhagic shock [30, 31] while ACE inhibitors that block AngII and aldosterone formation remain a standard treatment for hypertension [32].

1.3 Regulation of Fluid Balance

1.3.1 Osmosis in the Context of a Single Cell

The physico-chemical process of osmosis dictates that water will flow across a permeable membrane to equilibrate solute concentration on both sides. The force that drives this movement is referred to as hydrostatic pressure, and relates to the difference in solute concentrations on either side of a water-permeable membrane. Eukaryotic cells comprise such a water-permeable membrane via the expression of proteins such as aquaporins [33], and although water is a biological necessity for cell survival, too much or too little is lethal [34]. For example, if a cell is placed in a diluted environment where its internal solute concentration is greater than that of the extracellular fluid, water will flow into the cell in an effort to achieve solute equilibrium, causing the cell to swell and lyse. The opposite is true if a cell is placed into a concentrated solution, where the internal solute concentration is now less than the extracellular fluid. In this hypertonic state, water will be drawn from the cell to compensate for the higher extracellular solute concentration, causing the cell to shrink and perish.

1.3.2 Systemic Osmoregulation

Maintaining osmolality, a measure of total solute concentration, within ranges that favours cell survival is a prerequisite for mammalian life [35]. Healthy humans and rats strive to maintain the osmolality of their extracellular fluid (ECF) near the osmotic set-point of approximately 288 milliosmoles per kilogram (mosmol/kg) and 294 mosmol/kg, respectively [35]. Maintaining ECF osmolality near this value is critical to survival, since changes in ECF tonicity will quickly draw water into or out of tissues as dictated by osmosis (section 1.3.1). Mammals are able to correct for incessant osmotic perturbations faced in their environment in both a behavioural and neuroendocrine fashion. Specifically, any deviation from osmotic equilibrium adjusts the intricate

balance between water intake and excretion to stabilize ECF tonicity [34-37]. Indeed, even changes in plasma osmolality by a single percent has been shown to recruit homeostatic osmoregulatory mechanisms [38]. Environmental stimuli that create a hypertonic ECF, including the ingestion of salt or the evaporative loss of water, triggers osmoregulatory mechanisms that coordinate an increased desire for water (thirst), decreased salt appetite, increased retention of water

(antidiuresis) and decreased retention of sodium (natriuresis). In contrast, when faced with environmental stimuli that dilute the ECF such as consuming excess water or insufficient amounts of salt, osmoregulatory mechanisms will act to reduce the sensation of thirst, increase salt appetite, promote water excretion (diuresis) and increase sodium retention. Failure to properly osmoregulate can have lethal consequences [39]. Enclosed in a rigid cranium, the brain is particularly vulnerable to the swelling and shrinking effects of uncompensated changes in ECF osmolality. This includes death associated with excessive water intake [40, 41], severe dehydration [42] and abnormally excessive sodium intake [43].

1.3.3 The OVLT as the Central Osmoreceptor

The mammalian capacity to thrive in a perpetually variable osmotic environment suggests the evolution of sensory mechanisms capable of detecting osmotic perturbations of the ECF and coordinating timely compensatory responses. Seminal work by Verney helped cement the concept that a central detector for osmotic pressure, which he termed osmoreceptor, resides within the brain

[44]. Lesion studies followed his initial observations, and highlighted the anterior hypothalamus as a portion of tissue necessary for proper osmoregulation [45]. Investigations of the anterior hypothalamus revealed that a significant portion of the anteroventral region of the third ventricle

(AV3V) lacks a blood-brain barrier, and has the capacity to adequately sense the contents and osmolality of the body’s plasma. Further studies delineated the organum vasculosum laminae

terminalis (OVLT) as a key osmosensory site within the AV3V [46]. The mechanistic properties underlying osmoreception in the OVLT will be discussed in section 1.3.6.3a.

The OVLT’s ability to sense systemic changes in plasma osmolality is a key feature for its role in the initiation and maintenance of osmotic homeostasis [36, 47, 48]. Neurons within the

OVLT project to effector sites that coordinate thirst behaviour (section 1.3.2) and vasopressin secretion into the posterior pituitary (section 1.2.3). Our behavioural urge to drink in hypertonic states, known as osmotic thirst, relies extensively on neural projections from the OVLT to the median preoptic nucleus (MnPO) [48, 49]. The neural circuits underlying osmotic thirst have been reviewed extensively [36] and will not be further discussed in this thesis. The OVLT’s specific role in modulating vasopressin secretion will be discussed in section 1.3.6.3a after a brief introduction to the neuroendocrine mechanisms that underlie vasopressin secretion.

1.3.4 Neuroendocrine Mechanisms Underlying Vasopressin Secretion

Verney’s original observations that the amount of vasopressin (VP) liberated from the posterior pituitary (section 1.3.2) increases as a function of plasma osmolality motivated the investigation and characterization of the cellular entities responsible for its secretion [44, 50, 51].

Experiments by Bargman and Scharrer using a stain selective for neurosecretory products traced the fiber tracts of neurosecretory terminals in the posterior pituitary. They revealed that these terminals were in fact axonal projections of magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei [52]. Harris was the first to suggest that electrical signaling between magnocellular neurosecretory cells (MNCs) and their terminals mediated neurosecretion [53, 54].

His “neurohumoral transmission” hypothesis was proposed in a time of revolutionary neuroscientific findings, including the discovery of the ionic basis for action potential propagation and evidence of chemical neurotransmission [55]. Ultimately, Harris’ proposal underlined that

secretory neurons controlled the release of hormones from their terminals facing the posterior pituitary through the discharge of action potentials down their hypothalamo-hypophysial tracts.

This hypothesis was confirmed by Douglas and Poisner, who revealed calcium-dependent increases in VP concentration upon electrically stimulating the secretory terminals of isolated posterior pituitaries [56]. Their results support the “stimulus-secretion coupling” model of VP neurosecretion [57], which posits that action potentials emanating from MNCs depolarize the secretory terminals and open voltage-gated calcium channels to allow calcium influx and VP secretion.

1.3.5 Electrophysiological Characterization of VP-Containing MNCs

The combination of Verney’s observation that increases in ECF tonicity induce VP secretion and Douglas and Poisner’s stimulus-secretion coupling model (section 1.3.4) motivated investigations that characterized the electrical activity of VP-secreting MNCs (VP MNCs) in the

SON and PVN. Using antidromic stimulation [58], an electrophysiological identification tool through which the stimulation of a nerve ending can trigger a detectable back-propagating action potential in the cell soma, electrophysiologists were able characterize the electrical properties of identified mammalian MNCs that evoked antidromic action potentials upon posterior pituitary stimulation. Eric Kandel was the first to report intracellular recordings of action potentials in magnocellular cells of fish, and suggested that they behave similar to central neurons [59].

The electrophysiological characteristics of VP MNCs have been thoroughly investigated

[60-62]. The consensus reports that VP is released from VP MNCs in an activity-dependent manner, whereby the frequency and pattern of evoked action potentials directly correlates with the quantity of secreted VP [63]. VP MNCs demonstrate three consistent patterns of action potential discharge: slow irregular firing, fast continuous firing, or phasic firing [62, 64-68]. Slow irregular

firing is characterized by a low mean firing rate of 1-3 Hz, while fast continuous firing reflects a discharge frequency of generally more than 3 Hz. Additionally, VP MNCs also engage in phasic firing patterns, hallmarked by long bursts of discharge followed by silent periods, and significantly high intraburst discharge frequencies [68]. Although the complete ionic basis underlying burst firing remains unclear, these bursts are thought to favour spike broadening and enhanced calcium influx [69] that represent a physiological adaptation to maximize hormone release [70, 71].

1.3.6 Intrinsic and Extrinsic Modulation of Vasopressin MNCs

Similar to motor neurons in which action potential discharge elicits movement, MNCs represent a neural output that coordinates physiologically meaningful effects through hormone secretion. This “all-or-nothing” locus implies that these neurons represent the final integrative hub in coordinating feedback mechanisms using action potential discharge when the osmotic environment is perturbed via changes in plasma osmolality or volume. Evidence suggests that VP

MNCs are regulated by intrinsic mechanisms, glial interactions, and synaptic afferents originating from osmosensory and cardiovascular origin.

1.3.6.1 Intrinsic Osmosensitivity of VP MNCs

Evidence confirming that VP MNCs are intrinsically capable of detecting extracellular changes in osmotic pressure was first reported by Mason [65]. He demonstrated that VP MNCs embedded within acute hypothalamic slices could be depolarized in response to hyperosmotic solutions even in conditions where chemical neurotransmission is blocked. Although a portion of this depolarization could be mediated by extrinsic mechanisms including gliotransmission [72],

Oliet and Bourque undoubtedly demonstrated that hyperosmotic stimulation can depolarize acutely isolated MNCs that retain no glial or synaptic partners [73]. Initial electrophysiological evidence of hyperosmotically-induced depolarization was associated with a decrease in the

membrane resistance of isolated MNCs. This highlighted that these neurons’ osmoreceptive capabilities likely rely on cation influx [73]. Further studies by Oliet and Bourque revealed that this enhanced ionic conductance was mediated by the opening of single ion channels whose probability of opening were associated with decreases in cell volume, and thus were named

“stretch-inactivated” cation (SIC) channels [74, 75]. Reducing cell volume (cell shrinkage) can be achieved as a result of hyperosmotic stimulation, where osmosis dictates water should flow out of cells (section 1.3.1), or through the application of negative pressure through the recording pipette in a whole-cell recording configuration [76]. Pharmacological experiments causally demonstrated that this SIC bridged the conversion of the cell’s mechanical stimulation created by shrinking and osmosensitive depolarization. When isolated MNCs were hyperosmotically stimulated in the presence of gadolinium, a known blocker of SIC channels in yeast [77], gadolinium’s presence inhibited the characteristic osmosensitive currents normally evoked by hyperosmotic stimulation, but did not alter the expected reduction in cell volume. This result asserted the SIC channel as the molecular mechanotransducer underlying osmoreception in MNCs [78]. The SIC channel was later identified as a truncated form of the transient receptor potential vanilloid 1 receptor (TRPV1) [79] and cloned as the ΔN-TRPV1 receptor [80]. These channels were later found to interact with a calcium-dependent phospholipase C [81] and the MNC’s inherent cytoskeletal machinery [82, 83] to activate.

The role of intrinsic depolarization in response to hyperosmotic ECF as a result of ΔN-

TRPV1 receptor activation was first conceptualized as a mechanism to enhance the capacity of

MNCs to discharge action potentials upon receiving extrinsic excitatory postsynaptic potentials

(EPSPs) [38, 84]. Since the membrane potential of VP MNCs typically lies below the action potential threshold in resting osmotic conditions [38], increasing the conductance of ΔN-TRPV1

channels in hyperosmotic conditions could provide an additional depolarizing force to the VP

MNCs membrane potential, thus facilitating afferent EPSPs to reach the action potential threshold in an effort to liberate VP and promote antidiuresis.

1.3.6.2 Glial-MNC Interactions

While VP MNCs demonstrate a capacity to independently detect and respond to changes in ECF osmolality through the ΔN-TRPV1 receptor, gliotransmission can also modulate the

MNC’s resting membrane potential [85]. In hypotonic conditions, astrocytes embedded within the

SON release taurine via volume regulated anion channels (VRACs) that agonize inhibitory glycinergic receptors (GlyRs) on VP MNCs [72, 86]. Activated GlyRs induce chloride influx that hyperpolarizes the VP MNC and makes it harder for afferent EPSPs to reach the action potential threshold.

1.3.6.3 Afferent Modulation of VP MNCs

1.3.6.3a Osmotic Modulation: OVLT

As previously highlighted in section 1.3.3, the OVLT comprises the central osmoreceptor that can detect changes in plasma osmolality and project to effector sites to coordinate VP secretion. OVLT neurons, like VP MNCs, are intrinsically osmosensitive through the expression of mechanosensitive channels identical to VP MNCs [80, 87, 88], are osmotically controlled by gliotransmission [89-91], and also increase their rate of action potential discharge as a positive function of plasma osmolality [38]. Anatomical tracing [92] and electrophysiological [93, 94] studies have revealed excitatory glutamatergic and inhibitory GABAergic projections of OVLT neurons to VP MNCs of the SON (SONVP) that can evoke monosynaptic inhibitory and excitatory postsynaptic potentials. The OVLT→SONVP synaptic connection has been studied extensively and suggests that osmotically evoked changes in SONVP MNC firing rates are selectively mediated

through increases or decreases in the intensity of the excitatory component of OVLT-derived inputs [95-97]. Recent work has demonstrated that extrinsic factors can modulate the strength of the OVLT→SONVP synaptic connection. Central clock neurons presynaptically disinhibit the osmoreceptor-mediated synaptic excitation during the late sleep period to enhance VP release [98] while local application of AngII (section 1.2.3) can potentiate OVLT→SONVP synaptic excitation

[99].

1.3.6.3b Cardiovascular Modulation: Baroreceptors

As introduced in section 1.2.2, carotid baroreceptors activate in conditions that stretch blood vessels and initiate homeostatic mechanisms to lower blood pressure. These changes in blood pressure also modulate VP release via GABAergic baroreceptor projections to SONVP neurons. Studies elucidating the ascending pathway of the baroreceptor afferents highlight the locus coeruleus [100], diagonal band of Broca [101-103], and perinuclear zone [104] as essential relay nuclei for this circuit. This inhibitory baroreceptor input represents a negative feedback mechanism to decrease VP release in conditions of high blood pressure [102, 105]. Reducing circulating VP concentrations helps normalize high blood pressure in two ways. First, vasopressin promotes the increase of blood volume through its effects on the kidney (section 1.2.3), and in higher concentrations, vasopressin acts as a potent vasoconstrictor [47, 106, 107]. Therefore, reducing VP levels should alleviate a condition of high blood pressure (discussed in section 1.5.5).

1.3.7 Fluid Balance and Plasma Sodium

The core principle of fluid balance suggests that the amount of water and solute excreted from the body must equal the amount of water and solute consumed (section 1.2.3). Having evolved from seawater organisms, our ECF is rich in many electrolytes found in the ocean, none of which is more prevalent than sodium. As the main solute particle in our ECF, sodium is a chief

controller of osmotic pressure and blood volume. Additionally, sodium also plays life-sustaining roles in acid-base balance, cell metabolism and nervous system function. Sodium consumption is therefore critical to our survival. Estimates suggest that the minimum amount of sodium required for proper bodily function in humans is at least 500mg per day [108, 109]. Due to the importance of maintaining sodium homeostasis, mammals have evolved mechanisms distinct from osmoreception (introduced in section 1.3.2) to monitor the level of plasma sodium. Importantly, the subfornical organ (SFO) is thought to represent the primary locus for sodium-sensing and the control of salt-appetite [110-114]. Our sodium sensing mechanisms help initiate homeostatic responses when ECF sodium concentration is perturbed from the physiological set-point of 135-

145 milliequivalents (mEq) [114]. In conditions of low plasma sodium, or hyponatremia, the

RAAS (as explained in section 1.2.3) becomes activated to increase sodium retention. In conditions of high plasma sodium, or hypernatremia, the RAAS becomes inhibited to promote sodium excretion while osmoregulatory mechanisms (as explained in section 1.3.2) promote water retention to equilibrate ECF sodium levels and osmolality. Although these compensatory mechanisms are thought to allow for the normalization of plasma sodium in varying conditions of sodium intake, evidence from human [7, 115, 116] and animal [117-119] studies reveal that the consumption of excess dietary sodium can lead to small but sustained increases in the concentration of plasma sodium. While the previous two sections aimed to highlight the complex physiological mechanisms underlying the regulation of blood pressure (section 1.2) and fluid balance (section 1.3), the chronic increase in plasma osmolality as a result of consuming excess dietary sodium can lead to sustained increases in ECF volume, which may influence blood pressure

[7, 120].

1.4 High Dietary Sodium Intake as a Risk Factor for Hypertension

1.4.1 Changes in the Perception and Intake of Salt Throughout Human History

Sodium chloride (salt) has played significant roles in civilization throughout human history. It was once considered to have immense religious significance, and because it does not decompose, it was thought by some to represent life itself [121]. Salt was used in ancient times as a medical remedy for skin lesions, edema, and digestive troubles while also playing central roles in the economies of many regions [122]. The search for salt was a significant driving force in the creation of trade routes throughout the early world and was sometimes implicated in the outbreak of conflicts [123]. Indeed, it was a precious commodity and currency in ancient times; the term salary originates from salarium, or salt money, given to Roman soldiers [121].

Despite the important cultural roles that salt played in shaping human behaviour, nutritional anthropological models suggest that salt was not heavily consumed in ancestral human civilizations [124, 125]. Preagricultural humans were predicted to have consumed less than 800 milligrams of sodium each day [5, 109, 125], likely as a result of its scarcity in a diet composed mainly of vegetables and animal meats. The modern perception towards dietary sodium has radically changed in the last 250 years [126]. Our industrialized civilization has become capable of producing large-scale quantities of salt, making it an abundant and cheap substance [127].

Strikingly, food corporations ubiquitously add salt to processed foods in order to improve shelf- life, flavour and texture [128, 129], making industrialized humans the only species known to have acquired the habit of consuming excess salt [124]. Indeed, approximately 75% of sodium in foods are added during processing [130]. Current estimates suggest the average sodium intake in the industrialized world (further discussed in section 1.4.2) ranges from 3.5-5.5 grams, or 8-12.5 grams of sodium chloride [109, 131, 132], well in excess of physiological need (section 1.3.7).

1.4.2 Forming the Relationship between High Dietary Salt Intake and Hypertension

High dietary salt intake was first proposed to be harmful to the body in the early 20th century. Ambard and Beaujard suggested that excess sodium intake could increase arterial blood pressure [133], a claim that fueled mixed opinions of support [134, 135] and skepticism [136, 137].

Reducing salt intake as a treatment for hypertension was serendipitously revolutionized by Walter

Kempner in the 1940s in his treatment for renal failure. Kempner recognized that because renal failure impairs the kidney’s ability to excrete metabolic waste products, he placed patients suffering from renal failure on diets carefully designed to decrease waste product metabolism and hence, reduce demands on kidney function. Low-fat and low-sodium coupled with high-protein content characterized this approximately 2,000 calories per day diet, which was named the

Kempner rice diet [138]. Strikingly, patients suffering from renal disease and malignant hypertension placed on his diet had better therapeutic results than patients treated with standard antihypertensive treatments at the time; diuretics and herb extracts [139]. Although Kempner was not interested in the specific components of his blood-pressure reducing diet, Grollman was the first to suggest that the hypotensive locus of Kempner’s rice diet lied within its low sodium content

[140]. This observation helped motivate the first small-scale studies that surveyed the relationship of one’s sodium consumption and blood pressure.

In the 1950s, Louis Dahl, a physician and researcher, probed the possible relationship of salt intake and hypertension among every member of the Brookhaven national laboratory who reported for their annual physical examination. He and his colleagues surveyed their patients’ perception of how much sodium they consumed, and roughly classed their responses into low, average or high levels of intake. Complementing their survey data with blood pressure measurements revealed a significant and positive correlation between the perceived quantity of

salt consumption and mean blood pressure [141]. Further characterizing this association using sodium excretion, a physiological proxy for sodium intake, revealed that individuals who reported consuming larger amounts of dietary sodium in fact had both increased levels of sodium in their urine and mean blood pressures [142]. While this study probed subjects of the same society, inter- populational studies also began to associate higher salt intakes with higher blood pressures in various world regions [131, 143-148].

In the late 1980s, a systematically organized international study was performed to standardize the relationship between sodium excretion and blood pressure. The Intersalt initiative analyzed urine samples over a 24-hour period in relation to blood pressure recordings of over

10,000 subjects from 52 populations in 32 countries [149]. Importantly, 48 of these centers recruited subjects from the industrialized world, while four centers recruited subjects from isolated, indigenous populations whose dietary habits reflect those of Paleolithic humans, including a very low sodium intake. The standardized estimates of the Intersalt study revealed strikingly strong relationships that insinuate high salt intake as a detrimental factor to human health through its effects on blood pressure. Differences in sodium intake and effects on blood pressure between the industrialized and remote populations were enormous. Median daily salt intake in the industrialized populations was reported to be 9 grams while average blood pressures were 120 mmHg systolic 74 mmHg diastolic. In contrast, the remote population’s median salt intake ranged from 1-3 grams, while average blood pressures were reported to be 103 mmHg systolic 63 mmHg diastolic [130, 150, 151]. Within each industrialized population, the amount of sodium excretion increased with blood pressure and age, while in the four remote populations, average sodium excretion was significantly lower than industrialized societies and did not increase with blood pressure or age. Coincident with the standardized estimates reported by the Intersalt study and the

growing public health implications associated with a high dietary sodium intake, many governmental organizations and research societies began recommending sodium reduction across the world. The World Health Organization currently recommends the intake of no more than 2 grams of sodium, or 5 grams of table salt per day [8] while the American Heart Association advises the daily intake of less than 1.5 grams of sodium [152]. These recommendations suggesting an approximate 50% reduction in average sodium intake relative to current consumption (section

1.4.1) seem feasible when reconciling that the minimum sodium intake needed for survival ranges near 0.5 grams (section 1.3.7) and some indigenous peoples live on nearly 1 gram of sodium per day. However, less than one percent of Americans consume sodium below the AHA guidelines, while almost one third are hypertensive [153].

1.4.3 Sodium Reduction as a Strategy to Reduce Blood Pressure

The correlative relation between sodium intake and blood pressure motivated small- and large-scale clinical trials that ultimately formed a causal relationship between sodium intake and blood pressure. Restricting access to sodium reversibly reduced mean blood pressures of hypertensive patients in double-blind, randomized clinical trials [2, 154-157] and in community- based interventions [158]. One study even remarked that modestly reducing salt intake reduced blood pressure by the same magnitude as a frontline antihypertensive treatment [154]. Further studies that monitored acute changes in mean blood pressure in humans [159] and chimpanzees

[127] before and after eating a single meal containing excess salt revealed sustained increases in mean blood pressure that remained for hours after consumption.

The Dietary Approaches to Stop Hypertension collaborative research group, or DASH, is of special mention in that they performed two rigorously controlled clinical trials to cement the causality in how first modifying dietary patterns, and second in solely modifying levels of sodium

intake, could reduce blood pressure. Their first multicenter, randomized feeding study investigated changes in blood pressure and electrolyte balance when consuming a typical American diet versus the low-fat, vegetable-, mineral-, and fiber-rich “DASH” diet [160, 161]. They found that if sodium and alcohol intake were kept constant between groups, consuming the DASH diet significantly decreased average systolic blood pressure by 5.5 mm Hg and diastolic blood pressure by 3.0 mm

Hg compared to subjects remaining on the American diet. In their second study, they demonstrated that subjects consuming the DASH diet combined with a high, intermediate, or low level of sodium intake revealed significant dose-dependent reductions in average systolic and diastolic blood pressures [162]. Thus, even when consuming a vegetable-heavy diet low in saturated fat and high in magnesium, potassium, calcium and fiber, the hypotensive effect of reducing sodium intake is not masked. In consolidation of the DASH trials, meta-analyses of clinical trials (e.g. [6, 115, 163]) have further characterized the hypotensive effects of sodium reduction. Altogether, these results causally relate the hypertensive effect of high dietary sodium intake, and establish the need to catalyze initiatives with the goal of reducing sodium consumption not just in research studies but within the widespread general population.

1.4.4 Projected Effect of Reducing Sodium Intake on Cardiovascular Health

The implementation of effective campaigns aimed at reducing sodium intake at the population level would require action by many stakeholders including legislators and food corporations [164]. A hallmark predictive model estimates that by reducing the daily average sodium intake of Americans by 1.2 grams, 60 to 100 thousand new cases of coronary heart disease,

32 to 66 thousand stroke incidents and 54 to 99 thousand heart attack incidents could be prevented each year. Ultimately, this would prevent 44 to 92 thousand deaths in America and reduce healthcare costs by $10 to $24 billion annually [1]. These initiatives have yet to take place, likely

as a result of major food corporations who benefit financially from increased sodium contents in their products. Their vested economic interests and power in governmental lobbying against sodium regulations may represent a significant obstacle in the ability to initiate large-scale salt reduction programs [129].

While effective sodium reduction campaigns remain to be seen in the United States, more than thirty countries have begun some effort to reduce sodium intake within the population, the majority of which have been cost-effective [165]. Finland stands out in that it recently achieved large-scale reductions in population sodium consumption. Combining the Finnish media to emphasize the harmful effects of salt, and salt-labelling legislation that required food companies to clearly print the sodium content of their products fueled reports and public opinion on the availability of healthier, good-tasting alternatives [166, 167]. This initiative reduced the population’s average sodium intake to nearly 2 grams per day, increased average life expectancy, decreased average blood pressure and severely reduced the death rates due to stroke and heart attack [168-170].

While section 1.4 aimed to causally link high levels of sodium consumption with high blood pressure, and posit that reducing sodium intake can ultimately increase global health, the biological mechanisms by which salt raises blood pressure remain unclear. The evidence outlined in this section suggest that the incidence of hypertension among societies consuming low salt diets will be significantly less than in societies consuming high salt diets. However, these population- level observations do not reveal the individual physiological susceptibilities of developing hypertension as a result of high salt intake.

1.5 Mechanisms Underlying Salt Sensitive Hypertension

1.5.1 Salt Sensitivity: An Overview

The Darwinian principle of natural selection implies that genetic mutations capable of increasing an organism’s ability to prosper within its environment will inevitably produce and raise more offspring that can transmit its genetic material. Having lived in sodium-scarce environments for hundreds of thousands, if not millions, of years, the humans that were best able to cope with small quantities of this life-sustaining mineral were those who ultimately gave rise to modern day humans. The RAAS (section 1.2.3) represents the most powerful sodium-retaining mechanism evolved to survive in sodium-poor environments [126]. Investigating RAAS activity in relation to sodium excretion reveals that if average daily sodium intake exceeded 1.15 grams, the RAAS system would no longer promote sodium reabsorption [166]. Since prehistoric humans and those presently living in some remote populations continue to consume less than 1 gram of sodium per day [150], their RAAS likely remains active in an effort to combat the chronic state of low blood pressure associated with hyponatremia. In complement with the development of a powerful salt appetite equipped with salt-seeking taste buds [171], the perpetually low-sodium environment throughout human history gave rise to humans who are driven to consume salt and expertly capable at retaining the minute traces they find. This implies that early humans who developed genetic mutations which enhanced their capacity to excrete sodium were at a disadvantage in ancient times

[28], whereas they would likely thrive in today’s industrialized world. Indeed, modern society has left the sodium-retaining experts of the human race with diets that contain exorbitant quantities of sodium unimaginable to our human ancestors.

While section 1.4 focused on the effect of high dietary salt intake and its ability to increase blood pressure at the population level, investigations at the individual level reveal a continuous

variability in how sodium loads affect blood pressure [172]. This variability, known as salt sensitivity, implies that two individuals ingesting the same salt load can have drastically different blood pressure responses (further discussed in section 1.5.3.2). Although the concentration of sodium loads and blood pressure response criteria of what salt-sensitivity implies continue to differ in many studies [173], they are thought to yield congruent results [173]. Remaining conscious of this variable definition, it is thought that approximately 30% of the normotensive population is salt-sensitive, while the incidence of salt-sensitivity is significantly higher in individuals with hypertension [174]. Importantly, salt-sensitive normotensives have a greater risk of developing hypertension than salt-resistant normotensives [175].

While evolutionary perspectives may speculate that our expertise in retaining excess sodium could now represent a detrimental adaptation in the face of a high salt diet, the physiological basis for what characterizes an individual as salt-sensitive, and why high dietary salt intake can induce hypertension in these individuals more frequently than salt-resistant individuals remains unclear. In the late 1940s, the notion of a hereditary component underlying human essential hypertension was proposed. While secondary hypertension was then defined as hypertension resulting from an observable cause, including renal failure or an adrenal tumor, Louis

Platt suggested that cases of human essential hypertension, in which no cause for the hypertension could be found, represented the manifestation of hypertension genes that depend on other genetic or environmental factors to become expressed [176, 177]. In the 1960s, Louis Dahl recognized

Platt’s idea and subsequent studies that furthered his claim for heredity in hypertension [178, 179].

Dahl suggested that an environmental stimulus may induce essential hypertension in genetically susceptible individuals. This motivated his pursuit of animal studies aimed at characterizing the gene-environment interactions underlying salt intake and the genesis of essential hypertension.

1.5.2 Salt-Sensitive Hypertension as a Continuous, Multifactorial Disease

Human beings are the only terrestrial mammals that have evolved to consume sodium in levels well above physiological necessity (section 1.4.1). Even our most phylogenetically-related species, the chimpanzee, consumes sodium in amounts similar to those of stone-aged humans

[127]. In 1953, Meneely and colleagues first characterized the toxic effects of excess sodium intake in Sprague Dawley rats, where seven groups of animals were fed increasing concentrations of dietary sodium [180]. They reported an association between the quantity of dietary sodium ingested and increases in both morbidity and average blood pressure that varied in proportion to the quantity of ingested sodium. At the individual level, however, they reported a marked degree of variation in the response of each animal’s change in blood pressure to high sodium intake in all seven groups [180].

Louis Dahl recognized the potential for genetic interplay to be the source of variation in

Meneely’s experiments. Dahl reported similar observations demonstrating wide variations in blood pressure responses of Sprague-Dawley rats placed on a high salt diet [181]. Approximately

20% of animals exposed to chronic excess dietary salt for a full year remained normotensive while the others increased blood pressure ranging from slight increases to fulminating hypertension. Dahl reasoned that if an animal’s sensitivity to salt was genetically controlled, it should be possible to separate two strains that differ in the incidence and gravity of hypertension developed from excess salt consumption [182]. Through the selective inbreeding of animals that had the largest increases in blood pressure and animals that remained normotensive in response to a high sodium load, Dahl created two strains of rats that markedly differed in their sensitivity to salt [183, 184]. The strain most prone to developing hypertension from salt was named the sensitive (S) strain, while the strain least prone to developing hypertension from salt was termed the resistant (R) strain.

Importantly, he demonstrated that the S strain was not spontaneously hypertensive. Although genetically predisposed, the additive environmental factor of dietary salt was necessary for hypertension to manifest in the S strain.

Dahl’s studies provided the first genetic model of salt-sensitive hypertension, which suggested that complex genetic factors play a role in determining blood pressure responsiveness to a salt load. Nevertheless, new animal models continue to emerge that can consistently induce hypertension through the treatment of high salt intake that can be coupled with defined physiological manipulations. These include replacing an animal’s drinking water with 2% sodium chloride for 7 days, known as salt loading [107] or providing chow with high sodium content coupled with the systemic administration of deoxycorticosterone (DOCA) [182], or the subcutaneous infusion of AngII [185], further discussed in section 1.5.7. These distinct models capable of inducing salt-sensitive hypertension models in otherwise wild type, salt-resistant animals, highlight the multifactorial genetic complexities that likely underlie salt-sensitivity.

Initial investigation into the differentiation of salt-sensitive and salt-resistant animals highlighted the kidney’s compromised ability to excrete excess sodium. These observations were first made in renal cross-transplantation studies between Dahl S and R rats. Specifically, transplanting kidneys from R rats into S rats were shown to consistently ameliorate their regulation of blood pressure, whereas transplantation of S kidneys into R rats consistently induced hypertension [182]. Since the kidney represents the major long-term regulator of blood pressure through its regulation of sodium and water excretion (section 1.2.3), it is suggestive that the manifestation of salt-sensitive hypertension is a result of some sort of dysfunction in the renal handling of sodium.

1.5.3 Renal Dysfunction in Salt-Sensitive Hypertension

1.5.3.1 Overview of Pressure Natriuresis

The renal-body fluid feedback mechanisms proposed by Guyton highlight the kidney as a major regulator of blood pressure through its ability to normalize temporary imbalances in ECF volume via changes in natriuretic and diuretic capacities [20, 25]. The concept of pressure- natriuresis, implied from section 1.2.3, proposes that increases in blood pressure will cause the kidney to promote sodium excretion (natriuresis) [186]. Natriuresis effectively reduces ECF volume, which in turn reduces cardiac output and blood pressure. Pressure-natriuresis is largely mediated by the RAAS (section 1.2.3), where increased blood pressure inhibits the secretion of renin and thereby AngII [186]. Early studies that characterized the pressure-natriuresis response in normotensive subjects [187, 188] found that when healthy humans are placed on diets containing

0.2 to 10 grams of sodium per day, urinary sodium content increases proportionally, while mean blood pressure remains stable. However, when consuming extreme levels of sodium such as 20 or

34 grams per day, mean blood pressure significantly increases [188]. These studies purport that the pressure-natriuresis response to excess sodium intake grants salt-resistant subjects exceptional flexibility in daily fluid and electrolyte intake so long as daily sodium intake remains in the normal range of 0-10 grams. A net gain of plasma sodium and water will arise in conditions where sodium intake exceeds this range, which increases blood volume and pressure.

1.5.3.2 Impaired Pressure-Natriuresis in Salt Sensitive Hypertension

Guyton first suggested that some hypertensive disorders could be characterized by renal alterations that cannot compensate for enhanced sodium intake without increasing blood pressure, effectively predisposing them to have impaired pressure-natriuresis relationships [25]. This motivated investigation into the effects of salt loading on blood pressure responsiveness in

hypertensive patients [189-192]. Each of these studies revealed two clusters of salt-sensitive and salt-resistant pressure-natriuresis responses within the groups of hypertensive patients in response to a sodium load of 5-7 grams per day. The salt-resistant cluster acted like normotensive controls in that they remained capable of achieving blood pressure homeostasis in response to salt loading, while the salt-sensitive cluster took significantly longer to achieve sodium balance which was accompanied by at least a 10 mmHg increase in mean blood pressure [172]. Further investigations revealed that although both groups increased their cardiac output, the magnitude of increase was significantly greater in the salt-sensitive populations [191]. Altogether, these results suggest that salt-sensitive individuals cannot normalize their ECF volume in response to a salt load without a concurrent increase in blood pressure within the “normal" range of sodium intake (0-10 grams).

Ultimately, blood pressure homeostasis becomes sacrificed in salt-sensitive individuals to maintain sodium balance at levels of daily sodium intake common in the industrialized world. This leaves salt-sensitive individuals who consume a high salt diet at a magnified risk for developing hypertension [193, 194].

The exact combination of genetic and physiological alterations that play a causative role in the induction and maintenance of salt-sensitive hypertension continue to remain a hotly debated topic [9, 28, 126, 164, 172, 174, 182, 195-203]. A variety of studies have revealed pathophysiological alterations present in animal models of salt-sensitive hypertension, including gene mutations for ion channels and transporters implicated in sodium reabsorption [120, 174], renal vasodysfunction [196], renal inflammation [204], abnormally high levels of circulating AngII

[172, 205, 206], increases in total peripheral resistance [174], vascular endothelial dysfunction [9], impaired nitric oxide signaling [207], reduced number of functional nephrons [28, 182, 203], enhanced sympathetic nerve activities [201] and exaggerated pro-hypertensive neuroendocrine

mechanisms [107]. Although each of these identified alterations warrant further discussion of their physiology and pathophysiology, evidence suggests that most forms of hypertension include a significant contribution from enhanced sympathetic drive [208]. Therefore, this thesis will now focus on the effects of high salt intake on increasing blood pressure through sympathetically- mediated increases in peripheral resistance and neurogenically-mediated increases in water and sodium retention.

1.5.4 Role of the Autonomic Nervous System in Salt Sensitivity

1.5.4.1 Sympathetic Control of Cardiovascular and Renal Function

As introduced in section 1.2.1, the autonomic nervous (ANS) system plays a pivotal role in the regulation of blood pressure. It does so by integrating baroreceptive sensory information that initiate homeostatic mechanisms to control blood pressure through the regulation of sympathetic nerve activities (SNA). Blood pressure is ultimately a function of cardiac output and total peripheral resistance (TPR). The ANS, via its regulation of SNA, can modulate both of these factors, as well as fluid balance [209]. Sympathetic efferent nerves branch from thoracic/lumbar intermediolateral (IML) columns of the spinal cord and release norepinephrine to activate α and

β-adrenergic receptors on target organs [210]. Activation of the cardiac sympathetic nerves increases cardiac output, while the lumbar and splanchnic nerves increase TPR [119]. Enhanced

SNA (sympathoexcitation) occurs in response to a variety of environmental perturbations, including hypernatremia [119, 201, 211, 212]. In response to an acute hyperosmotic stimulus, osmoregulatory mechanisms (as explained in section 1.3.2) will act to increase blood volume to normalize plasma osmolality and maintain body fluid homeostasis. This increase in blood volume stretches baroreceptors which in turn enhance parasympathetic vagal responses and decreases SNA to reduce cardiac output to normalize blood pressure. However, recent evidence suggests that

chronic hypernatremia is associated with raised vasomotor tone and increased cardiac output as a result of chronically elevated SNA [208].

1.5.4.2 Abnormally Elevated SNA Following High Salt Intake

Both human and animal studies demonstrate that sympathetically-mediated increases in blood pressure are etiologically involved in the pathogenesis of hypertension [201, 204, 213, 214].

In animal studies, chronic hyperosmotic challenges increase peripheral SNA (section 1.5.4.1), and this increase is pathologically potentiated in animal models of salt-sensitive hypertension [119,

201, 202, 212, 215]. The significance of chronically enhanced sympathoexcitation in the pathogenesis of hypertension (first introduced in section 1.5.3.2) can be appreciated by reviewing the types of clinical interventions used to treat hypertensive patients. Common pharmacological treatment regimens include the use of first-line β-adrenergic receptor antagonists, or beta-blockers, while more complicated cases sometimes surgically remove specific sympathetic nerves in an attempt to normalize blood pressure [208]. These interventions have also been shown to reduce blood pressure in animal models of salt-sensitive hypertension [119, 201]. Recent work has suggested that elevated SNA observed in hypertension may result from the exaggerated activity of the autonomic neurons driving them. Post-ganglionic sympathetic efferents that arise from the

IML column of the spinal cord (section 1.4.4.1) are regulated primarily by cholinergic pre- ganglionic neurons in the rostroventrolateral medulla (RVLM) [209]. The RVLM is thought to represent the final integrative hub for the sympathetic control of blood pressure via its regulation of SNA. In animal studies, inhibiting RVLM neurons via microinjections of GABA decrease SNA and blood pressure, while chemical excitation of the RVLM via microinjections of glutamate increase SNA and blood pressure [201, 216]. Recent electrophysiological surveys of the RVLM suggests that a discrete population of neurons are responsible for the increase in SNA and ABP as

a result of glutamatergic receptor activation [216]. This suggests that the enhanced sympathoexcitation observed in salt-sensitive hypertension may result from chronic increases in

RVLM neural excitability (discussed in section 1.5.4.3). This hypothesis has motivated investigation into the electrophysiological correlates of cellular and synaptic plasticity that allow for these neurons to abnormally enhance and maintain elevated levels of SNA in neurogenic hypertension. As introduced in section 1.3.7, the consumption of excess sodium can lead to sustained increases in plasma osmolality and circulating levels of plasma sodium. Evidence suggests that detection of plasma hypernatremia is a key mediator that underlies the enhanced activity of both RVLM neurons and sympathoexcitation in animal models of salt-sensitive hypertension.

1.5.4.3 Detection of Plasma Hypernatremia as the Mediator of Sympathoexcitation

While the pressure-natriuresis response (section 1.5.3.1) allows for efficient sodium excretion in response to high sodium intake in the salt-resistant population, the salt-sensitive population’s impairment in this system may lead to small increases in the levels of circulating plasma sodium when consuming a high salt diet [120]. Recent work highlights the OVLT (section

1.3.3) as the mediator of both acute and chronic hyperosmolality-induced increases in lumbar and splanchnic SNA. Acute infusions of varying concentrations of hypertonic saline into the internal carotid artery of anesthetized animals caused dose-dependent increases in average blood pressure and SNA, which were markedly reduced upon chemically inactivating OVLT neurons with muscimol [216, 217] or completely lesioning the OVLT [211]. In animal models of salt-sensitive hypertension, chemically inactivating [218] or lesioning [219, 220] the OVLT prevented the induction of neurogenic hypertension. These results suggest that hypernatremia detected by the

OVLT may lead to the pathological excitation of RVLM neurons that in turn lead to the

sensitization of sympathoexcitatory reflexes. From a network perspective, the OVLT has been found to initiate the hyperosmotic sympathoexcitatory response through projections to the RVLM via a glutamatergic relay in the PVN [221]. Electrophysiological investigation suggests that

RVLM-projecting PVN neurons that receive OVLT input are more excitable in an animal model of salt-sensitive hypertension achieved though combining a high salt diet with a chronic infusion of AngII (animal model introduced in section 1.5.2). This increased excitability was found to be due to a reduced small-conductance calcium activated potassium (SK) current that unmasked the afterhyperpolarization potential into an afterdepolarization potential [222]. While increases in

SNA following hypernatremia mediated by the OVLT→PVN→RVLM pathway may represent a seminal contribution to the clinical understanding of salt-sensitive hypertension, blocking this sympathoexcitatory response with ganglionic antagonists does not fully restore blood pressure to normotensive levels [201, 202, 223]. This suggests that central osmoreceptor activation may also mediate salt-sensitive hypertension by other mechanisms.

1.5.5 Role of Vasopressin in Rat Models of Salt-Sensitive Hypertension

1.5.5.1 Exaggerated Release of Vasopressin in Models of Salt-Sensitive Hypertension

Hyperosmotic challenges recruit central osmoregulatory networks to increase the secretion of VP, whose actions on the kidney promote water retention via actions on the V2R (section 1.2.3).

At higher concentrations, vasopressin can also act as on VP-1-receptor (V1R) expressing vascular smooth muscle cells to mediate a potent vasoconstrictor effect (section 1.3.6.3b). While hyperosmotic stimulation, including hypertonic saline infusion [50, 224] and dehydration [50, 62,

67] have all been demonstrated to increase the firing rate and prevalence of phasic firing in VP

MNCs via extrinsic osmosensitive glutamatergic projections (section 1.3.4), VP MNCs also

receive extensive inhibitory GABAergic projections [225]. In conditions of high blood pressure and volume, GABAergic baroreceptor innervation provides a negative feedback mechanism to reduce the activity of VP MNCs (section 1.3.6.3b).

Evidence suggests that abnormal VP release is implicated in animal models of salt- sensitive hypertension [47]. Earlier studies noted that the concentration of circulating VP [226,

227] and the levels of VP mRNA in MNCs [228] are consistently elevated in animal models of neurogenic hypertension. Since VP is synthesized and released in an activity-dependent manner

(section 1.3.5), these observations suggest that these neurons are overly recruited in the hyperosmotic condition associated with salt-sensitive hypertension. Importantly, although high levels of circulating VP have powerful vasoconstrictive effects (section 1.2.3), basal levels of circulating VP are not thought to contribute to basal blood pressure tone [229]. A recent study reported that in the salt-loading animal model of salt sensitive hypertension (section 1.5.2), VP

MNCs are more electrically active in-vivo, and blocking VP signaling using a V1R antagonist significantly reduced their elevated blood pressure [107]. While the baroreceptor negative feedback response helps reduce the activity of VP MNCs in conditions of high blood pressure

(section 1.3.5.3b), this observation suggests that the inhibitory control of baroreceptors becomes reduced in salt-sensitive hypertension.

1.5.5.2 GABA is Excitatory in Salt-Sensitive Hypertension

The ionotropic GABAA receptor expressed on cell membranes allows chloride to flow across its electrochemical gradient when opened. Chloride’s reversal potential is usually more hyperpolarized than the resting membrane potential, such that when GABAA receptors open, hyperpolarization ensues as chloride fluxes into the cell. Few exceptions have been found to this observation, including reports that GABAA signaling is excitatory in development [230],

neuropathic pain [231], and epilepsy [232]. Two recent studies provide multiple lines of evidence to support the observation that GABAA receptor activation of SONVP MNCs is also excitatory in animal models of salt sensitive hypertension [107, 227]. First, gramicidin-perforated patch recordings of VP MNCs reveal that spontaneous and evoked postsynaptic currents in response to

GABAA receptor activation are depolarizing (i.e. excitatory) in SONVP MNCs in vitro. Second, in- vivo recordings of VP MNCs decrease their firing rates in response to the GABAA antagonist bicuculline. Third, in-vivo telemetry experiments reveal that agonizing GABAA receptor in the

SON increases blood pressure, which can be blocked by the systemic infusion of a V1R antagonist

(section 1.5.5.1).

Excitation as a result of GABAA receptor activation implies that chloride’s electrochemical gradient has depolarized such that chloride now effluxes from SONVP MNCs when GABAA receptors activate. Molecular investigations undertaken in the highlighted studies reveal altered expressions of chloride transporters that lead to increases in intracellular chloride concentrations of SONVP MNCs. One study revealed that increased intracellular chloride concentration was due to the downregulation of the chloride-exporting potassium-chloride cotransporter KCC2 [107].

The observation of KCC2 downregulation was found to be a consequence of locally secreted brain- derived neurotrophic factor (BDNF)-activation of the tropomyosin receptor kinase B (TrkB).

Selectively knocking down TrkB receptors in the SON during the induction of salt-sensitive hypertension preserved inhibitory GABAA receptor-mediated evoked postsynaptic potentials

[107]. The other study highlighted that the intracellular chloride concentration was increased as a result of upregulating the chloride-importing sodium-potassium-chloride cotransporter NKCC1

[233]. Together, these observations provide causal evidence that salt-sensitive hypertension increases intracellular chloride concentrations in SONVP MNCs, such that when GABAergic

projections activate GABAA receptors, they now excite SONVP neurons. Therefore, in conditions of high salt intake, the baroreceptor negative feedback control mechanism fails, and instead potentiates the activity of VP MNCs which promotes enhanced VP release and VP-mediated increases in blood pressure.

1.5.5.3 Increased Osmoreceptor Drive in Salt Sensitive Hypertension?

Accumulating evidence suggests that high dietary salt intake leads to neurogenically mediated increases in water retention in states of chronic hypernatremia. As explained in section

1.5.5.1, chronic exposure of rats to high dietary salt intake results in excessive activation of MNCs, leading to VP-mediated increases in blood pressure. Although this effect is associated with a reduction in the efficacy of inhibitory synaptic signaling by baroreceptors [47], it remains possible that a facilitation of excitatory osmoreceptor signaling and intrinsic osmosensitivity can also contribute to this process.

As introduced in section 1.3.6.3a, both GABA- and glutamate-releasing OVLT neurons make direct connections with SONVP MNCs that can evoke monosynaptic inhibitory and excitatory postsynaptic potentials. Importantly, OVLT neurons are intrinsically osmosensitive and consistently increase their firing rate in response to hyperosmotic stimulation (section 1.3.6.3a). In turn, hyperosmotic stimulation of the OVLT increases the frequency of AMPA receptor-mediated but not GABAA receptor-mediated spontaneous currents recorded in SONVP MNCs [96]. Recent work has demonstrated that extrinsic factors, including the central clock and AngII can increase the synaptic strength (i.e. gain) of the OVLT→SONVP synaptic connection in response to a hyperosmotic stimulus (section 1.3.6.3a). We suspect that the induction of salt-sensitive hypertension caused by high salt intake could also increase the synaptic gain at excitatory

OVLT→SONVP synapses. An increased synaptic gain could imply an increase in the number of

OVLT→SONVP synaptic connection through the growth of new synapses (synaptogenesis), as well as an increase in the probability of release of existing OVLT→SONVP synaptic connections.

Our motivation is also founded on studies that reveal the occurrence of new synapse formation in the SON following chronic dehydration [234-236].

1.5.6 Specific Aim

We will perform an electrophysiological study to assess the synaptic gain of the excitatory

OVLT→SONVP synaptic connection using an in vitro preparation using two distinct animal models of salt-sensitive hypertension introduced in section 1.5.2 that will be briefly described in sections 1.5.7 and 2.1. Using an acute angled hypothalamic slice that retains the OVLT→SONVP synaptic connection (see methods), we will first assess the osmoresponsiveness of SONVP neurons in response to an acute hyperosmotic stimulus. Since in vivo experiments have revealed that SONVP

MNCs are more active in salt-sensitive hypertension (section 1.5.5), we expect that the same hyperosmotic stimulus will cause a greater increase in the rate of action potential discharge of

SONVP MNCs obtained from hypertensive animals. This increased osmoresponsiveness could be attributed to either increases in the SONVP neuron’s intrinsic osmosensitivity (section 1.3.5.1) or increased osmoreceptor-mediated synaptic gain (section 1.3.5.3). To assess changes in synaptic gain, we will assess the synaptic responses of hyperosmotic stimulation of SONVP neurons through the frequency and magnitude of spontaneous excitatory postsynaptic currents (sEPSCs). If synaptic gain is found to be altered, we will assess whether increased OVLT→SONVP synaptic plasticity is mediated via effects on the pre- or post-synaptic side. This will be done through the assessment of miniature EPSC frequency and amplitude, paired pulse ratio and asynchronous

EPSC frequency and amplitude.

1.5.7 AngII-Salt Model of Salt-Sensitive Hypertension

The direct and indirect mechanisms of AngII action are varied. As introduced in section

1.2.3, AngII is a potent vasoconstrictor and promotes natriuresis in conditions of hypervolemia.

Additionally, hypertension can be induced in animals via chronic administration of AngII in part by activating the sympathetic nervous [185, 237]. AngII’s hypertensive actions are particularly associated with chronically elevated SNA to the splanchic circulation [238]. Recent evidence has demonstrated that the sympathoexcitatory actions of AngII are exacerbated when coupled with a high salt diet. Notably, systemic infusions of AngII at doses that alone cannot raise blood pressure

(i.e. sub-pressor) combined with a high salt diet, herein referred to as “AngII-salt”, consistently lead to the development of neurogenic hypertension maintained by elevated sympathetic nerve activities [218, 221, 238-240]. While the cellular mechanisms by which increased salt intake amplifies the neurogenic component of this response remain unclear, evidence points to a circumventricular organ (section 1.3.3) capable of detecting changes in plasma osmolality and blood-borne signals as a key integrative site [238, 240, 241]. Initial investigations highlighted the

OVLT (section 1.3.3 and 1.5.4) and the SFO (section 1.3.7) as primary candidates for the convergence of these signals since they both respond to changes in plasma osmolality [88, 242] and levels of circulating AngII [243, 244]. Lesion studies confirmed the OVLT as the important site of action for the induction of AngII-salt hypertension. Specifically, lesions to the SFO had only minor effects on blood pressure [240] in animals exposed to AngII-salt treatment, while the hypertensive effect of this treatment was abolished in OVLT-lesioned animals [241]. While these studies suggest that the OVLT plays a key role in the induction of AngII-salt hypertension’s enhanced sympathetic drive, it remains possible that this model also facilitates vasopressin release as revealed in the salt loading animal model of salt-sensitive hypertension [107]. Although the

levels of circulating VP has not been reported in AngII-salt model, a collaborator is currently investigating the possibility of VP-mediated increases in blood pressure in this model. Therefore, we also included this model in our study to examine whether there is electrophysiological evidence of exaggerated SONVP MNC osmoresponsiveness, and if such activity is due to an increase in osmoreceptor signaling. In conclusion, these experiments will enable us to monitor the impact of salt-sensitive hypertension on the synaptic gain of the osmoreceptor pathway underlying exaggerated VP release.

2. Methods

2.1 Animal Care

All procedures involving animals were performed according to protocol #1190 approved by the Facility Animal Care Committee (FACC) of McGill University. Experiments were performed on in-house bred transgenic male Wistar rats that express enhanced green fluorescent protein (eGFP) under the control of the VP promoter [245]. Rats were housed under 12 h light/dark conditions (light 7AM-7PM), and food and water were provided ad libitum, except when specified.

2.1.1 Salt Loading Model of Salt-Sensitive Hypertension

Salt-sensitive hypertension by salt loading was achieved by replacing the animals tap water by 2% NaCl for 7 days, as previously described [107].

2.1.2 AngII-Salt Model of Salt-Sensitive Hypertension

Salt-sensitive hypertension was induced by replacing animal’s regular chow (0.4% NaCl content) with mature rodent diet with high salt (4% sodium chloride content, Research Diets,

D17013i) coupled with a subcutaneous implantation of an Alzet Osmotic Minipump to deliver

Angiotensin II (Sigma Aldrich, A9525). This model was created and optimized by a collaborator and has been described previously [185, 221, 239]. Briefly, animals weighing 225-250g were placed on high salt chow for 5 weeks. After 3 weeks, animals were weighed and implanted with an osmotic minipump that allowed for subcutaneous infusion of angiotensin II at a rate of

150ng/kg/min. Animals remained on the high salt chow coupled with the minipump for two more

2 weeks. Surgeries were performed on animals anesthetized with vaporized isoflurane (5% induction, 2.5% maintenance) in a stream of O2 (1.5 L/min induction, 0.8 L/min maintenance).

Once fully anesthetized as indicated by a loss of hindpaw withdrawal reflex, the animal was placed

on a heating pad with nose inserted into an anesthesia cone piece connected to the isofluorane system on its stomach. 5-8 squared centimeters of hair were shaved between the animal’s shoulder blades and the shaved area was swabbed with 70% alcohol and betadine. Sterile surgical scissors created a vertical 3cm long incision between the scapulae to open the skin while avoiding underlying tissues. With one hand holding the edge of the incision with a tissue forcep, the other hand dissected the fascia connecting the skin from the underlying muscle tissue with blunt-blunt dissecting scissors to create a subcutaneous tunnel to insert the minipump. After ensuring the pump was fully inside the pocket area and no significant tension exists on the incision, the incision was closed with 3-0 Vicryl absorbable sutures using interrupted stitches. Anesthesia was then removed and animals are given subcutaneous injections of Penicilin G procaine (30,000 units), 0.05mg/kg buprenorphine, and 0.8mL isotonic saline. Once animals regained consciousness, they were placed in a recovery cage rack 2-3 hours post-operation before returning to their home rack. Another

0.05mg/kg buprenorphine injection was given 10 hours post-operation. Animals remained on the high salt chow coupled with the minipump for the final 2 weeks, whereby mean arterial pressure steadily increases for the first week following implantation [185]. Upon slice preparation, the pump was extracted and volume of angiotensin solution was quantified and validated to determine the quantity of angiotensin II infused into each animal.

2.2 Electrophysiology in Acute Hypothalamic Slices

2.2.1 Acute Slice Preparation and Recording Conditions

Acute angled horizontal hypothalamic slices (400 µm thick) were prepared from adult male

VP-GFP Wistar rats (280-400 g) as described previously [246]. Trunk blood was collected immediately following decapitation and placed in a 4°C cold room for 1 hour and then spun in a a centrifuge at 5000g for 5 minutes to separate serum from hematocrit. Using a freezing-point

depression osmometer (Model 3320, Advanced Instruments), serum osmolality was measured.

The acute slice was perfused at 1.5 ml/min with oxygenated (95% O2; 5% CO2) artificial cerebrospinal fluid (ACSF) comprising (in mM) NaCl (104), NaHCO3 (26), NaH2PO4 (1.23),

KCl (3), MgCl2 (1), CaCl2 (2), D-glucose (10), osmolality 297.5 ± 3 mosmol/kg.

Importantly, since the strength of the OVLT→SONVP synaptic connection has been shown to change throughout the day [98, 247], and that SONVP MNCs are intrinsically thermosensitive, whereby they increase their rate of action potential discharge with increased temperature [248], all recordings were performed in the daytime between 2PM and 7PM while the perfused ACSF was maintained at 31.5 ± 1°C. Patch pipettes were prepared from glass capillary tubes (1.2mm o.d., A-

M Systems Inc.) filled will a solution containing (in mM) K-gluconate (110), HEPES (10), KCl

(10), MgCl2 (2) and Na-ATP (0.5), internal solution osmolality was 275 mosmol/kg. Pipette resistance in the bath was 3-4 MΩ. Whole cell recordings were performed as described previously

[246]. Recordings where access resistance was larger than 25 MΩ were omitted. Briefly, recordings were performed using glass pipettes mounted to amplifier headstages driven by motorized micromanipulators to fluorescently validated GFP-expressing MNCs. Electrical signals were amplified using a multiclamp-700B amplifier. Data was acquired using Clampex 10 and subsequent analysis were processed using Clampfit 10.

2.2.2 Chemicals and Drugs

The ACSF described above was supplemented with various chemicals and drugs according to experiments described in section 3. Bicuculline methochloride (BIC) (Tocris,. Burlington, ON),

Tetrodotoxin (TTX) (Alamone Labs) and mannitol were dissolved directly into the aCSF at required concentrations.

2.2.3 Spontaneous Current Clamp Recordings

Current clamp recording traces were obtained from fluorescently validated SONVP MNCs.

No holding current was applied throughout any recording. Recording traces began with 10-12 minutes of baseline electrical activity and/or firing. Once basal activity was achieved, an acute hyperosmotic stimulus (15mosmol/kg mannitol) was perfused into the bathing solution for 10 minutes. A 10-minute wash period ensued. Firing rate and membrane potentials were analyzed for the final three minutes of the baseline and evoked segments of the recording trace. Events were detected using “Event Detection” in Clampfit 10.0.

2.2.4 Spontaneous Voltage Clamp Recordings

Voltage clamp recording traces were obtained from SONVP MNCs in a gap-free protocol with the addition of 5uM bicuculline methochloride infused into the ACSF to block fast inhibitory transmission. Membrane potential was clamped at -60mV to record spontaneous excitatory postsynaptic currents. Recording traces began with 10-12 minutes of baseline activity. Once basal activity was stabilized, an acute hyperosmotic stimulus (15mosmol/kg mannitol) was perfused into the bathing solution for 10 minutes. A 10-minute wash period ensued. Spontaneous EPSC amplitudes were analyzed for the final three minutes of the baseline and evoked segments of the recording trace. Additional experiments will infuse 0.5uM tetrodotoxin into the bath solution to block synaptic activity and isolate miniature excitatory postsynaptic currents. Events were detected using “Template Search” in Clampfit 10.0 using custom built templates.

2.2.5 Evoked Voltage Clamp Recordings

Bipolar stimulating electrodes were made from a pair of Teflon-coated platinum iridium wires (50 µm o.d.; A-M Systems Inc., Everett WA) as described in [246]. The electrode tips impaled tissue adjacent to the OVLT to activate OVLT afferents projecting to the SON. Two 1 ms

pulses spaced by 50 ms were delivered using an isolated stimulation device (DS2; Digitimer Ltd.,

Hertfordshire, England) to activate afferents of the OVLT that projected to SONVP MNCs to assess the paired pulse ratio. This was done by measuring the ratio of mean current evoked from the second EPSC over the mean current evoked by the first EPSC in the first 40 ms of the EPSC. The frequency and amplitude of the asynchronous component associated with electrical stimulation of

SONVP MNCs [249, 250] was analyzed using the template search strategy outlined in section 2.2.4.

2.3 Statistics

All values in this study are reported as mean plus or minus the standard error of the mean

(± s.e.m.). Statistical differences between mean values were tested using Student’s two-tailed, or paired t-test, Wilcoxon Signed Rank Test or Mann-Whitney U Test, as appropriate, with Sigmaplot

2.03 software (SPSS Inc., Chicago IL). Differences between values were considered to be significant when p < 0.05.

3. Results

3.1 Results Overview

As stated in section 1.5.6, the purpose of this thesis was to examine if osmoregulatory signaling is enhanced in SONVP MNCs in two models of salt-sensitive hypertension. The models were briefly described in sections 1.5.2 and 1.5.7 (see sections 2.1.2 and 2.1.3 for methods). We investigated this research question using an in vitro electrophysiological approach (see section 2.2. for methods) that first assessed whether the overall osmoresponsiveness of SONVP MNCs is enhanced in these models. The findings from these experiments are presented next.

3.2 Effects of Salt Loading (SL)

3.2.1 SL Increases SONVP MNC Osmoresponsiveness

To determine if SL treatment increased SONVP MNC osmoresponsiveness, we obtained whole-cell patch clamp recordings of fluorescently identified SONVP neurons in angled slices of the adult rat hypothalamus, which maintain OVLT→SONVP synaptic connectivity (see methods) from control animals (euhydrated, EU) and animals exposed to the SL protocol. SL treatment was associated with a significant increased in serum osmolality (from 298.7 ± 1.5 mosmol/kg in EU; n

= 12 animals; to 330.2 ± 6.6 mosmol/kg in SL; n = 12 animals; p = 0.000214; t test). We first compared the osmoresponsiveness of SONVP MNC between the EU and SL via hyperomostic stimulation (Figure 3.1), which has been previously shown to increase the rate of action potential discharge in SONVP MNCs relative to baseline in the EU condition [98, 99]. Specifically, bath application of hypertonic mannitol increased the firing rate of SONVP MNCs from both EU (from

2.6 ± 0.8 Hz to 3.7 ± 0.7 Hz; n = 12 cells from 9 animals; p = 0.0114, paired t test; Figure 3.2A,B)

and SL animals (from 1.7 ± 0.6 Hz to 5.8 ± 0.5 Hz; n = 12 cells from 9 animals; p = 0.000391, paired t test; Figure 3.2A,B).

To examine whether SL enhanced overall SONVP MNC osmoresponsiveness, we examined if the hyperosmotic stimulus caused a larger increase in the firing rate of SONVP MNCs of SL animals versus EU animals. Indeed, the osmotically-evoked increase in action potential firing was significantly greater in SONVP MNC recorded from SL animals compared to EU (1.1 ± 0.4 Hz in

EU versus 4.0 ± 0.8 Hz in SL; p = 0.003; Mann-Whitney Rank Sum Test; Figure 3.2C). Therefore,

SL treatment enhanced the overall osmoresponsiveness of SONVP MNCs following hyperosmotic stimulation, which in turn led to the generation of significantly more discharged action potentials.

As previously described, increased osmoresponsiveness could be attributed to enhancements of the SONVP MNC’s intrinsic osmosensitivity (section 1.3.6.1) or OVLT-mediated synaptic excitation (section 1.3.6.3). We tested both hypotheses and presented the results in sections 3.2.2 and section 3.2.3.

3.2.2 SL Increases SONVP MNC Intrinsic Osmosensitivity

To determine if the increased overall osmoresponsiveness observed in section 3.2.1 is associated with an increased intrinsic osmosensitivity of SONVP MNCs, we compared the change in membrane potential associated with hyperosmotic stimulation for the dataset of cells included in section 3.2.1. Within these cells, bath application of hyperosmotic mannitol significantly depolarized the resting membrane potential of both EU (from −46.3 ± 1.2 mV to −44.7 ± 1.1 mV; n = 12 cells from 9 animals; p = 0.042, Wilcoxon Signed Rank Test; Figure 3.2D) and SL SONVP

MNCs (from −47.1 ± 2.4 mV to −42.4 ± 1.9 mV; n = 12 cells from 9 animals; p = 0.00108; paired t test; Figure 3.2D). To examine whether SL treatment enhanced the intrinsic osmosensitivity of

SONVP MNCs, we examined whether the hyperosmotic stimulus caused a significantly larger

depolarization of the membrane potential in SL versus EU animals. Indeed, the osmotically- evoked membrane depolarization was significantly greater in SL animals compared to EU animals

(1.6 ± 1.0 mV in EU versus 4.7 ± 1.1 mV in SL; p = 0.0456; t test; Figure 3.2E). Therefore, the enhanced overall osmoresponsiveness of SONVP MNCs as a result of SL treatment is associated with increased SONVP MNCs intrinsic osmosensitivity.

3.2.3 Salt Loading Does Not Enhance Osmoreceptor Signalling

To determine if the increased osmoresponsiveness observed in section 3.2.1 was associated with an increase in the synaptic gain of osmoreceptor afferents, we compared the change in spontaneous excitatory postsynaptic current (sEPSC) frequency associated hyperosmotic stimulation while blocking fast inhibitory synaptic transmission with the bath application of 5 µM

BIC. We compared the rate of sEPSC frequency of SONVP MNCs between the EU and SL condition in response to hyperosmotic stimulation (Figure 3.3), which has been previously shown to increase relative to baseline in the EU condition [96, 98, 246]. Specifically, bath application of the hyperosmotic mannitol (15mM) significantly increased the sEPSC frequency recorded in

SONVP MNCs obtained from both EU (from 1.8 ± 0.3 Hz to 2.5 ± 0.3 Hz; n = 12 cells from 7 animals; p = 0.000226, paired t test; Figure 3.4A,B) and SL animals (from 1.9 ± 0.1 Hz to 2.5 ±

0.2 Hz; n = 12 cells from 5 animals; p = 0.00377, paired t test; Figure 3.4A,B). sEPSC amplitude was not changed in response to hyperosmotic stimulation in either the EU (from −18.6 ± 0.6 pA to −18.2 ± 1.2 pA; p = 0.695, paired t test; Figure 3.4D) or SL condition (from −22.9 ± 0.9 pA to

−22.0 ± 1.1 pA; p = 0.461, paired t test; Figure 3.4D). To examine whether SL treatment enhanced osmoreceptor signalling, we examined if the hyperosmotic stimulus caused a significantly greater hyperosmotically induced increase in sEPSC frequency within SONVP MNCs obtained from SL animals versus EU animals. We found that the osmotically-evoked increase in sEPSC frequency

was not significantly different in SONVP MNC obtained from SL animals compared to EU animals

(0.6 ± 0.2 Hz in EU versus 0.6 ± 0.1 Hz in SL; p = 0.921, t test; Figure 3.4C). Importantly, the basal amplitude of sEPSCs recorded in the SL condition was significantly greater than those recorded in the EU condition (−18.6 ± 0.62 pA in EU versus −22.9 ± 0.9 pA in SL; p < 0.001;

Mann-Whitney Rank Sum Test; Figure 3.4D). Therefore, SL treatment is associated with an increased amplitude of sEPSCs but not with a hyperosmotically-induced increase in sEPSC frequency compared to the EU condition.

3.2.4 SL Discussion

3.2.4.1 Summary of SL Results

The results presented in section 3.2 suggest that responsiveness of SONVP MNCs to hyperosmotic stimuli (i.e. osmoreceptor gain) is potentiated in angled horizontal hypothalamic slices prepared from SL treated animals. In response to the same hyperosmotic stimulus, we saw a significant increase in the enhancement of action potential discharge frequency of these cells, coupled with a significantly greater depolarization of the membrane potential. We then decided to investigate if SL treatment altered the strength of OVLT signaling to these neurons and found that

SL treatment did not alter osmoreceptor signalling in this pathway.

3.2.4.2 Possible Mechanisms Underlying Increased Osmoresponsiveness in SL

As described in section 1.3.5, both intrinsic and extrinsic factors modulate the activity of

VP MNCs. Here, we reveal that SONVP MNCs demonstrate enhanced intrinsic osmosensitivity following SL treatment. Although the cellular basis for this effect was not investigated in this thesis, we highlight possible intrinsic mechanisms that could underlie the enhanced overall osmoresponsiveness of SONVP MNCs in response to SL treatment.

As described in section 1.3.6.1, the ΔN-TRPV1 receptor represents the molecular osmoreceptor and opens in response to reductions in cell volume associated with hypertonicity through interactions with the cell’s cytoskeletal machinery, including actin and tubulin filaments.

Previous work has demonstrated that a dense layer of filamentous actin (F-actin) lies beneath the plasma membrane of MNCs and plays an important role in the generation of intrinsic osmosensory response that can be pharmacologically modulated [251]. Indeed, pharmacological treatment with a compound that promotes F-actin polymerization enhanced intrinsic osmosensitivity of isolated

MNCs, indicating that MNC osmoresponsiveness varies in proportion with the density of polymerized actin at the cell surface [82]. Second, microtubules play an essential role in the osmotic and mechanical activation of SON MNCs [252]. Importantly, microtubules have been shown to directly interact with the ΔN-TRPV1 receptor. Recent work has shown that obstructing this interaction impairs the mechanical activation of MNCs, while enhancing interactions between the ΔN-TRPV1 receptor and microtubules at the cell surface increases intrinsic osmoresponsiveness [83]. As outlined in section 1.5.6, SL increases the levels of BDNF-signalling, which, in fact, has also been shown to promote both actin polymerization [253] and microtubule stabilization [254]. Therefore, along with altering the expression of chloride transporters that effectively abolish baroreceptor-mediated inhibition (section 1.5.5.2), increased BDNF signalling may strengthen the cytoskeletal components of SONVP MNCs and thereby make these cells more sensitive to reductions in cell volume caused by hyperosmotic stimuli. Preliminary investigations by a colleague support this hypothesis. In isolated SONVP MNC obtained from animals exposed to the SL treatment, reducing cell volume through the application of negative pressure in a whole- cell configuration (previously described in section 1.3.6.1) enhances osmoreceptor gain and is associated with smaller reductions in cell volume (data not shown). Lastly, a report suggests that

inhibitory gliotransmission following SL is altered in SON MNCs [86]. As introduced in section

1.3.6.2, taurinergic gliotransmission provides an inhibitory tone to SON MNCs via GlyR signalling. Following SL treatment, the glial processes that normally envelop SON MNCs retract, thereby abolishing inhibitory glycinergic tone in hypothalamic explants. Although the SL treatment would now have made glycinergic activation excitatory, this alteration in the glial microenvironment increases SON MNC excitability by removing a tonic inhibitory input. To summarize section 3.2, we reveal that SL treatment significantly enhances osmoreceptor gain of

SONVP MNCs, which was associated with an increase in these neuron’s intrinsic osmosensitivity and increased sEPSC amplitude. In section 3.3, we present and discuss the effects of AngII-salt induced hypertension on SONVP MNC osmoresponsiveness.

3.3 Effects of AngII-Salt Hypertension

3.3.1 AngII-Salt Hypertension Increases SONVP MNC Overall Osmoresponsiveness

To determine if AngII-salt treatment increased SONVP MNC osmoresponsiveness, we obtained whole-cell patch clamp recordings of fluorescently identified SONVP neurons in angled slices of the adult rat hypothalamus, which maintain OVLT→SONVP synaptic connectivity (see methods) from control animals (euhydrated, EU) and animals exposed to the AngII-salt protocol.

AngII-salt treatment did not significant increase serum osmolality (from 298.7 ± 1.5 mosmol/kg in EU; n = 12 animals; to 300.7 ± 1.0 mosmol/kg in AngII-salt; n = 12 animals; p= 0.278; t test).

Bath application of a hyperosmotic stimulus (+15mM mannitol) significantly increased the rate of action potential discharge in SONVP MNCs relative to baseline in both EU and AngII-salt conditions (Figure 3.5). Bath application of hypertonic mannitol increased the firing rate of SONVP

MNC obtained from both EU (from 2.6 ± 0.8 Hz to 3.7 ± 0.7; n = 12 cells from 9 animals; p =

0.0114, paired t test; Figure 3.6A,B) and AngII-salt animals (from 3.3 ± 0.9 Hz to 6.2 ± 0.6, n =

12 cells from 9 animals; p = 0.000378, paired t test; Figure 3.6A,B). To examine whether AngII- salt treatment enhanced the overall osmoresponsiveness of SONVP MNCs, we examined if the hyperosmotic stimulus caused a larger increase in the firing rate of SONVP MNC in response to the hyperosmotic stimulus in AngII-salt animals versus EU animals. Indeed, the osmotically-evoked increase in action potential firing rate was significantly greater in SONVP MNC obtained from

AngII-salt animals compared to EU (1.1 ± 0.4 Hz in EU versus 2.9 ± 0.5 in AngII-salt; p = 0.0116; t test; Figure 3.6C). These results confirm that AngII-salt treatment enhances the overall osmoresponsiveness of SONVP MNCs in response to hyperosmotic stimulation, which in turn leads to more action potentials discharged than in the EU condition. This increased osmoresponsiveness could be attributed to either increases in the SONVP neuron’s intrinsic osmosensitivity (section

1.3.5.1) or increased osmoreceptor-mediated signaling (section 1.3.5.3). We investigated both hypotheses and present the results in sections 3.3.2 and section 3.3.3.

3.3.2 AngII-Salt Does Not Increase SONVP MNC Intrinsic Osmoresponsiveness

To determine if the increased osmoresponsiveness of SONVP MNCs exposed to the AngII- salt treatment observed in section 3.1.1 was associated with increases in their intrinsic osmosensitivity, we compared the effect of hyperosmotic stimulation on the membrane potential of the dataset of cells included in section 3.3.1. Recordings from identified SONVP MNCs confirmed that bath application of hypertonic mannitol significantly depolarized the resting membrane potential of neurons obtained from EU animals (from −46.3 ± 1.2 mV to -44.7 ± 1.1 mV; n = 12 cells from 9 animals; p = 0.042, Wilcoxon Signed Rank Test; Figure 3.6D) and AngII- salt animals (from −47.0 ± 1.6 mV to −44.7 ± 1.5 mV; n = 12 cells from 9 animals; p = 0.047; paired t test; Figure 3.6D). To examine whether AngII-salt treatment enhanced SONVP MNC intrinsic osmosensitivity, we examined if the hyperosmotic stimulus caused a larger membrane depolarization in AngII-salt animals versus EU animals. Interestingly, the osmotically-evoked depolarization in the membrane potential was not significantly different in SONVP MNC obtained from AngII-salt animals compared to those obtained from EU animals (1.6 ± 1.0 mV in EU versus

2.3 ± 1.1 in AngII-salt; p = 0.626; t test; Figure 3.6E). Therefore, while AngII-salt treatment enhances the overall osmoresponsiveness of SONVP MNCs in response to hyperosmotic stimulation, this increase is not associated with a change in the intrinsic osmosensitivity.

3.3.3 AngII-Salt Increases Synaptic Gain in the OVLT→SONVP Pathway

To determine if the increased osmoresponsiveness of SONVP MNCs exposed to the AngII- salt treatment observed in section 3.3.1 is due to enhanced osmoreceptor signaling, we performed whole cell patch-clamp recordings to compared the change in spontaneous excitatory postsynaptic current (sEPSC) frequency associated with a hyperosmotic stimulus while blocking fast inhibitory synaptic transmission with the bath application of 5 µM BIC in the angled horizontal slice preparation. Hyperosmotically stimulating the slice revealed increases in sEPSC frequencies in both conditions (Figure 3.7). Recordings from identified SONVP MNCs confirmed that bath application of hypertonic mannitol (+15mM) significantly increases sEPSC frequency of SONVP

MNCs obtained from EU (from 1.8 ± 0.3 Hz to 2.4 ± 0.3 Hz; n = 12 cells from 7 animals; p =

0.000226, paired t test; Figure 3.8A,B) and AngII-salt animals (from 3.0 ± 0.5 Hz to 4.2 ± 0.6 Hz; n = 12 cells from 6 animals; p = 0.00122; paired t test; Figure 3.8A,B). sEPSC amplitude was not changed in response to hyperosmotic stimulation in either the EU (from −18.6 ± 0.6 pA to −18.2

± 1.2 pA; p = 0.695, paired t test; Figure 3.8D) or SL condition (from −21.6 ± 0.8 pA to −21.3 ±

1.0 pA; p = 0.613, paired t test; Figure 3.8D). To determine whether AngII-salt treatment enhanced osmoreceptor synaptic gain, we examined if the hyperosmotic stimulus induced a greater increase in the frequency of sEPSCs recorded in SONVP MNCs obtained from AngII-salt versus EU animals. Indeed, the osmotically-evoked increase in sEPSC frequency was significantly greater in

SONVP MNC obtained from AngII-salt animals compared to those obtained from EU animals (0.6

± 0.2 Hz in EU versus 1.2 ± 0.2 Hz in AngII-salt; p = 0.0284, t test; Figure 3.8C). We also noticed significant differences in both the frequency and amplitude of basal sEPSCs between the EU and

Salt-AngII condition. The basal sEPSC frequency in the AngII-salt treatment was significantly greater in the AngII-salt condition (1.8 ± 0.3 Hz in EU versus 3.0 ± 0.5 Hz in AngII-salt; p =

0.0473; t test; Figure 3.8B). The basal amplitude of sEPSCs in the AngII-salt treatment was also significantly greater than in the EU condition (−18.6 ± 0.6 pA in EU versus −21.6 ± 0.8 pA in Salt-

AngII; p = 0.00777; t test; Figure 3.8D). These observations suggest that AngII-salt treatment enhances the overall osmoresponsiveness of SONVP MNCs is associated with enhanced osmoreceptor signalling, which in turn leads to the generation of more action potentials than in the

EU condition. Increased osmoreceptor signalling observed in animals exposed to the AngII-salt treatment could be attributed to effects on the pre- or post-synaptic side. The increased change in sEPSC frequency could be due to multiple factors including increased firing by OVLT neurons that project to SONVP MNCs, increased synaptic contacts between OVLT→SONVP MNCs, or an increased probability of release. While we did not assess changes in OVLT firing activity or changes in synaptic contacts, possible differences in presynaptic probability of release were examined using the paired pulse ratio.

3.3.4 AngII-Salt Increases Probability of Release of the OVLT→SONVP Connection

The chance that an action potential evoked from a central neuron’s cell soma can induce calcium-dependent neurotransmission at one of its presynaptic terminals is known as its probability of release (PR). The ratio of two consecutively evoked postsynaptic currents is defined as the paired pulse ratio (PPR), and increases in PPR are associated with decreases in PR [255].

Therefore, we investigated alterations in excitatory osmoreceptor signalling by monitoring changes in the PPR of OVLT afferents that synapse onto SONVP MNCs. To do this, we performed whole-cell patch clamp recordings while blocking fast inhibitory synaptic transmission with the bath application of 5 µM BIC in the angled horizontal slice preparation. Pairs of electrical stimuli applied 50 ms apart were delivered to the OVLT (see methods, Figure 3.9A) and the PPR was

calculated from the mean current elicited by two consecutive pulses recorded in SONVP MNCs

(Figure 3.9B). The PPR value obtained from SONVP MNCs of EU animals is in agreement with previous studies [98, 99]. Importantly, AngII-salt treatment significantly reduced PPR in SONVP

MNCs (from 1.93 ± 0.08 in EU; n = 12 from 5 animals; to 1.65 ± 0.06 in AngII-salt; n = 12 from

3 animals, p = 0.00523; t test; Figure 3.9C). Since a decrease in PPR implies an increase in PR, this suggests that the glutamatergic axon terminals in the OVLT→SONVP pathway becomes altered to enhance synaptic vesicles release in response to the arrival of an action potential. Therefore, AngII- salt hypertension increases the synaptic gain of the OVLT→SONVP synaptic connection, whereby action potentials generated in the OVLT are more likely to excite SONVP MNCs.

3.3.5 AngII-Salt Increases Quantal Size of SONVP MNCs

The magnitude of EPSCs are determined in part by the number of synaptic contacts and and quantal size. While the results in section 3.3.3 reveal that both the baseline sEPSC frequency and amplitude are significantly greater in SONVP MNCs of the AngII-salt treatment compared to

EU, this may not necessarily be a result of increased synaptic contacts or quantal size. Indeed, sEPSCs represent synaptic events mediated by both action-potential dependent vesicle fusion and stochastic fusion of single vesicles, herein referred to as miniature EPSCs (mEPSCs). As well, increased sEPSC amplitudes can represent the spontaneous summation of multiple vesicles fusing to the postsynaptic density at the same time. Indeed, electron microscopy analysis has shown that single axons can form twin synaptic terminals onto MNCs [256], such that spontaneous action potentials propagating to such synapses could potentially elicit sEPSCs whose amplitude could be twice the normal quantal size. Patch clamp recordings of SONVP MNCs were performed while blocking both fast inhibitory transmission using 5 µM BIC and action-dependent excitatory synaptic events using 0.5 µM TTX. Using this pharmacological combination, we were able to

isolate and compare the frequency and amplitude of mEPSCs recorded from SONVP MNCs obtained from EU (Fig 3.10A) and AngII-salt (Fig 3.10B) animals. Both the mEPSC frequency and amplitude were significantly different in the AngII-salt condition compared to EU.

Specifically, SONVP MNCs obtained from AngII-salt animals demonstrated a significantly greater mEPSC frequency compared to EU (1.6 ± 0.2 Hz in EU; n = 12 cells from 5 animals; versus 2.6 ±

0.4 Hz in AngII-salt; n = 12 cells from 2 animals; p = 0.023, Mann-Whitney Rank Sum Test;

Figure 3.10C). As well, mEPSC amplitude was significantly greater in SONVP MNCs obtained from AngII-salt treatment compared to EU (-14.2 ± 0.8 in EU; n = 12 cells from 5 animals; versus

-19.2 ± 1.5 in AngII-salt; n = 12 cells from 2 animals; p = 0.014, Mann-Whitney Rank Sum Test;

Figure 3.10D). While this experiment confirms that quantal size is increased in SONVP MNCs following AngII-salt hypertension, this experiment does not confirm that increased quantal size originates from OVLT afferents.

3.3.6 AngII-Salt Increases the Frequency and Amplitude of Asynchronous EPSCs

Previous studies have demonstrated that electrically evoked EPSCs in the SON are followed by an increased rate of stochastically released quanta released due to the buildup of residual calcium [99], and are called asynchronous EPSCs (aEPSCs) [249, 250]. aEPSCs are a very powerful measure of synaptic activity because they represent quantal events that occur specifically at the synapses activated by the electrical stimulus. This provides an opportunity to measure changes in the quantal size (i.e. aEPSC amplitude) and release probability (estimated as aESPC frequency) of unitary events at these sites. We therefore examined the frequency and amplitude of aEPSCs detected during a 250 ms window immediately following the decay of the second OVLT-evoked EPSC in the same set of experiments outlined in section 3.3.4 (Figure

3.11A). We aimed to measure the difference in aEPSC frequency and amplitude measured in

SONVP MNCs of EU animals and those exposed to the AngII-salt treatment (Figure 3.11B,C). The measured aEPSC frequency and aEPSC amplitude were both significantly different in cells obtained in AngII-salt hypertension compared to EU. Specifically, SONVP MNCs obtained from

AngII-salt animals demonstrated a significantly greater aEPSC frequency (31.7 ± 2.8 in EU; n=12 cells from 3 animals; versus 41.0 ± 1.3 Hz in AngII-salt; n = 12 cells from 3 animals; p = 0.00603, t test; Figure 3.11D). As well, aEPSC amplitude was also significantly greater in SONVP MNCs obtained from AngII-salt compared to EU animals (-20.7 ± 1.5 in EU; n = 12 cells from 3 animals; versus -28.5 ± 2.7 in AngII-salt; n = 12 cells from 3 animals; p = 0.0208, t test; Figure 3.11E).

3.3.6 AngII-Salt Discussion

3.3.6.1 Summary of AngII-Salt Results

Previous studies have revealed the OVLT as a key mediator in the AngII-salt animal model of salt-sensitive hypertension (section 1.5.7). The results presented in section 3.3 are the first to suggest that this model of salt-sensitive hypertension also increases osmoreceptor gain in SONVP

MNCs in horizontal hypothalamic slices prepared from AngII-salt treated animals. In response to the same hyperosmotic stimulus, we saw a significant increase in the enhancement of action potential discharge frequency of these cells, which was not associated with a significantly different depolarization of the membrane potential. However, we found that the increased osmoresponsiveness of MNCs was associated with a strengthening of the excitatory synaptic afferents to these neurons from the OVLT. Further characterization of this enhancement revealed an increase in the synaptic gain of SONVP MNC-projecting OVLT neurons. Specifically, AngII- salt treatment caused an increase in PR and quantal size.

3.3.6.2 Possible Mechanism Underlying Increased Synaptic Gain in AngII-Salt

Although the cellular basis that could underlie the enhanced overall osmoresponsiveness of SONVP MNCs in AngII-salt hypertension was not investigated in this thesis, we highlight potential mechanisms that may be responsible for this effect. Our results in section 3.3.3 reveal an increase in PR in the OVLT→SONVP connection following AngII-salt treatment. This observation furthers a previous observation that reported acute bath applications of AngII increased PR of the

OVLT→SONVP connection in vitro [99]. We highlight the possibility that an increased PR may be associated with an increase in the density or activity of voltage gated calcium channels located at the presynaptic terminals of the OVLT→SONVP synapses. This would allow an action potential emanating from the OVLT cell soma to cause more calcium influx in these presynaptic terminals and lead to more calcium-dependent neurotransmission events. Further studies are required to test this hypothesis.

Other mechanisms that we did not explore may also mediate the observed increase in

OVLT→SONVP synaptic gain. As indicated in the results section, there were significantly more sEPSCs recorded in basal conditions, highlighting that perhaps its afferents were more active.

Specifically, we highlight the possibility that in AngII-salt hypertension, OVLT neurons that project to SONVP MNCs are more active through increased osmosensitivity and excitability. It is also possible that AngII-salt treatment increased the number of OVLT synaptic contacts onto

SONVP MNCs.

As mentioned in section 1.5.7, OVLT neurons are excited by circulating AngII. Although the basis for such effects remain unclear, it is possible that elevated levels of circulating AngII for prolonged periods can enhance the intrinsic osmosensitivity of OVLT neurons. This is based on observations from isolated osmosensitive neurons in the SON that express the same molecular

osmoreceptor as osmosensitive neurons in the OVLT (section 1.3.6.3a). Specifically, isolated SON neurons were found to increase their osmosensitivity in response to acute applications of AngII

[257]. Although the downstream effects of AngII receptor activation remain unknown, they were shown to strengthen the cytoskeletal machinery in SONVP MNCs [257]. Therefore, chronic elevations of circulating AngII may lead to lasting increases in the intrinsic osmosensitivity of

OVLT neurons through cytoskeletal plasticity.

Another factor that might increase OVLT activity could be changes in the expression of ion channels or transporters contributing to neural excitability. As introduced in section 1.4.5.3,

AngII-salt hypertension has been shown to increase the excitability of RVLM-projecting PVN neurons by reducing SK currents that unmask the afterdepolarization current, which has recently been shown to have direct effects on sympathetic tone [258]. Since chronic hyperosmotic challenges have been previously shown to alter the transcriptome of osmosensitive neurons [259], it is possible that increases in OVLT activity in AngII-salt hypertension could be due to altered expression of ion channels or ion transporters that modulate the cell’s excitability. The epithelial sodium channel (ENaC) is sensitive to changes in extracellular sodium concentration and is highly localized in the OVLT [260]. Recent evidence highlights the possibility that AngII-salt hypertension may alter the expression or activity of epithelial sodium channels (ENaCs) in the

OVLT. This hypothesis is made following a report that intracerebroventricular infusions of benzamil, a potent ENaC antagonist, attenuated the induction of AngII-salt hypertension [218] in the same way that that OVLT lesions did (as explained in section 1.5.6). Therefore, AngII-salt hypertension may cause a variety of alterations in ion channel expression and activity on OVLT neurons, which may lead them to increase their neural excitability.

The results of experiments examining aEPSCs described in section 3.3 highlight that the

aEPSC frequency in SONVP MNCs that originate from OVLT afferents is significantly increased following AngII-salt hypertension. While this observation could be attributed to the increased PR at OVLT→SONVP synaptic terminals as described in section 3.3.3, it remains possible that AngII- salt hypertension increases the number of OVLT→SONVP synaptic connections. This is founded on observations that reveal the occurrence of new synapse formation in the SON following both dehydration and lactation [256, 261]. In summary, we propose that these changes may contribute to the hyperactivation of SONVP MNCs and lead to the exaggerated release of VP in AngII-salt hypertension.

4. General Discussion and Conclusion

4.1 Summary of Thesis Motivation and Results

While the combination of genetic and physiological alterations that induce salt-sensitive hypertension remain unclear, this thesis highlighted neural mechanisms that play potentially etiological roles in its development and maintenance. In section 1.5.4, central osmoreceptor activation was shown to pathologically enhance sympathoexcitation in salt-sensitive hypertension.

However, blocking the enhanced sympathoexcitatory response did not fully restore blood pressure to normotensive levels, indicating that central osmoreceptor activation may also mediate salt- sensitive hypertension by other mechanisms. This thesis aimed to further reports suggesting that the mechanisms underlying VP release are exaggerated in salt-sensitive hypertension. In this thesis, we performed an electrophysiological study to assess whether VP-releasing neurons in the

SON are more osmoresponsive, and if this is due to increased osmoreceptor signaling from the

OVLT and/or increases in intrinsic osmosensitivity of SONVP MNCs. Specifically, we investigated these properties using an in vitro preparation in two distinct animal models of salt-sensitive hypertension. In both models, we found osmoresponsiveness to be enhanced via distinct mechanistic differences.

4.2 Comparison of the Salt-Sensitive Hypertension Models

It is striking to note that although both animal models reveal enhanced osmoresponsiveness of SONVP MNCs in vitro, the increase seems to be mediated by different mechanisms. Notably, in the SL model, this was associated with increased intrinsic SONVP MNCs osmosensitivity, while in the AngII-salt model, this was associated with increased osmosensitive synaptic gain from the

OVLT, specifically, an increased probability of release and quantal size. Importantly, as mentioned

in section 2, we used the same type of animals and maintained the same recording environments for both conditions. Therefore, the observed mechanistic differences leading to enhanced SONVP

MNC osmoresponsiveness likely rely on inherent differences between the animal models used to induce salt-sensitive hypertension. We highlight two major differences that could account for our contrasting results.

First, the SL model restricted animals from drinking tap water. Therefore, the hypernatremic stimulus was also a potent hyperosmotic stimulus. As a result, their serum osmolalities were significantly higher than those of euhydrated animals. As osmosis dictates

(section 1.3.1), chronic dehydration draws water from the animal’s cerebrospinal fluid into the plasma and therefore establishes a chronic hypertonic environment. Since SON MNCs do not undergo regulatory volume decreases in response to hyperosmolality [262], they may instead compensate for the chronic hyperosmotic state by strengthening their cytoskeleton. In contrast, the intrinsic osmosensitivity of MNCs was unaltered in the AngII-salt model. The absence of a significant change in systemic osmolality in this model provides support for the suggestion that the increased osmosensitivity of MNCs observed in SL results specifically from an exposure to chronic hyperosmolality. Further studies are required to test this hypothesis.

Second, in our opinion, AngII-salt hypertension represents a more physiologically relevant animal model. AngII-salt hypertension has the advantage of being similar to human scenarios that lead to salt-sensitive hypertension, in which the consumption of large quantities of salty food occurs on a much longer timescale than the SL protocol, and does not cause a significant increase in systemic osmolality. In addition, in humans and other mammals, although hypernatremia should effectively inhibit RAAS signalling (section 1.2.3), there is evidence revealing that salt-sensitive hypertensives display abnormally elevated levels of circulating AngII despite elevated levels of

plasma sodium, which has been characterized as the AngII escape phenomenon (highlighted in section 1.5.2). Therefore, a model that provides animals with increased sodium intake and elevated levels of circulating AngII for a prolonged period of time may be considered more clinically important than an extreme hypernatremic stimulus alone, and thus has more translational appeal to modelling human disease.

4.3 Perspectives of Thesis Results in Regards to Global Health

Hypertension is and will continue to remain a significant global health concern for our indefinite future. It is recognized as a leading risk factor for human mortality and morbidity and is associated with staggering healthcare costs on a global scale. Epidemiological estimates also suggest that its incidence is only expected to rise in the coming years. The pathophysiological underpinnings of hypertension vary extensively across patient populations, and likely explain why a significant portion of the hypertensive population does not respond to any specific or combination of treatment regimens. Section 1.1 of this thesis highlighted that hypertension is associated with a combination of genetic and environmental factors, few of which are modifiable.

Notably, the habit of increased dietary sodium consumption in the industrialized world has been shown to pose a significant hypertensive threat. However, while section 1.4 established high dietary sodium intake as an etiological factor in the pathogenesis of hypertension, section 1.5 reveals that this is only a risk factor in salt-sensitive individuals. With this in mind, global campaigns that aim to reduce the population level of sodium intake may only have protective effects in the salt-sensitive population. On an individual level, assessing salt-sensitivity in patients that are likely to develop hypertension may be useful in determining whether sodium reduction might be effective in preventing the disease. Importantly, the incidence of salt-sensitivity increases with age, which implies the importance of remaining mindful of one’s sodium consumption for a

healthier future even if not presently salt-sensitive. As highlighted in section 1.5.3, a variety of studies have revealed pathophysiological alterations implicated in human and animal studies of salt-sensitive hypertension. While circulating VP does not contribute to basal blood pressure tone

(section 1.5.5.1), the evidence presented in this thesis corroborates reports that suggest salt- sensitive hypertension can increase the activity of VP-releasing neurons, which can cause VP- mediated increases in blood pressure. Specifically, we demonstrate that the osmoreceptor gain of

VP-releasing neurons in the supraoptic nucleus is enhanced in two animal models of salt-sensitive hypertension, each associated with distinct electrophysiological signatures. This thesis therefore provides a better understanding of how high salt intake alters signalling pathways that control VP secretion and lead to VP-mediated increases in blood pressure, and may provide a lead towards the innovation of novel therapeutic approaches for the treatment of hypertension.

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