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NEED-INDUCED AND NON-NEED-INDUCED SODIUM APPETITE BY RATS IN A SIMULATED FORAGING PARADIGM

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NEED-INDUCED AND NON-NEED-INDUCED SODIUM APPETITE BY RATS IN A SIMULATED FORAGING PARADIGM
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2008

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Appetite ( jstor )
Available water content ( jstor )
Body weight ( jstor )
Commodities ( jstor )
Food ( jstor )
Food intake ( jstor )
Foraging ( jstor )
Paradigms ( jstor )
Rats ( jstor )
Sodium ( jstor )
City of Miami ( local )

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NEED-INDUCED AND NON–NEED-INDUCED SODIUM APPETITE BY RATS IN A SIMULATED FORAGING PARADIGM By CONNIE L. COLBERT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2002

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Copyright 2002 by Connie L. Colbert

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This thesis is dedicated to my wonderful family and friends. I thank all of them.

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ACKNOWLEDGMENTS I gratefully acknowledge the help and guidance that I received from my thesis advisor, Dr. Neil Rowland. Dr. Rowland believed in my abilities and gave me independence when I was learning to stand on my feet. I truly appreciate the freedom that he gave me to learn from my mistakes. I also appreciatively acknowledge the guidance and friendship that I found in Kimberly Robertson. Without her constant reassurance that I would get the hang of things, I do not think my work in the lab would have progressed as it did. Kimberly and Dr. Rowland offered strong support for which I will always be thankful. Perhaps most important, however, are my family and friends over the many years it has taken to reach this point. They provided me with many outlets through which I could celebrate my victories and vent my frustrations. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... xi CHAPTER 1 LITERATURE REVIEW................................................................................................1 Preference.......................................................................................................................2 Appetite....................................................................................................................... ....2 Hormones of Sodium Appetite.......................................................................................3 Methods of Inducing Sodium Appetite...........................................................................4 Furosemide...............................................................................................................4 Hydrochlorothiazide.................................................................................................4 Deoxycorticosterone Acetate...................................................................................5 Typical Protocols for Examining Sodium Appetite........................................................7 An Alternative Protocol: Simulated Foraging................................................................8 2 EXPERIMENT 1: SIMULATED FORAGING AND SODIUM PREFERENCE UNDER NEED FREE AND DOCA TREATMENT CONDITIONS..........................10 Introduction................................................................................................................... 10 Hypotheses....................................................................................................................10 Methods.........................................................................................................................11 Animals..................................................................................................................11 Apparatus...............................................................................................................11 Training..................................................................................................................11 Testing....................................................................................................................12 Results........................................................................................................................ ...13 Discussion.....................................................................................................................14 3 EXPERIMENT 2: SIMULATED FORAGING AND SODIUM APPETITE UNDER BASELINE AND NEED-INDUCED (H C Z) CONDITION S ...................................... 21 Introduction................................................................................................................... 21 v

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Methods.........................................................................................................................22 Animals..................................................................................................................22 Apparatus...............................................................................................................23 Training..................................................................................................................23 Testing Procedures.................................................................................................23 Baseline............................................................................................................23 Hydrochlorothiazide (HCZ) phase...................................................................24 Results........................................................................................................................ ...24 Discussion.....................................................................................................................25 4 EXPER I MENTS 3a TO 3d: PHYSIOLOGICAL M EASUREMENTS .........................28 General Introduction.....................................................................................................28 General Aims for Experiment 3....................................................................................28 Experiment 3a: Physiological and Behavioral Characterization of HCZ-Induced Salt Appetite ( S D Rats and 0.1 M NaHCO 3, 0.3 M NaHC O3, and 0.3 M NaCl) ........... 29 Methods and Procedures........................................................................................29 Animals............................................................................................................29 Procedures........................................................................................................29 Results of Experiment 3a.......................................................................................30 Discussion of Experiment 3a.................................................................................31 Experiment 3b Physiological and Behavioral Characterization of HCZ-Induced Salt Appetit e .................................................................................................................. . .. 31 Introduction to Experiment 3b...............................................................................31 Methods and procedures........................................................................................32 Animals............................................................................................................32 Procedures........................................................................................................32 Assays....................................................................................................................33 Statistics.................................................................................................................34 Results of Experiment 3b.......................................................................................34 Discussion of Experiment 3b.................................................................................35 Experiment 3c Physiological and Behavioral Characterization of HCZ-Induced Salt Appetite ( L E Hooded Rats and 0.3 M NaCl ) ........................................................... 36 Methods and Procedures........................................................................................36 Animals............................................................................................................36 Procedures........................................................................................................36 Results of Experiment 3c.......................................................................................36 Discussion of Experiment 3c.................................................................................37 Experiment 3d Physiological and Behavioral Characterization of HCZ-Induced Salt Appetite ( L E Hooded Rats a nd 0.3 M NaCl or 0.3 M NaHCO 3 ) ... ........................... 37 Methods and Procedures ... ....................................................................................... 37 An i m al s ............................................................................................................37 Procedures........................................................................................................37 Results of Experiment 3d.......................................................................................38 Discussion of Experiment 3d.................................................................................38 5 GENERAL DISCUSSION............................................................................................51 vi

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LIST OF REFERENCES...................................................................................................57 BIOGRAPHICAL SKETCH.............................................................................................60 vii

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LIST OF TABLES Table page 2-1. Order of Stimuli Presentation....................................................................................17 4-1 Urine volume excreted................................................................................................44 viii

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LIST OF FIGURES Figure page 2-1 Average meal number Experiment 1...........................................................................18 2-2 Average meal size Experiment 1.................................................................................19 2-3 Preference-aversion data Experiment 1.......................................................................20 3-1 Mean number and size Experi m e nt 2. .. .........................................................................26 3-2 Mean bout nu m ber and size Experi m ent 2 .. .................................................................27 4-1 Mean b ody w eight Experi m ent 3a ... .............................................................................40 4-2 Mean f ood i ntake E xperi m ent 3a ... ...............................................................................40 4-3 Mean w ater i ntake Experi m ent 3a ... .............................................................................41 4-4 Mean s alt i ntake Experi m e nt 3a .... ................................................................................41 4-5 Mean b ody w eight Experi m ent 3b .... ............................................................................42 4-6 Mean f ood i ntake E xperi m ent 3b .... ..............................................................................42 4-7 Mean w ater i ntake Experi m ent 3b .. . .............................................................................43 4-8 Mean s odium b icarbonate i ntake Experi m ent 3b . . . . .....................................................43 4-9 Mean s odium and p otass i um o utput Experi m e nt 3b . .. . . ................................................45 4-10 Plas m a a ldosterone Experi m e nt 3b . . ..........................................................................46 4-11 Plas m a r enin a ctivity Experiment 3b . . . ......................................................................46 4-12 Mean b ody w e ight Experi m e nt 3c .. .. ..........................................................................47 4-13 Mean f ood i ntake Experi m e nt 3c. . .. ............................................................................47 4-14 Mean w ater i ntake Experi m e nt 3c . . . ...........................................................................48 4-15 Mean NaCl i ntake Experi m e nt 3c . . . ............................................................................48 ix

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4-16 Mean body weight Experiment 3d...........................................................................49 4-17 Mean food intake Experiment 3d.. ...........................................................................49 4-18 Mean water intake Experiment 3d............................................................................50 4-19 Mean salt intake Experiment 3d.... ...........................................................................50 x

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science NEED-INDUCED AND NON–NEED-INDUCED SODIUM APPETITE BY RATS IN A SIMULATED FORAGING PARADIGM By Connie L. Colbert August 2002 Chair: Neil Rowland Department: Psychology Sodium-replete rats prefer sodium solution to water when it is offered at concentrations near isotonicity. They do not drink large amounts of sodium solution when it is hypertonic unless a physiological need is aroused. Additionally, rats treated with the hormones that are typically elevated in times of sodium deficit will consume large quantities (relative to need-free conditions) of hypertonic sodium solution. Pattern analysis has been used to describe the behavioral strategy rats use to maintain sodium homeostasis. However, all studies examining sodium appetite, to date, use the ad libitum or brief duration access protocols. The ad libitum paradigm consists of rats having continuous access to one or more commodities. In contrast, the brief access protocol consists of limited access, but during the time available, the animal is free to consume as much of a commodity as it wishes. Neither of these approaches simulates the conditions of a rat’s niche particularly well. A better alternative exists. xi

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The simulated foraging paradigm is a method of simulating environmental availability using the price (effort and/or time) necessary to obtain the commodity; lever press is often the currency used. Because sodium is not very abundant in many habitats, when attempting to uncover the strategies that a rat uses to maintain sodium balance, it is not really informative to offer sodium on a schedule that the species did not encounter during its evolutionary history. The simulated foraging paradigm allows the researcher to simulate a sparse (realistic) environment in which a rat would have to invest energy/time to gain access to the given commodity. We have found that the amount of effort/time required to obtain salt has a profound impact on the patterning (strategy) of a rat’s behavior. It reduces the total number of episodes when sodium solution is expensive, but there is also an increase in the size of each bout as compared with the less expensive condition. Primarily, the present set of experiments attempted to characterize rats’ behavioral strategies for procuring and consuming sodium solution in a simulated plentiful or sparse environment. Additionally, we described the sodium appetite and the associated behavioral strategies of rats given false signals of deficit (through administration of the mineralocorticoid, DOCA), and those actually depleted of sodium (through use of a natriuretic agent, HCZ). We demonstrated a robust appetite for both NaCl and NaHCO3 in rats required to work (at procurement fixed ratio of 1, 80, and 300 lever presses) for access to sodium solution. xii

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CHAPTER 1 LITERATURE REVIEW Sodium is an essential element to almost every terrestrial animal’s diet. One of the major symptoms and signs of sodium deficiency is a decrease of extracellular fluid volume, or hypovolemia (Denton, 1982). Most animals maintain the sodium content and therefore the extracellular fluid content of their bodies at a rather constant level (Goodman & Gilman, 1980; Fregly & Rowland, 1986). Sodium content in the body is regulated by coordination of intake and excretion. Sodium intake is most often studied in domestic rats, but it has also been documented in various other species (including dog, sheep, monkey, baboon, rabbit, and man) both inside the lab, and in the field (Denton, 1982). The rat is used as a model to potentially understand human salt-intake behavior in part because of convenience, but also because both species are omnivorous. The remainder of the discussion will focus on sodium appetite in the lab rat. There are two aspects of sodium-salt intake: preference and appetite. A preference for sodium entails the rat consuming a sodium containing solution based, in part, on taste (believed to be hedonically rewarding; see Schulkin, 1991), and is characterized by behavioral responding to the palatability of sodium-solution concentrations (discussed below). An appetite for sodium is demonstrated when a rat ingests ‘unpalatable’ solutions (palatability being assessed by basal intake or preference data). Typically, a high (> 0.3 M) concentration of NaCl is used because it drives down intake during baseline conditions (Fregly & Rowland, 1986). These two aspects of sodium intake can 1

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2 be further separated by physiological need for sodium: preference often occurs even when the animal is sodium replete, while an appetite involves a physiological need. The concentration and form of salt is an important determinant of voluntary sodium intake in laboratory rats. Rats will not regulate sodium intake (or they will do so poorly) when sodium is offered in food (Fregly et al., 1965); though, they prefer to regulate sodium when it is offered in solution (Bertino & Tordoff, 1988). Preference Many strains of rat show a sodium preference when different concentrations of NaCl are offered concurrently with distilled water. The preference for sodium-solution in sodium-replete rats is maximized at approximately 0.15 M and decreases to either side of that concentration forming what many have termed an inverse U-shaped preference-aversion function (Fitzsimons, 1979, pp. 459). Concentrations > 0.3 M appear to be aversive to the animals, while concentrations < 0.03 M are consumed with indifference by the animals. Surprisingly, the anion that sodium is paired with does not seem to make a large difference. Appetite Need-induced sodium appetite (or salt appetite) is initiated through the loss of sodium. It can be illustrated by a condition such as hypovolemia. An animal in the hypovolemic-state needs both sodium and water to restore blood volume and osmotic balance (Stricker & Wolf, 1969). In contrast, non–need induced sodium appetite can be induced through the manipulation of hormones that have been identified in the control of sodium intake (Wolf, 1965). In this situation, the animal is sodium replete, but it is thought that the addition of exogenous hormones of salt appetite generates a false signal of deficit (Wolf, 1964). In this situation, the animal does not need to consume sodium,

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3 but it does so preferentially to water, even at concentrations observed to be aversive to normal animals. Hormones of Sodium Appetite The renin-angiotensin-aldosterone axis is thought to be a major player in organizing and initiating salt intake (for review see Stricker and Verbalis, 2000). Briefly, baroreceptors within the kidneys detect hypovolemia and cause the kidneys to secrete renin enzyme. Renin cleaves endogenous angiotensinogen, producing angiotensin I. A reaction in the lungs involving angiotensin-converting-enzyme (ACE) further cleaves angiotensin I to form angiotensin II (ANG II). Angiotensin II is a potent dipsogen, vasoconstrictor, and stimulant of aldosterone secretion. Aldosterone (a mineralocorticoid), together with ANG II, promotes renal sodium conservation. Increased production of aldosterone inhibits renal renin secretion, which reduces circulating levels of ANG II. In addition to the renal sodium conservation effects of the two hormones, ANG II and aldosterone are believed to work together, centrally, to initiate salt seeking and ingestive behavior. The synergy hypothesis, first introduced by Fluharty and Epstein (1983), posits that ANG II and aldosterone work synergistically to promote sodium intake. It is known that ANG II alone is sufficient to induce an appetite for sodium. Chronic intracerebroventricular infusions of ANG II induce sodium intake in rats (Buggy & Fisher, 1974). Moreover, adrenalectomized animals (without the ability to produce aldosterone) show a vigorous sodium appetite. In fact, without the opportunity to consume sodium, adrenalectomized rats will die in a matter of days. It is also known that mineralocorticoid treatment, alone, can stimulate sodium intake. When high doses of either deoxycorticosterone acetate (DOCA), a synthetic mineralocorticoid, or aldosterone

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4 are given to adrenalectomized rats, renin secretion is decreased. Thus, the salt appetite that occurs under these conditions may be driven by mineralocorticoid alone (Fregly and Rowland, 1986). When the two signals (ANG II and aldosterone) work together, however, much lower amounts are needed to produce an even higher sodium-solution intake than is seen when either signal is increased independently. Methods of Inducing Sodium Appetite There have been many procedures used to promote sodium intake in rats. The following is a select overview of some of those methods. Of interest to the present discussion are the furosemide, hydrochlorothiazide, and DOCA methods of inducing salt appetite. Furosemide Furosemide is an injectable diuretic often used in conjunction with a low-sodium diet. It is classified as a high-ceiling diuretic; this class of agents inhibits chloride and sodium reabsorption in both the ascending limb of the loop of Henle and also in the proximal tubule of the kidney (Goodman and Gilman, 1980). Furosemide has been used to promote acute water and electrolyte loss in the rat (Denton, 1982). Jalowiec (1974) did not report adverse behavioral effects of furosemide, but Wolf et al. (1974) found that it induced anorexia in his animals. Hydrochlorothiazide Hydrochlorothiazide (HCZ) is classified as a thiazide diuretic. Thiazides are considered a separate class from high ceiling diuretics on the basis of chemical and physiological criteria (Goodman and Gilman, 1980). Thiazides increase renal excretion of sodium and chloride along with water. They work through inhibiting the reabsorption of sodium and chloride in the distal segment of the kidney. Thiazides have a diuretic

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5 effect within an hour of oral administration, and are rapidly excreted (within 3 to 6 hours) (Goodman and Gilman, 1980). Fregly (1967) performed a series of experiments exploring the sodium appetite induced by both chronic and acute administration of HCZ. Twenty-four hour intake tests revealed that rats increased NaCl (0.15 M) consumption comparably with both dosages of HCZ used (0.3 and 0.6 g/kg diet). He found that neither food nor water intakes were significantly affected by HCZ treatment. In a later study, Fregly and Kim (1970) showed that rats given HCZ (0.6 g/kg diet) drank approximately 10 times the amount of sodium-salt solution (salts tested ranged from 0.05 to 0.15 M) than they consumed of water. Although these studies did not look at the pattern of intake, they do provide us with information about a possible alternative method of inducing a need-based sodium appetite that has remained relatively unexplored. To our knowledge, no one has used HCZ to induce a sodium appetite in rats since Fregly. There may be some advantages to using the HCZ method of inducing salt appetite, namely its ease of administration. The powdered form of HCZ can easily be mixed into powdered chow. Fregly and Kim (1970) reported that there were no adverse effects on food intake. The diuretic furosemide must be delivered by injection, which may add an unnecessary confound to the study of salt appetite. Moreover, there are complications of anorexia associated with furosemide (Wolf, 1974) that might be especially problematic with chronic administration. Deoxycorticosterone Acetate The primary endogenous mineralocorticoid, aldosterone, and the much less physiologically abundant deoxycorticosterone act on the distal tubules of the kidney to enhance the reabsorption of sodium ions (Goodman and Gilman, 1980). The renal

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6 tubules ordinarily reabsorb nearly all the sodium filtered at the glomerulus. Deoxycorticosterone and aldosterone are identical in their actions; however, deoxycorticosterone is not as potent as aldosterone. Deoxycorticosterone acetate (DOCA), a synthetic mineralocorticoid, administered at higher doses (> 1 mg/day) increases intake of sodium solutions in intact animals. Daily treatment with these higher dosages of DOCA results in an elevated intake of NaCl solution. Water intakes are secondarily elevated to compensate for the osmotic load caused by increased sodium intake and renal sodium retention. The classic studies of salt appetite in rats, induced by DOCA, monitored intakes once daily. Stricker, Gannon, and Smith (1992) went a step further to obtain detailed analyses of the patterns of food and fluid ingestion in rats after DOCA (2 mg/ day) treatment using a specially designed cage linked to microprocessors. They found that daily intakes of concentrated (0.5 M) NaCl and water increased progressively when rats were treated with DOCA, but that food intakes were unchanged. During control and experimental conditions, the rats ate and drank mainly during the 12 h dark phase (which began 9 h after the animals were injected in the DOCA phase). During control periods the rats (on average) were initiating a new episode of food and/or fluid every 10-20 min. When DOCA was administered, the rats had a comparable number of food and water ingestive bout episodes, but saline intake was four times greater during DOCA treatment than in the control period. The 0.5 M NaCl bouts were the same size as control NaCl bouts, and were much smaller than water bouts. Pattern analysis revealed that the DOCA-induced NaCl bouts were incorporated into existing food and water bouts, resulting in more complex ingestive episodes (Stricker, Gannon, and Smith, 1992).

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7 Typical Protocols for Examining Sodium Appetite Under both need and non–need related conditions intake tests are typically performed using either ‘free access’ or ‘brief access’ protocols. The first often uses the 24 h two bottle choice paradigm that consists of the ani m al being exposed to two different solutions for one or more periods of 24 h. The locations of the drinking tubes are often reversed daily to avoid a selection of drinking tube based on habit alone. This type of study yields total amount of solution consumed but does not reveal the pattern of intake. A substantial advantage is offered by the use of a lickometer and interfacing computer, which allows a record of the exact temporal sequence of fluid bouts. The data so obtained are, however, often quite complicated because of multiple switching between solutions creating "mixed" bouts, as the rats create isotonic cocktails of hypertonic sodium solution and distilled water. The second major protocol used, the brief access paradigm, consists of depriving animals for some experimenter-determined amount of time, of a salt-containing solution and later allowing access to the solution for a specified amount of time. The purpose of the deprivation is to motivate drinking behavior to occur. This is often done to accommodate the researcher, and so, many times the period of observation is during daylight hours and not during the dark phase, when drinking normally occurs in the rat. Neither of the above protocols is particularly closely related to the environmental context in which salt consumption might normally occur. Animals do not live in niches where salt is consistently abundant, nor do they live in a world where they are unable to seek out the mineral they need. In other words, providing rats with unlimited salt or preventing them from having access for a period of time fails to mimic the conditions an

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8 animal would either encounter or create in their natural environment. This means that any data we glean from these types of studies are limited in their scope and interpretation. An Alternative Protocol: Simulated Foraging Collier and his colleagues introduced an alternative to the two previously discussed protocols, a simulated foraging task. It combines operant methodology and a free-feeding paradigm to simulate the availability, or the cost of foraging. Animals live in an operant chamber environment and must “work” for access to commodities. Working consists of some energy/time-consuming task such as pressing a lever or running in a wheel a certain number of times. When the experimenter-determined requirement is met, the animal has unlimited access to the commodity (for example, food or water), but only for a specified amount of time. After the animal pauses for a period of time (i.e., 5 minutes), the commodity is withdrawn. Once this occurs, the animal must again carry out the requirement to obtain access. In the following, the amount of work is termed a procurement fixed ratio (PFR), indicating there is a set amount of work they must do in order to obtain the commodity. This approach has advantages over the usual method of observing feeding and drinking behavior. One advantage is that the animal will initiate its own meals. This results in clearly defined meal episodes. However, the experimenter-determined period of time that must elapse before the drinking/feeding apparatus is retracted is still an important consideration and could effect meal size and number. That is, if the ‘time elapsed since last lick’ criterion is shorter than a rat’s normal interbout interval, then the meal will have been cut short. Another advantage is that the amount of work can be experimentally determined so that there can be different costs associated with commodities overall, and even within

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9 individual commodities. In other words, the experimenter can explore differences in meal patterns when a commodity is ‘inexpensive’ (i.e., 1 lever press) versus when they are ‘expensive’ (i.e., 300 lever presses). In fact, the change in commodity cost results in an interesting relationship between meal size and number. An ‘expensive’ PFR results in few, but large meals being ingested at one time, whereas an ‘inexpensive’ PFR results in many, smaller meals being consumed. Varying commodity cost is a worthwhile way of exploring salt-obtaining strategies because it mimics scarce or unpredictable environments. Collier and Rovee-Collier (1980) point out that behavior tends to be expansive in times of plenty, which obscures strategies that have evolved to see species through scarce times. Studying sodium appetite in an open economy (where the experimenter provides unlimited access to sodium) reveals little about how an animal would behave in an appropriate niche. A third, related, advantage of using Collier and colleagues’ protocol is that the cost of commodities could be manipulated independently, so that relative costs are different. In this way, salt can be relatively more expensive than water, for example. This added level of complexity would allow one to more closely mimic the relative availability of sodium and water that is characteristic of a given niche. Finally, another advantage of this paradigm is that the animal will experience only self-imposed periods of deprivation, thus presumably avoiding any possible side effects associated with experimenter-induced periods of deprivation. These things considered, the record of behavior is a better approximation of how an animal normally feeds-that is, episodically (and exclusively of other commodities).

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CHAPTER 2 EXPERIMENT 1: SIMULATED FORAGING AND SODIUM PREFERENCE UNDER NEED FREE AND DOCA TREATMENT CONDITIONS Introduction The aim of Experiment 1 was to examine the sodium consumption patterns of rats in a simulated foraging paradigm and the differences in patterning resulting from DOCA-induced sodium appetite. Adding an environmental context to studies of sodium appetite should reveal that environmental availability of commodities (such as sodium) is an important aspect of sodium preference and appetite that should not be ignored. Though this experiment is the first of its kind regarding sodium appetite, the idea is not new. In fact, Rowland (1990a) appealed to researchers studying ‘ingestive behavior’ for “more complex paradigms that entail some kind of evaluation of the total environmental situation by the organism and the production of a strategy” to be applied to hydromineral studies as early as 1990. Hypotheses Experiment 1 was designed to explore whether the following hypotheses would be supported: Consumption patterns of salt solution will change as a result of imposing procurement fixed ratio cost. Intake of NaCl solution will be governed by the same principle that Collier and colleagues identified for food and water. That is, as the PFR increases, the salt bout size will increase, while the frequency decreases. Addition of daily DOCA administration will result in increased salt bouts compared with baseline conditions. 10

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11 Imposition of PFR will not change the preference aversion curve (for three selected concentrations of NaCl: 0.04 M, 0.15 M, and 0.4 M). Imposition of PFR will not alter the nocturnal salt-bout pattern observed in DOCA-treated rats under free access studies. Methods Animals Four male Sprague Dawley rats (Harlan Labs) weighing approximately 400 g at the start of experiment were used. They had previously been in an unrelated flavor conditioning experiment that did not involve manipulation of fluid balance. Apparatus A standard operant chamber (Med Associates) with inner dimensions measuring 34 X 24.5 X 29.5 (cm, L x W x H), was used throughout the experiment. The chamber was fitted with two response levers on one wall that were located 11.7 cm apart from each other. The animals lived in the chambers for 23.5 hours a day. During testing, they received distilled water or NaCl solution after completing an experimenter-determined procurement fixed ratio (PFR) on the response levers. The fluid reinforcers were delivered through bottles with spouts on a motorized platform that could be moved to be either accessible or inaccessible to the rats. When in the reinforcement position, the tip of the spout was 2 mm outside of the cage and accessible through a vertical slot (1 X 1.5 cm) in the cage wall. Training Animals were placed into operant chambers for one week prior to the beginning of lever training. Pellet food (Purina 5001), from a jar remote from the drinking spouts and distilled water were both available ad libitum. Lever training consisted of equal training on the left and right lever. The PFR was increased in the following order: 1, 5, 10, 20,

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12 40, 60, 80, 100, 120, 140, 160, 200, 240, 280, 300. The lever press requirement was needed in order to procure a drinking commodity (H2O during training). The animal could then consume as much as it wished, but when 5 minutes elapsed in which no spout contact occurred the spout was retracted. Every 100th lick was recorded as it occurred in real time [rats lick 5-7 times per second, so each recorded instance corresponds to approximately 15 seconds of continuous licking]. Testing Two odors (lemon or peppermint) were added as consistent cues with either distilled water or different concentrations of salt (0.04 M, 0.15 M, or 0.4 M NaCl). The odor-solution combination was alternated on average every 24 hours. Thus, a given rat always received, say, lemon paired with NaCl, but the left/right position alternated daily. The odor, presented on a cotton swab (70 P L/ day) in a plastic dish beneath the reinforce m ent slot (outside of the cage), was added with the intent that it would serve as a discriminative stimulus for the solution. Procurement-fixed-ratio schedules were first presented in an ascending, then in a descending order (1, 80, 300, 80, 1). In the first part of the study, the performance on the descending limb matched closely the performance on the ascending limb, so subsequent studies used only the ascending order. The criterion for moving onto the next PFR was initially set at stable 2-day intake (for NaCl and H2O) in terms of number of bouts taken. Unfortunately, that was not always possible since at some concentrations, the rats maintained a side bias and drank equally from the water and salt solution. When this occurred, we waited until the total number of meals was approximately equal for two consecutive days. Therefore, the data for each solution will be presented as means of all

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13 days. Both PFR and NaCl concentration were presented in ascending order (See Table 2-1). At the end of the 0.40 M series, the rats were put on a concurrent schedule of PFR 1 H2O and PFR 80 NaCl. During the DOCA phase, the sa m e presentation order was used, except rats received daily injections of DOCA (6 mg/kg B.W.). Results The results of the 0.04 M NaCl phase are presented in Figures 2-1 and 2-2 (panels A and D). It can be seen that the rats consumed equal amounts of 0.04 M NaCl and distilled water as the position of the fluid was alternated from day to day. If anything, 0.04 M NaCl was consumed at a slightly higher amount than water. However, with the addition of DOCA injections, the rats initiated more NaCl bouts (Figure 2-1, compare panels A and D); water bouts were slightly decreased. The mean fluid bout size of NaCl was also increased compared with the baseline condition, while bout size for water was unaffected (Figure 2-2, compare panels A and D). The results of the 0.15 M NaCl phase can be seen in Figures 2-1 and 2-2 (panels B and E). During the 0.15 M NaCl condition, rats took slightly more water than NaCl bouts in the baseline phase. However, this pattern was reversed during DOCA administration at PFR 1 (Figures 2-1 and 2-2, panels E) when NaCl was taken in preference to water. Figures 2-1 and 2-2 (panel B) shows that water and saline were initiated equally during the baseline phase when PFR was raised to 80 and 300, but that water was consumed in slightly higher bout sizes than NaCl as the “cost” increased. On the other hand, during DOCA administration at PFR 80 and 300, rats initiated more 0.15 M NaCl bouts compared with water bouts (Figure 2-1, compare panels B and E). Moreover, the number of bouts initiated under DOCA conditions exceeded the number initiated during baseline conditions. It can be seen in Figure 2-2 (panels B and E) that the size of distilled water

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14 bouts remained approximately the same for the DOCA injection phase when comparing within PFRs. It can be seen in Figure 2-1 (panel C), that the baseline intake of 0.4 M NaCl was low. The number of NaCl bouts were similar (approximately 1 bout/day) across all PFR values implemented. The number of water bouts decreased from PFR 1 to PFR 80, and only slightly more from PFR 80 to PFR 300 (Figure 2-1, panel C). DOCA administration raised the number of 0.4 M bouts initiated at all PFRs (Figure 2-1, panels C and F). Water bout size remained stable within the PFRs while 0.4 M NaCl bouts increased in size (Figure 2-2, panels C and F). Note when DOCA was given the number of water bouts was also increased (Figure 2-1, panels C and F), but NaCl bout size increased while water bout size remained the same (Figure 2-2, panels C and F). This pattern resulted in rats consuming salt more often and in larger portions during the DOCA phase. The same pattern was also seen in all other concentrations of NaCl presented (Figure 2-1, panels A to C). The preference for the three concentrations of NaCl can be seen in Figure 2-3. Discussion The results are interesting in comparison with Stricker, Gannon, and Smith’s (1992) study; they found that DOCA-treated rats added many small salt (0.5 M NaCl) bouts to their established feeding and drinking routine. In the present experiment, rats in the PFR 1 condition initiated and took many saline bouts, but at the higher PFR conditions (i.e., PFR 80 and 300) the rats did not initiate as many bouts. This suggests that in times of commodity scarcity (simulated by high PFRs), Sprague Dawley rats will not use the same strategy to consume sodium as in times of plenty (i.e., free access). Adding an environmental context to studies of sodium appetite reveals that

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15 environmental availability of sodium is an important aspect of sodium preference and appetite that should not be ignored. A second dimension on which the present experiment and past studies can be compared is the time of day that the salt bouts are initiated. Twenty-four hour free-access studies using DOCA-induced salt appetite show that a majority of the salt is consumed during the dark phase (Schulkin, 1991). Even if the DOCA injection is given early in the light cycle, rats will often wait until the lights shut off to begin consuming great quantities of saline (Stricker, Gannon, and Smith, 1992). The rats in Experiment 1 also consumed the majority of fluids, including sodium-solution during the dark cycle (data not shown). This fact leads to interesting questions about what is controlling the sodium intake behavior (both procurement and intake). Rowland (1990a) emphasized this curious finding seen in studies of DOCA-induced salt appetite as a means for looking at salt appetite in a more complex fashion. Because rats are willing to sustain large variations in sodium balance in the day versus the night, he stated that at the very least there is a nycthemeral-gating factor controlling sodium consumption. The methodology used in Experiment 1 is amenable to answering some of those questions. The lever-pressing task extends the period of time in which the rat is signaling the experimenter that it is about to engage in a bout of sodium-solution. Rather than trying to sample plasma at the moment in which the animal approaches the spout (free-access protocols), a lever-press protocol would allow time to elapse between the point at which the animal initiates the behavior and its gaining access to the sought commodity. Moreover, with an ‘expensive’ PFR, the rat’s motivation to work for the commodity guarantees the rat is about to engage in a large salt bout, which is not certain in a free

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16 access study. The period of lever pressing would be the optimal time to look at the hormonal profile of the rat to determine the chemicals involved in salt appetite. It is conceivable that experiments using agonists and antagonists could be designed to determine a cause-effect relationship between the chemicals involved in organizing and guiding this sodium regulatory behavior. The details of such an investigation remain to be worked out, but this methodology certainly opens doors for further study.

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17 Table 2-1. Order of Stimuli Presentation. The PFR was presented in ascending order within a concentration of NaCl. Concentrations of NaCl were also presented to rats in ascending order. BaselineDOCA 0.04M PFR1 PFR80 PFR3000.04M PFR1 PFR80 PFR300 0.15M PFR1 PFR80 PFR3000.15M PFR1 PFR80 PFR3000.40M PFR1 PFR80 PFR3000.40M PFR1 PFR80 PFR300

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18 Average Meal Numbe r PFR 1PFR 80PFR 300 02468 1012141618 Average Meal Number (with Daily DOCA Injection) (N=4) PFR 1PFR 80PFR 300 PFR 1PFR 80PFR 300 02468 1012141618 Water NaCl 0.04 MA C DF0.40 M 0.40 M 0.15 MB 02468 1012141618 0.15 ME Procurement Fixed Ratio Schedule0.04 MAverage Meal Number (Baseline) (N=4) Figure 2-1 Average meal number initiated at different procurement fixed ratios and with different concentrations of NaCl (0.04 M, 0.15 M , or 0.40 M) available. The left panels are Baseline intake and the right panels are the sa m e rats under 0.6 m g/kg /day DOCA administration.

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19 PFR 1PFR 80PFR 300 0 500 100015002000250030003500 Average Meal Size (In Licks) Average Meal Size (Baseline) (N=4) 0.15 M 0.04 M 0.04 MA B D PFR 1PFR 80PFR 300 0 500 100015002000250030003500 Water NaCl 0.40 MC0.40 MFAverage Meal Size (with Daily DOCA Injection) (N=4) Procurement Fixed Ratio Schedule 0 500 100015002000250030003500 0.15 ME Figure 2-2 Average meal size of rats under different procurement fixed ratios and with different NaCl concentrations available. The left panels are Baseline intake and the right panels are the sa m e rats under 0.6 m g/kg /day DOCA ad m i nistration.

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20 0102030405060708090100 NaCl Preference (in %) 0102030405060708090100 Preference for NaCl (Baseline) (N=4) PFR 1PFR 80PFR 300PFR 1PFR 80PFR 3000.04 M0.15 M NaCl 0.40 MPFR 1PFR 80PFR 300PFR 1PFR 80PFR 300 0.04 M Procurement Fixed Ratio Schedule 0.15 M 0.40 MPFR 1PFR 80PFR 300PFR 1PFR 80PFR 300Preference for NaCl (with Daily DOCA Injection) (N=4)************ Figure 2-3 Preference-aversion data for the three concentrations of NaCl by rats in a simulated foraging paradigm. An asterisk indicates a significant difference from 50%, or non-preference.

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CHAPTER 3 EXPERIMENT 2: SIMULATED FORAGING AND SODIUM APPETITE UNDER BASELINE AND NEED-INDUCED (HYDROCHLOROTHIAZIDE) CONDITIONS Introduction The DOCA method of inducing a sodium appetite does not reproduce the physiological state of a naturally occurring (sodium) deprived animal. DOCA mimics the elevated mineralocorticoid signal observed in rats with low body sodium content. The rats, however, are not sodium deprived; in fact, when given access to saline they have an excess of sodium because of the role that mineralocorticoids play in signaling the kidneys to conserve sodium (Guyton and Hall, 1980). A better characterization of the chemical signals that occur in a diet-induced sodium appetite (natural appetite) would come from placing the rats on a low sodium diet. Unfortunately (but fortunately for rats), the kidneys are very proficient at conserving sodium so that a small amount of concentrated sodium solution is all that is needed to restore a sodium deficit. This would make studying the behavior very difficult. A second option is to chronically deplete the rats with a diuretic so that sodium is continuously lost in the urine. In this way, one might induce a robust appetite that is not easily satiated. That was the approach taken in Experiment 2. The sodium appetite that occurs in HCZ-treated rats using hypertonic concentrations of salt has not been well characterized. As mentioned previously, a sodium bicarbonate solution was selected based on Fregly and Kim’s 1970 work. They found that rats treated with HCZ consumed three times the amount of 0.10 M NaHCO3 21

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22 than untreated rats did (19.25 3.49 versus 6.47 0.65 mL per 100 g B.W., respectively). However, when 0.15 M NaCl was available, the intakes of both groups were not different (13.92 0.63 versus 17.58 0.96 per 100 g B.W., respectively), presumably because the isotonic saline used was so highly preferred, even in the sodium-replete state, that a further increase was obscured. Sodium appetite is most convincingly demonstrated when the baseline consumption of salt solution is low. When a hypertonic sodium solution is used, rats consume very little during the need-free state; this makes it all the more apparent when sodium is consumed once an appetite is induced. It was this basic premise that led to the selection of 0.3 M sodium bicarbonate as the sodium solution in Experiment 2. In addition, we wanted to replicate the work of Fregly and Kim (1970) and show that rats drink copious amounts of sodium bicarbonate when HCZ is administered. Experiment 2 tested the following hypotheses: Consumption patterns of sodium bicarbonate solution will change as a result of imposing PFR cost. Specifically, they will be consistent with the findings in Experiment 1. Adding the diuretic hydrochlorothiazide to the diet will increase the amount of sodium salt consumed at all PFR costs. The majority of sodium intake will occur during the dark phase. Methods Animals Four male Long Evans rats (Harlan Industries), weighing 350 g at the start of the experiment, were used. Food and water were available as detailed in procedures.

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23 Apparatus The operant chambers used in Experiment 2 were the same as those used in Experiment 1. Training Rats were allowed to habituate to living in the operant chambers 23.5 h for 4 days prior to the start of lever training. During this time, animals had ad libitum access to sodium deficient powdered chow and distilled water. Training consisted of left and right levers being removed on alternating days. The PFR schedule was increased every two days so there was equal training opportunity on both levers. No special lever-press shaping procedure was used during training. The animals received distilled water as a reinforcer for successful completion of the imposed PFR. Testing Procedures Two of the four rats had 0.3 M NaHCO3 (based on work by Fregly and Kim (1970), see Discussion section below for explanation) permanently presented on the left, and the other two had it on the right. Water was available on the other side. A fixed position was used in an attempt to mimic permanent locations of fluid commodities (such as a watering hole). A natural ingredient sodium deficient powdered chow (Teklad, WI; TD 90228, sodium content of diet typically about 0.01-0.02%) was available ad libitum from a jar fixed to the center of the wall directly opposite the levers and spouts. Baseline The PFR was initially set at 1 until two subsequent days of stable performance occurred. The criterion for moving on was set as the same bout number initiated two days in a row and total licks being within 1000 licks of each. Therefore, each animal progressed at different rates throughout the experiment. Baseline consisted of four

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24 phases: PFR 1 for both response levers, then PFR 80 (both levers), next PFR 300 (both levers), and finally PFR 1-H20 & PFR 80-NaHCO3 offered concurrently. This last condition was intended to mimic differential cost for procuring salt and water in an environment in which salt is more difficult to obtain. Hydrochlorothiazide (HCZ) phase The HCZ phase consisted of adding 0.6 g hydr ochlorothiazide (Sig m a, St. Louis, MO) to every 1 kg of sodium deficient powdered chow. During this phase, rats were exposed to the same conditions as during baseline, with the exception that HCZ was added to their diet. Results The results of Experiment 2 are shown in Figure 3-1 and 3-2. It can be seen that rats consumed 0.3 M NaHCO3 when PFR was 1, but not when 80 or 300 lever presses were required (Figure 3-1, Panel A). The number of distilled water bouts consumed decreased as the PFR increased (Figure 3-1, panel A). During the experimental phase, when HCZ was added to the diet, NaHCO3 bouts were initiated more frequently in PFR1; bout numbers initiated for sodium bicarbonate we re increased from approximately 4 to 15 (Figure 3-1, panel B). Hydrochlorothiazide administration stimulated an increase in NaHCO3 bouts at PFR 80 and PFR 300, as well. During the experimental phase, NaHCO3 was consumed at each PFR requirement (Figure 3-1, panel B). Animals drank noticeably less water (a function of fewer bouts initiated and less consumed each time) when HCZ was administered (Figure 3-1, compare panels A & B and C & D). Figure 3-2 contains data for the different salt and water PFRs offered concurrently. During the baseline phase, when NaHCO3 was made relatively more expensive than water, the rats did not consume any salt meals. However, once HCZ was added to their diet, they all worked for

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25 access to the sodium solution. Interestingly, the bout patterns still followed the expected patterns from Marwine and Collier’s (1979) original work. The number of NaHCO3 (PFR 80) bouts consumed was slightly less than the number that occurred with the lower ‘priced’ water, but each time salt was initiated, much larger bouts were consumed than were seen with water. Discussion The HCZ-induced appetite persisted throughout this experiment. Rats were willing to work for access to salt at each of the PFRs, including when water could be obtained with only one lever press. These results differ from DOCA-treated animals (Experiment 1, data not shown) in which only one of the rats gained access to salt solution when water was the cheaper commodity. Though these comparison data are not conclusive, they are one possible dimension on which the motivation to consume salt can be assessed. Moreover, juxtaposing different costs more closely mimics the kind of variable environment a rat would likely encounter as a part of its niche. Assuming that salt and water would be available at the same energy/time cost really limits what we can learn about a rat’s strategy for procuring sodium which is often a scarce commodity.

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26 Baseline Phase Mean Meal SizeProcurement Fixed Ratio Requirement PFR 1PFR 80PFR 300 Mean Meal Size (in Licks) 025050075010001250150017502000 HCZ Phase Mean Meal Size PFR 1PFR 80PFR 300 Baseline Phase Mean Meal Number PFR 1PFR 80PFR 300 Mean Meal Number 0510152025 NaHCO3 (n=3) dH2O (n=3) HCZ Phase Mean Meal Number PFR 1PFR 80PFR 300 Figure 3-1 Mean number (top panels) and size (bottom panels) of sodium bicarbonate or water bouts by three rats in a simulated foraging task. The left panels show Baseline intake and the right panels show intake after treatment with the diuretic HCZ (0.6 g/kg food).

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27 Baseline Phase Mean Meal SizeProcurement Fixed Ratio Requirement PFR 1, 80 Mean Meal Size (in Licks) 025050075010001250150017502000 HCZ Phase Mean Meal Size PFR 1, 80 Baseline Phase Mean Meal Number Mean Meal Number 024681012141618 NaHCO3 (n=3) dH2O (n=3) HCZ Phase Mean Meal Number Figure 3-2 Mean bout number (top) and size (bottom) of sodium bicarbonate and water when differential procurement fixed ratios are implemented. Water was made “inexpensive” at PFR 1, while salt was relatively “expensive” at PFR 80. The left panels show Baseline intake and the right panels show intake after treatment with the diuretic HCZ (0.6 g/kg food).

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CHAPTER 4 EXPERIMENTS 3A TO 3D: PHYSIOLOGICAL MEASUREMENTS General Introduction The methodology used in Experiment 2 revealed the behavioral effects of HCZ on sodium bicarbonate intake, however, the physiological effects of the diuretic were not well understood. Therefore, the studies in Experiment 3 were carried out in order to characterize the physiological effects of HCZ. Primarily we were interested in the ability of HCZ to sustain a long-term appetite. The robust sodium appetite-stimulating effects of chronic administration were confirmed by Experiments 3a, 3b, and 3d. We also wanted to know the natriuretic capacity of HCZ in order to validate our claim that it induced a need-based appetite. Those data were obtained in Experiment 3b. However, during the course of our investigation of the physiological consequences of chronic HCZ treatment, we found an unexpected effect on body weight that occurred when NaHCO3 was the salt available, but not when NaCl was offered. These interesting results were the impetus behind Experiments 3c and 3d. General Aims for Experiment 3 The series of studies in Experiment 3 were done in parallel with Experiment 2 in order to explore the sodium appetite elicited by HCZ for hypertonic salt solutions (NaHCO3 and later NaCl) under free-access conditions. Experiment 3 had the following objectives: 1. Compare HCZ method of sodium depletion with furosemide method of inducing appetite. 28

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29 2. Characterize the intake of sodium in HCZ-treated animals. 3. Characterize the hormonal profile (specifically, plasma aldosterone and plasma renin activity) of rats treated with HCZ. 4. Examine the urinary sodium output of animals treated with HCZ and compare with control animals. Experiment 3a: Physiological and Behavioral Characterization of HCZ-Induced Salt Appetite (SD Rats and 0.1 M NaHCO3, 0.3 M NaHCO3, and 0.3 M NaCl) Methods and Procedures Animals Eighteen male Sprague Dawley rats (Harlan Industries, Indianapolis, IN) weighing approximately 470 g at start of experiment were used. All rats were housed individually in hanging wire cages with ad libitum access to sodium deficient powdered chow, dH20, and in different phases of the study, one of the following salt solutions 0.1 M NaHCO3, 0.3 M NaHCO3, or 0.3 M NaCl. The two solutions (salt and water) were presented in 100 mL inverted graduated cylinders with a rubber stopper and metal sipper spout. Procedures The rats were divided into three groups: one control and two experimental groups (HCZ and Furosemide). Rats in the HCZ group received 0.6 g of HCZ/kg food, daily. Furosemide (Abbott Labs, Abbott Park, IL) was injected (10 mg/kg B.W., subcutaneously) during routine maintenance handling at about 09:00 daily. Control animals received the sodium deficient powdered chow but no injections or HCZ. Salt solution was varied according to the following schedule: Days 1-7, 0.1M NaHCO3; Days 8-12, 0.3 M NaHCO3; Days 13-16, 0.3 M NaCl; Days 17-25, 0.3 M NaHCO3; Days 26-32, 0.3 M NaCl; and Days 37-43, 0.3 M NaHCO3. Rats in the Furosemide group were discontinued on day 11 because of weight loss (see results).

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30 Results of Experiment 3a The results for body weight can be seen in Figure 4-1. It can be seen that during exposure to 0.1 M NaHCO3, body weight differed among the three groups [F(2,15) = 8.77, p<0.01]. Treatment with furosemide resulted in a significant reduction in weight compared with control rats (p<0.05). Differences in body weight between HCZ-treated rats and controls appeared when the HCZ-treated rats had access to 0.1 M NaHCO3 and lasted for the duration of the experiment (p<0.05). The results for food intakes can be seen in Figure 4-2. There was a significant difference in food intakes for the control, HCZ, and furosemide treated rats under 0.1 M NaHCO3 condition [F(2,15)=6.56, p<0.01]. Rats receiving daily injections of furosemide ate on average 50% less than the control group (p<0.05). In contrast, food intakes were not statistically different between HCZ-treated and control groups throughout most of the experiment. However, toward the end, HCZ-treated rats consumed slightly more food than controls (p<0.05). The results from water intake are presented in Figure 4-3. Water intake was similar in all three groups during the initial 0.1 M NaHCO3 period [F(2,15)=1.23, p>0.05] Figure 6, condition A). Water intake was also statistically similar when 0.3 M NaHCO3 was the salt available (p>0.05), except for the last presentation of 0.3 M NaHCO3 (Figure 4-3, conditions B). However, when 0.3 M NaCl was offered, HCZ-treated rats consumed less water than control rats (p<0.01) (Figure 4-3, conditions C). The results for salt consumption are shown in Figure 4-4. The 0.1 M NaHCO3 intakes differed significantly between groups [F(2,15)=19.2, p<0.01]. Intake was highest for HCZ-treated rats (p<0.05), followed by furosemide-treated (p<0.05) and then control rats (Figure 4-4, condition A). When 0.3 M NaHCO3 was offered, controls ceased

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31 drinking the salt (Figure 4-4, condition B). All groups’ 0.3 M NaHCO3 intakes differed from one another [F(2,15)=19.8, p<0.01]. Again, HCZ-treated rats’ intake was greater than furosemide-injected rats and control rats (p<0.05). Until the last exposure to 0.3 M NaHCO3 (p>0.05), HCZ-treated rats consumed more salt solution than controls (p<0.05 for all t tests) (Figure 4-4, the first two occurrences each of conditions B and C). Discussion of Experiment 3a Experiment 3a revealed that SD rats would consume more sodium solution than controls even when a hypertonic NaHCO3 or NaCl solution was offered. This extends the work of Fregly and Kim (1970) that showed an appetite using 0.1 M or 0.15 M solution, respectively. Intake of sodium bicarbonate appeared to be more robust, making it a candidate salt solution for exploring sodium appetite. Traditionally, sodium chloride solutions are used with rats (Denton, 1982). Results of this study also support the use of HCZ as a chronic method of inducing a sustained salt appetite; furthermore, it is a better method compared with daily furosemide injections because there is less severe anorexia associated with HCZ treatment. Experiment 3b Physiological and Behavioral Characterization of HCZ-Induced Salt Appetite Introduction to Experiment 3b Experiment 3b was designed to explore whether salt intake was increased as a result of urinary sodium loss. Moreover, we wanted to know if there would be a change in sodium output as the animals were chronically exposed to HCZ. Additionally, we wanted to examine the effect of HCZ on 0.3 M NaHCO3 intake in rats of the Long Evans strain because this was the strain used in Experiment 2.

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32 Methods and procedures Animals Fifteen male Long Evans Hooded rats (Harlan Industries, Indianapolis, IN) were used. They weighed approximately 380 g at the start of experiment. Procedures Rats were housed individually in hanging wire cages with ad libitum sodium deficient powdered chow, dH20, and 0.3 M NaHCO3, except where noted. The two solutions (salt and water) were presented in 100 m L inverted graduated cylinders with a rubber stopper and metal sipper spout. The rats were divided into two groups: one control and one experimental group (HCZ). Rats in the HCZ group received 0.6 g of HCZ/kg food, daily. Amounts of water, salt, and food (plus spillage) were assessed daily during routine maintenance at approximately 10:00 h. All rats were transferred to individual metabolic cages for 24 h on days 3, 8, 13, 17, 20, 23, and 25. They had access to both food and water during this time. At the end of the 24 h period (which started at about 09:00 h) the urine of each rat was collected and 1 m L was frozen (4 C) for subsequent analysis of sodium and potassiu m . On day 20 (at approxi m ately 13:00 h with salt available), 70 L of blood was withdrawn from each rat into a capillary tube from a small nick on the tip of the tail. On day 23 (at approximately midnight with no salt available), blood was withdrawn from each rat in the same manner. On day 26 (at approximately noon, with no salt available), another blood sample was obtained. On day 28 (at approximately midnight, with salt available) a final sample of blood was withdrawn from the tail. On each occasion, the

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33 blood was placed into an ice-chilled 0.5 m L m i crocentrifuge tube containing 6 L EDTA. These samples were later analyzed for plasma renin activity (PRA). On day 29, the two groups were further subdivided into groups. Half the rats (of each group) received salt and the other half did not. At approximately 01:00 h rats in both groups were briefly anesthetized with isoflurane and a 1mL blood sample taken by cardiac puncture using a 23 g needle. Each rat’s sample was divided into two m i crocentrifuge tubes surrounded with ice water: 200 L were placed into a 0.5 m L tube containing 20 L EDTA and later analyzed for PRA. The re m aining 800 L were placed into a 1 m L tube (no EDTA) and later analyzed for plas m a aldosterone activity. Two rats from the HCZ group died from the procedure (no blood was obtained from them). On day 40 (01:00 h), blood was taken in the same manner. Each rat received the opposite salt condition as occurred on day 29. On day 56 (01:00 h), rats received salt under the same conditions as day 29 (opposite of day 40); blood was withdrawn in the manner described previously. In instances where two measurements from the same rat (under the same salt condition) occurred, the values were averaged together. Assays Blood samples were immediately centrifuged (4,000 rev/min at 4 C) and plasma removed and frozen at C. At a later time, samples were thawed on ice and plasma renin activity (PRA) was measured using the NEN Life Sciences angiotensin I (ANG I) radioi mm unoassay kit, m odified to use s m all sa m ple volu m es (10 L ) in the incubation phase. Plasma aldosterone concentration was measured using the Cambridge Diagnostics Coat-a-Count radioimmunoassay kit and duplicate 100 L samples. Urinary sodium and potassium concentrations were measured by flame photometry and, using the urine volumes for 24 hours, the sodium excretion was calculated.

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34 Statistics Analysis of variance (ANOVA) statistics (SigmaStat PC version 1.0) were run with main factors (where relevant) of drug treatment, time of sampling (day versus night), and availability of salt. Post hoc comparisons were by Student-Newman-Keuls method. Significance levels () were set at 0.05. Results of Experiment 3b As time passed, the separation in weight for the two groups grew, with HCZ-treated rats weighing less than controls (Figure 4-5). A difference in body weight was apparent for HCZ treated rats compared with controls (p<0.01). However, this difference in weight could not be accounted for by food intake. HCZ-treated rats consumed similar amounts of food to controls (p>0.05) (Figure 4-6). Overall, water intake for HCZ-treated rats was similar to that of controls (p>0.05) (Figure 4-7). In contrast, sodium bicarbonate (0.3 M) intake was dramatically higher (> 100 mL/day) in HCZ-treated rats than controls and it was stable across the 56 day period (p<0.01) (Figure 4-8). HCZ had an expected natriuretic effect that was sustained. HCZ-treated rats excreted a higher volume of urine compared with normal rats (p<0.05) (Table 4-1). Urine excretion occurred mostly during the dark phase, which coincides with drug ingestion via food intake (data not shown). It can be seen in Figure 4-9 (panel A) that rats treated with HCZ lost significantly more Na+ in their urine than controls (p<0.01). Potassium was excreted to a lesser extent, however the reverse relationship existed: control rats excreted more K+ than experimental rats (p<0.01) (Figure 4-9, panel B). Results for plasma aldosterone are shown in Figure 4-10. It can be seen that levels were higher in rats when they did not have access to 0.3 M NaHCO3 than when they did (p<0.05). Results for PRA are shown in Figure 4-11. The PRAs for HCZ-treated rats

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35 were significantly higher (p<0.05) than control levels under all four conditions tested (viz: During day with NaHCO3 available, during day without NaHCO3 available, during night with the salt available, and during the night without the salt available (Figure 4-11, panels A to D, respectively). Discussion of Experiment 3b The body weight and salt intake results from Experiment 3b differ from those obtained in Experiment 3a. Rats continued to lose weight in Experiment 3b, though they ate as much as controls. The decrease in body weight may have been partially accounted for by the diuretic/natriuretic effect. Another possible explanation comes from work by Cassis et al. (1998). They characterized the impact of ANG II infusion on body weight and found that rats infused over two weeks with ANG II (175 ng/kg/min) had lower body weights than controls, but that food intake was not different between the groups. ANG II infusion increases abdominal body temperature suggesting body weight is affected through mechanisms related to increased peripheral metabolism (Cassis et al., 1998). The rise in PRA resulting from HCZ treatment (See Figure 4-11) is an indirect measure of ANG II levels. Perhaps the increase in ANG II seen in HCZ-treated rats can explain the weight differences between groups. Rats in Experiment 3a did not show such a dramatic weight difference. There were some important differences between the two experimental protocols, which included strain and age differences. However, inspection of the impact of salt exposure on the food intake of rats in Experiment 3a suggested that the salt offered may have had an effect. To address whether the weight loss was a consequence of sodium bicarbonate we presented 0.3 M NaCl alongside water in Experiment 3c.

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36 Experiment 3c Physiological and Behavioral Characterization of HCZ-Induced Salt Appetite (LE Hooded Rats and 0.3 M NaCl) Methods and Procedures Animals Twelve male Long Evans Hooded rats (Harlan Industries, Indianapolis, IN) were used. They weighed approximately 470 g at the start of the experiment. Procedures The rats were housed individually in hanging wire cages with ad libitum access to sodium deficient powdered chow, dH20, and 0.3 M NaCl. The two solutions (salt and water) were presented in 100 mL inverted graduated cylinders with a rubber stopper and metal sipper spout. The rats were divided into two groups: one control and one experimental group (HCZ). Rats in the HCZ group received 0.6 g/kg of HCZ in their food, daily. On day 8 (at approximately 24:00 h with salt available), 70 L of blood was withdrawn from each rat into a capillary tube from a small nick on the tip of the tail. The blood was placed into an ice-chilled 0.5 mL microcentrifuge tube containing 6 L (100 mg/mL) EDTA and 10 L (1 mg/mL) heparin. These samples were later analyzed for PRA as before. Results of Experiment 3c The results of Experiment 3c can be seen in Figs 4-12 through 4-15. Body weight (Figure 4-12) for the two groups did not differ significantly for the 15 days of the experiment (p>0.05). Unlike animals in experiment 3b where HCO3 is the anion, HCZ-treated animals given access to 0.3 M NaCl did not lose weight. Food intake (Figure 4-13) for both groups were similar (p>0.05), as was water intake (p>0.05) (Figure 4-14).

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37 There was, however, a separation of NaCl intake for HCZ-treated animals compared with control animals (Figure 4-15), wherein HCZ-treated rats consumed noticeably more NaCl than controls (p<0.01). PRA in HCZ-treated animals was higher (20 pg/mL/min) than controls (2 pg/mL/min) when tested during the dark cycle with salt available (p<0.05). Discussion of Experiment 3c The results from Experiment 3c are in support of the notion that the salt available is an important factor in maintenance of body weight for the rats in these experiments. The rats in this experiment did not lose weight in the 15 days of the experiment; note this is beyond the time course they would have been expected to have lost weight (12 days) based on results from Experiment 3b. To further explore the effect sodium and its accompanying anion have on body weight Experiment 3d was done. We decided to contrast NaHCO3 with NaCl. Experiment 3d Physiological and Behavioral Characterization of HCZ-Induced Salt Appetite (LE Hooded Rats and 0.3 M NaCl or 0.3 M NaHCO3) Methods and Procedures Animals Twelve male Long Evans Hooded rats (Harlan Industries, Indianapolis, IN) were used. They weighed approximately 470 g at start of experiment. Procedures Rats were housed individually in hanging wire cages with ad libitum access to dH20, and either 0.3 M NaCl or 0.3 M Na NaHCO3. All rats received sodium deficient powdered chow with 0.6 g/kg HCZ added. The two solutions (salt and water) were presented in 100 mL inverted graduated cylinders with a rubber stopper and metal sipper

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38 spout. The rats were divided into two groups: group 1 received NaCl and group2 received NaHCO3. On day 12, because their body weights had been steadily decreasing, rats in group 2 were switched from NaHCO3 to 0.3 M NaCl (group 1 continued on NaCl). Two of the group 2 rats became ill and stopped drinking altogether and were euthanized because of their condition. All rats received 0.3 M NaCl for the rest of the experiment. Results of Experiment 3d Mean body weight (Figure 21) for HCZ-treated rats given initial access to 0.3 M NaHCO3 (group 2) decreased steadily as the experiment progressed and were different from rats in group 1 that had NaCl throughout (p<0.01). On day 12, When NaHCO3 was switched to 0.3 M NaCl the rats (group 2) began showing an increase in body weight. By the end of the experiment (Day 32), body weights were not different between the two groups (p>0.05). Food intake (Figure 22) was much lower in HCZ-treated animals receiving NaHCO3 access (p<0.01). Once the salt was switched, food for group 2 was consumed in amounts similar to group 1 (p>0.05). Water intake (Figure 23) for group 2 was considerably higher than group 1 while NaHCO3 was present (p<0.05), but followed that of group 1 when NaCl was the salt presented (p>0.05). Sodium bicarbonate (Figure 24) was consumed at higher amounts than NaCl (p>0.01). When group 2 was switched to NaCl, NaCl intake was comparable to group 1 (p>0.05). Discussion of Experiment 3d The results from Experiment 3d convincingly show that rats on HCZ-adulterated chow and consuming NaHCO3 are adversely affected. The rats’ body weights decreased when consuming the bicarbonate solution and subsequently increased when the anion paired with Na+ was switched to Cl-. It is interesting that the rats in group 2 were able to

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39 catch up to the body weights of the rats receiving NaCl throughout, this is evidence that HCZ is not anorexigenic. Results from the present experiment support the idea that the weight loss occurred because of the impact it had on food intake. The overall finding of these studies is that if HCZ is used as a chronic means of inducing a sodium appetite, sodium bicarbonate is not a good solution to use. The mechanism through which HCZ administration and sodium bicarbonate consumption leads to decreased food intake is not well understood.

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40 Figure 4-1 Mean body weight. Body weights differed between all groups. The group receiving furosemide (Lasix) was removed from the experiment because of anorexia. Control rats (filled circles) maintained body weight throughout the experiment, while HCZ-treated rats (unfilled circles) maintained a lower weight. A, B, and C refers to the salt available: 0.1 M NaHCO3, 0.3 M NaHCO3, or 0.3 M NaCl respectively. Days 02468101214161820222426283032343638404244 Average Weight (g) 0100200300400500600 C ontrol ( n= 6) ABCBCB Figure 4-2 Mean food intake. Food intakes were significantly different for rats treated with furosemide, but not those treated with HCZ. At the end of the experiment, HCZ-treated rats actually consumed significantly more food than controls. A, B, and C refers to the salt available: 0.1 M NaHCO3, 0.3 M NaHCO3, or 0.3 M NaCl respectively. Days 02468101214161820222426283032343638404244 Average Food Intake (g) 05101520253035 ABCBCB Treatment Legend:A 0.1M NaHCO3B 0.3M NaHCO3C 0.3M NaCl Control (n=6) HCZ (0.6g/KG Food) (n=6) Furosemide (10mg/KG B.W.)

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41 Figure 4-3 Mean water intake. Water intakes were similar for all three groups when NaHCO3 was the salt available. Water intake was lower in HCZ-treated rats than in controls when NaCl was offered. A, B, and C refers to the salt available: 0.1 M NaHCO3, 0.3 M NaHCO3, or 0.3 M NaCl respectively. g Days 02468101214161820222426283032343638404244 Average Water Intake (ml) 020406080100120140160180 Control ( n=6 ) ABCBCB Figure 4-4 Mean salt intake. HCZ-treated rats consistently consumed the highest amounts of NaHCO3 (both concentrations) and NaCl. Controls did not drink any of the hypertonic salt concentrations. The letter (A, B, or C) corresponds to the salt solution offered (see legend). HC / d) 6 Treatment Legend:A 0.1M NaHCO3B 0.3M NaHCO3C 0.3M NaCl Control (n=6) HCZ (0.6g/KG Food) (n=6) Furosemide (10mg/KG B.W.) Treatment Le g end: Control (n=6) Z ( 0 6 K G F ( ) Days 02468101214161820222426283032343638404244 Average NaHCO3 Or NaCl Intake (in ml) 020406080100120140160180 ABCBCB

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42 Days 0510152025303540455055 Body Weight 280300320340360380400420440460480 Control (n=7) HCZ (n=8) Figure 4-5 Mean body weight. The HCZ-treated rats (unfilled circles) lost significant amounts of body weight throughout the experiment, while the controls (filled circles) gained weight. The weight loss did not appear to be linked to food intake, at least after the first two weeks. 02468101214161820222426283032343638404244464850525456 Food Intake (g) 051015202530354045 Control (n=7) HCZ (n=8) Days Figure 4-6 Mean food intake. HCZ-treated rats (unfilled circles) ate less than controls (filled circles) for approximately the first two weeks, but then ate comparable amounts thereafter. The weight separation (see above) continued beyond the point at which their food intake became the same.

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43 dWater Intake (mL)Days 02468101214161820222426283032343638404244464850525456 Water Intake (mL) 020406080100120140160180 Control (n=7) HCZ (n=8) Figure 4-7 Mean water intake. Rats in both groups drank similar amounts of water. If anything, HCZ-treated (unfilled circles) rats drank more water than controls (filled circles). y 0.3M NaHCO3 Intake (mL)Days 02468101214161820222426283032343638404244464850525456 NaHCO3 Intake (mL) 020406080100120140160180 Control (n=7) HCZ (n=8) Figure 4-8 Mean sodium bicarbonate Intake. HCZ-treated rats (unfilled circles) drank significantly more NaHCO3 than controls. Intake was stable at 100 mLs across the entire 55 days of measurement for the HCZ-treated rats.

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44 Table 4-1 Urine volume excreted. On the days listed, 24 h of urine excretion was collected and measured. The day number refers to the number of days passed since treatment with HCZ began. The total volume excreted for each group remained relatively stable over time. DayUrine Volumes (ml) +/-SE Day 3 Control15.8 1.1 HCZ39.5 3.6Day 8 Control15.8 1.9 HCZ24.4 2.6Day 13 Control16.5 2.3 HCZ27.5 1.9Day 17 Control12.5 1.7 HCZ26.0 3.2Day 20 Control11.8 1.5 HCZ23.8 6.2Day 23 Control14.6 1.8 HCZ22.4 3.3

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45 Figure 4-9 Mean sodium and potassium output. HCZ-treated rats excreted more Na+ (top), but less K+ (bottom) than controls. Their output was consistent at each sampling. Total Na+ Excreted over 24 hour BlocksDays Day 2Day 8Day 14Day 18Day 20Day 23 Mean Na+ Excreted (meq) 0.00.51.01.52.02.53.03.54.0 Control Na HCZ Na Total K+ Excreted over 24 hour BlocksDays Day 2Day 8Day 14Day 18Day 20Day 23 Mean K+ Excreted (meq) 0.00.51.01.52.02.53.03.54.0 Control K+ HCZ K+

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46 Plasma Aldosterone ActivityGroups CNTL w/ SaltCNTL w/o SaltHCZ w/ SaltHCA w/o Salt Aldosterone, pg/ml 0100200300400500 CNTL with NaHCO CNTL w/o NaHCO3 HCZ with NaHCO3 HCZ w/o NaHCO3 Figure 4-10 Plasma aldosterone. Only significant differences in the levels of circulating aldosterone were seen between treatments when NaHCO3 was unavailable. There were no overall differences between control and HCZ-treated rats. Day with 0.3M NaHCO3 access ControlHCZ Night Without NaHCO3 2D Graph 4Group ControlHCZ 010203040506070 y Control HCZ Angiotensin I, ng/mL/min 010203040506070 Day with NaHCO3Night With NaHCO3Day Without NaHCO3 Figure 4-11 Plasma renin activity. HCZ-treated rats had higher PRA in all conditions tested. PRA is indirectly measured by amount of ANG I measured.

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47 Days 0246810121416 Body Weight (g) 420440460480500520540 Control (n=6) HCZ (0.6g/kg food) (n=6) Figure 4-12 Mean body weight. Rats in both groups maintained starting body weight for the duration of the experiment. HCZ-treated rats did not lose any weight, in contrast to HCZ-treated rats in Experiment 3b. Days 0246810121416 Sodium Deficient Powdered Chow Intake (g) 16182022242628303234 Control (n=6) HCZ (0.6g/kg food) (n=6) Figure 4-13 Mean food intake. There was no difference in mean chow intake between HCZ-treated and control rats.

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48 Days 0246810121416 Water Intake (ml) 01020304050607080 Control (n=6) HCZ (0.6g/kg food) (n=6) Water only fluid available this day Figure 4-14 Mean water intake. HCZ-treated rats drank less than controls, except when water was the only fluid available (Day 10). Days 0246810121416 NaCl Intake (ml) 020406080 Control (n=6) HCZ (0.6g/kg food) (n=6) Water only fluid available this day Figure 4-15 Mean NaCl intake. HCZ-treated rats consumed more NaCl than controls. After NaCl deprivation for 24 hours (Day 10), the HCZ-treated rats consumed over 20 mLs more than their previous total.

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49 Days 05101520253035 Body Weight (g) 100150200250300350400 0.3M NaCl Group (n=6) 0.3M NaHCO3 Group (n=4) NaHCO3 changed to 0.3M NaCl Figure 4-16 Mean body weight. All rats received HCZ (0.6 g/kg food) in their food. The group receiving NaHCO3 and water lost a significant amount of weight compared with those rats receiving NaCl and water. On Day 12, the NaHCO3 was switched to NaCl and rats’ weights increased immediately. Days 05101520253035 Sodium Deficient Powdered Chow Intake (g) 5101520253035 NaCl Group (n=6) NaHCO3 Group (n=4) NaHCO3 changed to 0.3M NaCl Figure 4-17 Mean food intake. Rats on NaHCO3 ate significantly less than those rats given NaCl. When the salt was switched, rats in both groups ate comparable amounts.

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50 Days 05101520253035 Water Intake (mL) 0102030405060708090100 NaCl Group (n=6) NaHCO3 Group (n=4) NaHCO3 changed to 0.3M NaCl Figure 4-18 Mean water intake. Rats receiving 0.3 M NaHCO3 consumed more water than the rats receiving 0.3 M NaCl. When NaHCO3was switched to NaCl, all rats drank comparable amounts. Days 05101520253035 Salt Intake (mL) 020406080100 NaCl Group (n=6) NaHCO3 Group (n=4) NaHCO3 changed to 0.3M NaCl Figure 4-19 Mean salt intake. Rats consistently drank more NaHCO3 compared with rats drinking NaCl. When rats were switched from drinking NaHCO3 to NaCl, both groups consumed comparable amounts of NaCl.

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CHAPTER 5 GENERAL DISCUSSION Sodium appetite is a phenomenon that occurs in many animals both in the lab and in the field. The belief that the ability to detect a sodium deficit and then to seek out and consume the needed substance is adaptive is widely held. Such an adaptive system must have evolved in an environmental context. The simulated foraging paradigm is a useful way of observing the strategies that have evolved to handle sodium need in a plentiful, scarce, or variable environment. The simulated foraging task in the present set of studies places the rat in control of its own drinking schedule. Rats typically would have to make decisions about when to take a meal and which commodity to consume, and these decisions usually carry important consequences. Current free-access methods of studying sodium appetite do not take this into account when they offer both ad libitum sodium solution and water to drink. Testing rats in an environment they would never encounter obscures the behavioral strategies that would normally be active when faced with sodium deficit (which incidentally would likely be caused by low sodium in the diet). The results from Experiment 1 and 2 are not directly comparable because of important methodological differences. There were differences in the: training procedures, testing procedures (including the rats’ inability to terminate a meal early in Experiment 1), sodium solution (i.e., NaCl versus NaHCO3), amount of concentrations encountered, the overall molarity of the hypertonic solution, sodium placement 51

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52 (alternating sides versus fixed), and route of administration of pharmacological agent. Nevertheless, the objectives of the two studies were different. A principal aim of Experiment 1 was to examine three specific concentrations from the normal preference aversion curve (those concentrations that define the parameters of the inverted U-shaped function normally yielded from free-access studies); we wanted to contrast the measurements of such a curve obtained in a simulated foraging paradigm. The results of Experiment 1 did not replicate findings from studies using the free-access protocol. Moreover, the post-experiment free-access preference test did not reveal the anticipated inverted U-shaped findings either. There are many plausible explanations for this contradictory finding that highlights some fundamental issues in the study of sodium preference and appetite. One difficulty in examining sodium preference is the unstable nature of the behavior. Prior sodium exposure has a considerable impact on preference behavior. That is, prior exposure to a palatable solution of sodium can increase the intake of unpalatable solutions. This has been used as a strategy by many researchers to ensure intake of very high molarity sodium solutions (i.e., Stricker, Gannon, & Smith, 1993). On the other hand, prior exposure of hypertonic solutions can also decrease preference of typically highly preferred concentrations (Davenport, 1973). In Experiment 1, the rats encountered an ascending series of NaCl concentration and then were exposed to that same ascending series again during DOCA treatment and again at the end of the experiment. Perhaps experiencing higher concentrations previously is the reason rats did not prefer 0.15 M when in the free-access condition.

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53 A second explanation could be that the rats formed an aversion to saline because of forced exposure to the concentrated saline. Devenport (1979) found th at rats given only a hypertonic solution of NaCl to drink for 24 hours did not prefer 0.15 M NaCl when later given a choice between it and water. Surprisingly, the effect was also apparent when 0.15 M was the only drinking solution for 24 hours! It is unexpected that the rats, which typically will consu m e approxi m ately 100% of their solution from the 0.15 M container with water available did not prefer 0.15 M saline in a two-bottle choice test if they had been offered only the isotonic solution for 24 hours. There were instances in Experiment 1 in which the rats only consumed drinking flui d from the salt container. Therefore, the “Devenport phenomenon” may also explain the lack of preference for saline during the post-test. A final explanation of why the rats in Experiment 1 showed abnormal preference behavior in our experiment could be related to the program settings of the mechanical spout. After the rats finished the procurement ratio, the spout became accessible and would remain accessible until 5 minutes had elapsed without a lick. Since the olfactory cues did not seem to serve as discriminatory cues, especially early on, and the location of the salt alternated daily, it is possible that the rats procured the ‘wrong’ solution and continued to drink it because it was the only solution available. It was because of this possibility that the protocol in Experiment 2 differed in two ways. In Experiment 2, the rats had a permanent side for which salt was assigned, and they could ‘turn off' the spout if they pressed the opposite lever two times to end a session early if they chose to procure the other drinking commodity.

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54 In stark contrast with our results is the evidence that claims that one prior episode of sodium depletion, or experience with the elevated hormones of salt appetite, will increase later need-free consumption of NaCl (Falk, 1966; Sakai, Fine, Epstein, & Frankmann, 1987; and Sakai, Frankmann, Fine, & Epstein, 1989). The researchers have even showed elevation in NaCl consumption even if the tests are separated by 4 months (Sakai et al., 1987). The results of Sakai and colleagues would support seeing an increased preference in our rats for NaCl after they experienced the DOCA treatment. However, Rowland (1990b) showed that a simple change in the type of food available would cause this phenomenon to disappear, indicating the elevation is dependent on the environmental context remaining stable. A change from the operant context to hanging cages may also have disrupted the conditions under which we would have seen the increased preference. Experiment 1 revealed that rats would work for access to sodium in a simulated foraging paradigm and they would regulate their intake according to the principles that Marwine & Collier (1979) found operating for food and water intake. The protocol used in Experiment 1 places us one step closer to piecing together the puzzle of sodium appetite as it occurs in a natural context. Experiment 2 was designed to study sodium appetite as it occurs in animals with a physiological need. It can be argued that this would be most like the natural mode of appetite induction in rats. In Experiment 2, the rats received a low sodium diet, but it was further necessary to deplete them of sodium because of the body’s capability of conserving sodium. We wanted to induce a robust behavior in order to study the rats’ strategy for procuring sodium in times of need. Still, in order to study sodium appetite in a realistic manner, it would be optimal to induce it

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55 through a low sodium diet after the rat was familiar with the source of sodium. According to the literature, this method of induction would take a minimum of 8 days, but the rats would become replete after consuming a small amount of hypertonic saline (Stricker, Thiels, & Verbalis, 1991). Administration of a diuretic such as HCZ is a good alternative to waiting the long period of time for an appetite to develop using a sodium deficient diet. When NaCl is used, instead of NaHCO3, the drawbacks of using HCZ are not apparent (see results on food intake from Experiments 3c & 3d). The benefits of using HCZ are that it is easy to administer, it does not impair food intake or growth, it has robust effects that are long lasting, and it is inexpensive. Furthermore, our study (part of Experiment 3b) exploring the time of day that HCZ stimulates sodium intake is consistent with other methods and with the usual nocturnal behavior of rats. The results of each of the studies presented in the present paper are the first of their kind in the field of sodium appetite. The methodology offered in these series of operant experiments and the characterization of hydrochlorothiazide’s chronic behavioral and physiological effects extend previous literature. Simulating an environment in which sodium is not constantly available adds a more practical dimension that challenges the rats to make decisions about consuming sodium as they might in a sodium-limited environment. Additionally, the foraging paradigm can be used with any method of inducing sodium-appetite, and it can (through using separate commodity costs) serve to characterize the differences of rats’ motivation to work for sodium under different methods of induction. But possibly the most important benefit that the foraging protocol offers is that a high procurement cost dramatically cuts down the number of unnecessary

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56 (secondary) bouts that rats take in the free-access paradigm (see Fitzsimons (1979) for explanation of primary and secondary drinking). These methodologies will likely open doors to many interesting questions.

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LIST OF REFERENCES Berridge, K.C., Flynn, F.W., Schulkin, J., & Grill, H.J. (1984). Sodium depletion enhances salt palatability in rats. Behavioral Neuroscience, 98(4), 652-60. Bertino, M., & Tordoff, M.G. (1988). Sodium depletion increases rats’ preferences for salted food. Behavioral Neuroscience, 102(4), 565-73. Buggy, J., & Fisher, A. E. (1974). Evidence for a dual central role for angiotensin in water and sodium intake. Nature, 250(5469), 733-5. Cassis, L.A., Marshall, D.E., Fettinger, M.J., Rosenbluth, B., & Lodder, R.A. (1998). Mechanisms contributing to angiotensin II regulation of body weight. American Journal of Physiology (Endocrinology & Metabolism, 37), 274, E867-E876. Collier, G. H., & Rovee-Collier, C. K. (1980). A comparative analysis of optimal foraging behavior: laboratory simulations. In. A. Kamil, J. Krebs, and R. Pulliam (Eds.), Foraging Behavior (pp 479-496). New York: Plenum Press. Davenport, l.D. (1973). Aversion to a palatable saline solution in rats: interaction of physiology and experience. Journal of Comparative and Physiological Psychology, 83, 98-105. Denton, D.A. (1982). The Hunger for Salt: An Anthropological, Physiological, and M edical A nalysis. New York : Springer-Verlag. Falk, J.L. (1966). Serial sodium depleti on and NaCl solution intake. Physiology and Behavior, 1 , 75-77. Fitzsimons, J.T.(1979). The Physiology of Thirst and Sodium Appetite. New York: Cambridge University Press. Fluharty, S.J., & Epstein, A.N. (1983). Sodium appetite elicited by intracerebroventricular infusion of angiotensin II in the rat: II. Synergistic interaction with systemic mineralocorticoids. Behavioral Neuroscience, 97(5), 746-58. 57

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58 Fregly, M.J. (1967). Effect of hydrochlorot hiazide on preference threshold of rats for NaCl solutions. Proceedings of the Society for Experimental Biology and Medicine, 125 , 1079-1084. Fregly, M.J., Harper, J.H., & Radford, E.P. ( 1965). Regulation of sodium chloride intake by rats. American Journal of Physiology, 209, 287-92. Fregly, M.J., & Kim, K.J. (1970). Specificity of the sodium chloride appetite of hydrochlorothiazide-treated rats. Physiology & Behavior, 5, 595-599. Fregly M.J., & Rowland, N.E. (1986). The renin-angiotensin-aldosterone system and sodium appetite in rats. In G. de Caro, A.N. Epstein, and M. Massi (Eds.). The Physiology of Thirst and Sodium Appetite (pp. 441-446). New York: Plenum Publishing Corporation. Fregly M.J., & Rowland, N.E. (1992). Comparison of preference thresholds for NaCl solution in rats of the Sprague-Dawley and Long-Evans strains. Physiology & Behavior, 51 , 915-918. Goodman, J., & Gilman, J. (1980). The Pharmacological Basis of Therapeutics . New York: Macmillan Publishing Company. Guyton, A.C., & Hall, J.E. (2000). Textbook of Medical Physiology . Philadelphia : A. Harcourt Healt h Sciences Co m pany. Jalowiec, J . (1974). Sodium appetite elicited by furose m i de: Effects of differential dietary maintenance. Behavioral Neurological Biology, 10 , 313-27. Marwine, A., & Collier, G. (1979). The rat at the waterhole. Journal of Comparative and Physiological Psychology, 93(2), 391-402. Rowland, N. E. (1990a). On the waterfront: predictive and reactive regulatory descriptions of thirst and sodium appetite. Physiology & Behavior, 48 , 899-903. Rowland, N. E. (1990b). Sodium Appetite. In E.D. Capaldi, & T.L. Powley (Eds.) Taste, Experience and Feeding . American Psychological Association. Pp. 94104. Rowland, N.W., & Fregly, M.J. (1990). Thirst and sodium appetite in Dahl rats. Physiology & Behavior, 47 , 331-335. Sakai, R.R., Fine, W.B., Epstein, A.N., & Frankmann, S.P. (1987). Salt appetite is enhanced by one prior episode of sodium depletion in the rat. Behavioral Neuroscience, 101(5) , 724-731.

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59 Sakai, R.R., Frankmann, S.P., Fine, W.B., & Epstein, A.E. (1989). Prior episodes of sodium depletion increase the need-free sodium intake of the rat. Behavioral Neuroscience, 103(1), 186-192. Schulkin, J. (1991). Sodium Hunger: The Search for a Salty Taste. New York: Ca m bridge University Press. Stricker, E.M., Gannon, K.S., & Smith, J.C. (1992). Salt appetite induced by DOCA treatment or adrenalectomy in rats: analysis of ingestive behavior. Physiology & Behavior, 52 , 793-802. Stricker, E.M., Thiels, E., & Verbalis, J.G. (1991). Sodium appetite in rats after prolonged dietary sodium deprivati on: a sexually dimorphic phenomenon. American Journal of Physiology (Regulatory Integrative Comparative Physiology, 29), 260, R1082-R1088. Stricker, E.M., & Verbalis, J.G. (2000). Water Intake and Body Fluids. In M.J. Zigmond, F.E. Bloom, S.C. Landis, J.L. Roberts, and L.R. Squire (Eds)., Funda m ental N euroscience (pp. 1111-1126). San Diego: Acade m ic Press. Stricker, E.M., & Wolf, G. (1969). Behavior al control of intravascular fluid volume: thirst and sodium appetite. Annals of New York Academy of Sciences, 157(2), 553-68. Wolf, G. (1964). Sodium appetite elicited by aldosterone. Psychonomic Science, 1 , 211212. Wolf, G. (1965). Effect of deoxycorticoste rone on sodium appetite of intact and adrenalectomized rats. American Journal of Physiology, 208(6), 12811285. Wolf, G., McGovern, J. F., & DiCara, L. V., (1974). Sodium appetite: Some conceptual and methodologic aspects of a model drive system. Behavioral Biology, 10, 27-42. Wolf, G. & Quartermain, D. (1966). Sodium chloride intake of desoxycorticcosteronetreated and of sodium-deficient rats as a function of saline concentration. Journal of Comparative and Physiological Psychology, 61(2), 288-91.

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BIOGRAPHICAL SKETCH Connie Lynn Colbert was born in Miami, Florida on May 5, 1977 to Robert and Debora Colbert. She has a twin brother and had an older brother (deceased), both of whom fostered a healthy competitive spirit regarding school. Connie always enjoyed school and knew at a very young age that she would enjoy a career in the sciences. She graduated high school in 1995 with an AA degree from Miami Dade Community College and then attended Florida International University in Miami and received her Bachelor of Science in Psychology in 1998. She spent the next year continuing her research at F.I.U., and in 1999, Connie started graduate school in Psychobiology at the University of Florida, where she continues on in pursuit of a Ph.D. 60