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Satiation of Need Related Sodium Appetite Studied Using Progressive Ratio Schedules

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Satiation of Need Related Sodium Appetite Studied Using Progressive Ratio Schedules
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STARR, LAURA J. ( Author, Primary )
Copyright Date:
2008

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Appetite ( jstor )
Blood ( jstor )
Food ( jstor )
Foraging ( jstor )
Motivation ( jstor )
Physiology ( jstor )
Plasmas ( jstor )
Rats ( jstor )
Satiation ( jstor )
Sodium ( jstor )

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University of Florida
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University of Florida
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Copyright Laura J. Starr. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/31/2006
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436098717 ( OCLC )

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SATIATION OF NEED RELATED S ODIUM APPETITE STUDIED USING PROGRESSIVE RATIO SCHEDULES By LAURA J. STARR 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 2005

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Copyright 2005 by Laura J Starr

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This thesis is dedicated to my husband, Jason, who has given me love and support during our years together, and for my parents, Beverly and Max for all their love and support throughout my life.

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ACKNOWLEDGMENTS I would like to thank my advisor and mentor, Dr. Neil E. Rowland, who has helped me significantly to accomplish these experiments and thesis, and has supported me greatly throughout my academic career. I would also like to acknowledge my committee members for all of their knowledge and contributions to my research at the University of Florida. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 Sodium Appetite......................................................................................................1 Mechanisms of Fluid Balance...............................................................................2 Methods for Studying Sodium Appetite................................................................2 Methods of depletion......................................................................................2 Testing methods.............................................................................................4 Overconsumption..................................................................................................4 Assessing Behavioral Motivation............................................................................5 Operant Conditiong and Foraging.........................................................................5 Progressive Ratios.................................................................................................6 Patch Theory..........................................................................................................7 Aim of Study............................................................................................................7 2 METHODS...................................................................................................................9 Experiment 1: Simulated Work for 0.45M NaCl in Daily Progressive Ratio Sessions .......................................................................................................9 Animals and Housing.........................................................................................9 Chronic Sodium Depletion Protocol..................................................................9 Testing Chambers and Procedures.....................................................................9 Data Analysis...................................................................................................11 Experiment 2: Simulated Work for 0.3M NaCl in Daily Progressive Ratio Sessions......................................................................................................11 Animals and Housing.......................................................................................11 Chronic Sodium Depletion Protocol and Test Procedures..............................11 Effects of Losartan on Operant Behavior........................................................11 Operant Behavior of Non-depleted Animals...................................................12 Data Analysis...................................................................................................12 v

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Experiment 3: Physiological Changes at Satiation of Operant Responding for NaCl...........................................................................................................12 Animals and Housing.......................................................................................12 Testing Chambers and Protocol.......................................................................12 Blood Sampling and Assay..............................................................................12 PRA Assay.......................................................................................................13 Data Analysis...................................................................................................14 3 RESULTS...................................................................................................................16 Experiment 1: Simulated Work for 0.45M NaCl in Daily Progressive Ratio Sessions......................................................................................................16 Experiment 2: Simulated Work for 0.3M NaCl in Daily Progressive Ratio Sessions......................................................................................................16 Experiment 3: Physiological Changes at Satiation of Operant Responding for NaCl...........................................................................................................17 4 DISCUSSION.............................................................................................................25 LIST OF REFERENCES...................................................................................................29 BIOGRAPHICAL SKETCH.............................................................................................33 vi

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LIST OF FIGURES Figure page 2-1. Differences between PR rates.....................................................................................15 3-1. Number of earned reinforcements in Experiment 1...................................................20 3-2. Number of bar presses in last completed ratio schedule in Experiment 1..................21 3-3. Number of earned reinforcements in Experiment 2...................................................22 3-4. Number of bar presses in last completed ratio schedule in Experiment 2..................23 3-5. Physiological measures of chronically depleted rats..................................................24 vii

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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 SATIATION OF NEED RELATED SODIUM APPETITE STUDIED USING PROGRESSIVE RATIO SCHEDULES By Laura J. Starr May, 2005 Chair: Neil E. Rowland Major Department: Psychology Numerous studies have examined powerful mechanisms for induction of need-related sodium appetite in mammals. In contrast, the existence of mechanisms for satiation has been a matter of debate. This is in part because, following acute sodium depletion, rats often consume hypertonic NaCl solution in considerable fold excess of their physiological deficit. In the present study, we have examined the motivational characteristics of need-induced sodium appetite using operant schedules. Male Long-Evans rats (a strain that we have shown to consume 2-3 fold above their deficit when access to NaCl is cost-free) were given ad libitum access to distilled water and a natural ingredient low-sodium (~0.015%) diet, and received a daily injection of furosemide (5 mg/kg). These conditions induced a chronic sodium appetite that was stable from day-to-day. Depleted rats were first trained to lever press for access to NaCl solution (0.3 or 0.45M) from a sipper spout that was retractable. They were then assigned to one of four groups such that completion of a progressive ratio (PR) yielded either short or long (7.5 viii

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or 15 sec) availability of the spout and the PR itself had either low or high steps (~1.25 vs 1.5-fold increments). The group given long salt access time (15 sec) pressed significantly less (i.e., their breakpoint was lower) than the group given short salt access time (7.5 sec). The total amount of NaCl consumed in each condition was close to the estimated physiological depletion. Additionally, the physiological measures of depletion correspond to their behavioral satiation. So, unlike free access conditions, the PR protocols reveal an accurate mechanism for satiation of sodium appetite. ix

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CHAPTER 1 INTRODUCTION The cells in our bodies all contain a mixture of water and solutes, sodium being the most abundant. Interstitial fluid and plasma (i.e., extracellular fluid, or ECF) are also a mixture of water and solutes, and any change in either can result in a number of consequences. Unlike with nutrients for which there are substantial corporeal reserves (e.g., adipose tissue), there is no way for the body to store extra fluid so there must be precise mechanisms by which the body can keep track of and control its body fluid. For example, when an organism loses water and solutes through normal activities such as respiration, sweating, or elimination of feces or urine, that loss must be replaced in order for the organism to maintain what is called fluid homeostasis. Fluid homeostasis refers to the observed balance of water and solutes in the body that reflects a balance between the amount of water and solutes taken in and the quantity of water and solutes lost. This balance is regulated by a variety of hormones, which will be discussed later in detail. Sodium Appetite Sodium appetite, or the behaviors associated with the voluntary consumption of sodium salts by an organism, is usually studied in animals that have been deprived of sodium in some way. Richter (1936) was one of the first researchers to study sodium appetite, and he did so by means of giving adrenalectomized rats access to a hypertonic NaCl solution in addition to their normal chow and water. He saw that these rats displayed a large increase in their intake of the hypertonic salt solution. Sodium appetite is the only specific appetite that organisms display. That is, if an organism is deficient in 1

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2 a vitamin or mineral, sodium is the only one in which the organism will actively seek out to replenish itself, using gustatory cues and previous experiences in order to do so. Mechanisms of Fluid Balance Body fluid balance is maintained primarily by the renin-angiotensin-aldosterone system. Hypovolemia, or the state of volume deficit, can be caused by any of the methods mentioned earlier, or by pharmacological agents, or surgical methods (e.g., adrenalectomy). This negative fluid balance is sensed by cardiopulmonary baroreceptors. This state causes a secretion of antidiuretic hormone (ADH, or vasopressin) from the posterior pituitary gland. ADH then acts on the kidney to increase water reabsorption. Also, renin is secreted from the granular cells of the kidney. Renin then causes the release of aldosterone from the adrenal cortex, which acts on the kidney by increasing sodium absorption. Additionally, renin cleaves the protein, angiotensinogen to form angiotensin I, which is converted to angiotensin II (Ang II) by angiotensin converting enzyme (ACE). Ang II stimulates the release of aldosterone from the adrenal cortex, stimulates the posterior pituitary to release ADH and activates hypothalamic neurons to stimulate fluid (i.e., both water and sodium) intake. Methods for Studying Sodium Appetite Sodium appetite has normally been studied using sodium depleted rats or mice given free access to hypertonic (i.e., with osmolarity greater than that of ECF) salt solutions. The various methods of depletion and testing are described in the following sections. Methods of depletion When an animal such as a rat is deprived of salt in its diet, aldosterone is secreted from the adrenal gland and acts to reabsorb sodium as described above. Because of this

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3 efficient conservation mechanism, it is difficult to induce a sodium appetite by altering the diet alone. As described above, Richter (1936) depleted rats of sodium using surgical adrenalectomy. This renders the animals unable to concentrate urinary sodium, and the unavoidable urinary sodium losses result in a strong and reliable sodium intake. The major disadvantages of this method are that it is unphysiological and irreversible. In 1974, Jalowiec showed that rats given a sodium deficient diet along with subcutaneous injections of furosemide would increase their intake of a hypertonic salt solution. Furosemide is a so-called loop diuretic that acts on the kidney by inhibiting reabsorption of solutes in the loop of Henle. Much work on sodium appetite has been done using furosemide in combination with sodium deficient diets, and in many laboratories including our own has been very effective in eliciting a quick and strong sodium appetite (Bertino & Tordoff, 1988; Coldwell & Tordoff, 1993; Han & Rowland, 1995; Lundy, Blair, Horvath & Norgren, 2003; Rowland, Goldstein & Robertson, 2003; Rowland, Farnbauch & Crews, 2004; Thunhorst, Xu, Cicha, Zaredetto-Smith & Johnson, 1998; Tordoff, Fluharty & Schulkin, 1991; Tordoff & McCaughey, 2001). Other methods of inducing sodium appetite include administration of mineralocorticoids (e.g., DOCA), ACE inhibitors, formalin, adrenocorticotrophic hormone (ACTH), Ang II, hydrochlorothiazide (HCZ; a diuretic that acts on the kidney by increasing the secretion of sodium chloride and water) and polyethylene glycol (PEG; a colloid that produces non-hypotensive hypovolemia) (Blackburn, Samson, Fulton, Stricker & Verbalis, 1993; Caputo, Rowland & Fregly, 1992; Colbert & Rowland, in press; Denton et al., 1999; Fregly & Kim, 1970; Lewis, 1960; McCutcheon & Levy, 1972; Quartermain, Miller &

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4 Wolf, 1967; Rowland, 1998; Rowland & Fregly, 1988; Rowland, Morien & Fregly, 1996). Testing methods Most experiments studying sodium appetite will often give free access of a salt solution to sodium depleted animals. Salt is therefore not presented in a pure form, like a salt lick, but in more palatable forms, such as in a salt solution. This salt solution is usually a hypertonic solution, which is a substance not normally preferred by sodium replete rats, and thus may serve as a marker of motivation. Additionally, our lab has presented NaCl solution in a gelatin form, of which both sodium depleted rats and mice will readily consume up to 1.5M (Rowland et al., 2004). Again, these salt solutions (or other forms) are available freely to the sodium depleted animal, with no cost (except perhaps the “cost” of consuming a normally unpalatable concentration). However, there have been a few studies that have offered salt solutions to sodium deprived animals only after completion of an operant response (Quartermain et al., 1967; McCutcheon & Levy, 1972), which may be thought of as a better indicator of motivation for salt. This topic will be discussed in more detail in the following section. Overconsumption Most experiments of sodium appetite have focused on the onset and not the satiety of this appetite. Indeed, most diuretic-treated rats consume substantially more NaCl than their deficit when given free access to hypertonic NaCl. For example, Jalowiec (1974), who pioneered this model, found that rats given furosemide and a low sodium diet consumed 200% of their deficit very rapidly, often during the first 30 minutes of access, from which he concluded that satiation mechanisms were unnecessary. Other studies examining sodium appetite have also shown this overconsumption

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5 (Rowland & Fregly, 1992; Sakai, Fine & Epstein, 1987; Stricker, Thiels & Verbalis, 1991). This result could occur because the rate of solute that can be consumed from a hypertonic solution is less than the time course of normal feedback or satiation mechanisms derived from that intake. If this hypothesis is correct, then if the rate of consumption of hypertonic solution were slowed then satiation should occur with lower net intakes and in particular intakes matched more precisely to the physiological deficit. Assessing Behavioral Motivation The term motivation implies the purposive exertion of an animal against environmental adversity (e.g., Epstein 1982) and some early studies of sodium appetite in rats modeled adversity in the form of operant lever pressing (McCutcheon & Levy, 1972; Quartermain et al., 1967). Operant conditioning is the method in which behavior can be reinforced or punished in order to change the occurrence of that behavior in the future. One of the most widely used methods of operant conditioning is that of bar-pressing in an operant chamber in order to gain access to a reinforcer, such as food. Operant conditioning is shown in this situation when an animal will continue to bar press when given a food reinforcer, thus strengthening the behavior of bar-pressing. Often times, animals will be food deprived before testing so that the food reinforcer will be stronger. Operant Conditioning and Foraging In studies of ingestive behavior, and especially sodium appetite, the commodity is usually available at no cost. However, animals foraging in nature will always have a cost in order to get food, be it searching for and traveling to food, preparing the food for ingestion, and possibly fighting off other animals interested in that food. Because of this, operant conditioning methods have been developed in order to mimic the costs of obtaining food. By using a bar-press operant as mentioned earlier, Kanarek and Collier

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6 (1979) simulated foraging in the laboratory by imposing a cost on a food reinforcer. Once the current cost (i.e., fixed ratio) was completed, the animal would have access to a food reinforcer. If the animal ceased to consume the food reinforcer for a fixed amount of time (e.g., 5 minutes), the animal was denied access to the reinforcer and could only obtain it again if they completed the current fixed ratio cost. By varying the cost required in order to obtain the food reinforcement, they showed a very reliable and interesting result. When the cost was low, the animal took many small meals, the same as seen in free feeding conditions. However, when the cost was made higher, the animals took very few large meals. This result was also shown for water reinforcement (Marwine & Collier, 1979) and more recently for sodium solution in sodium deprived rats (Colbert & Rowland, in press). Progressive Ratios Progressive ratios are a specific type of operant schedule, in which the operant cost is increased with each successive reinforcement, unlike that of a fixed-ratio cost, where the operant cost remains the same for each reinforcement. Progressive ratio schedules have been designed to establish an organism’s motivation for whatever commodity is being tested, and the researcher can establish a “breaking point” in which the organism will no longer increase their operant cost in order to obtain reinforcement and thus establish an index of reinforcement strength (Hodos, 1961; Hodos & Kalman, 1963, Stafford, LeSage, & Glowa, 1998). Progressive ratio schedules are often used in drug reinforcement studies (Arnold & Roberts, 1997) and also to evaluate gustatory stimuli (Reilly, 1999) and the reinforcement value of solutions (Sclafani & Ackroff, 2003).

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7 Patch Theory In nature, food is not often available randomly, but more likely in what are called patches, or clumped areas of food, such as a swarm of insects or a pasture laden with earthworms (Barnard, 1983). While foraging for food, an animal may come across any number of patches and will have to make a decision on which patch to forage in. There can be differences in how much food is available in each patch, how often the patch resources are repleted, how far away a patch is from the animal’s niche, and if another animal is currently at that same patch. Since the animal may not be able to see all patches at once, they will need to sample from various patches in order to decide which patch may be the most sufficient. Once a patch is chosen however, there will not always be a limitless supply of food available there. The more the animal consumes from the patch, in addition to that consumed by other animals that also visit the patch, the more the patch will become depleted of resources. Patch depletion may then lead to the animal traveling to another less-depleted patch (Barnard, 1983). However, many operant foraging protocols do not take this into consideration. In most experiments, as long as the animal will work for the commodity, that commodity will be available for it to consume. In order to mimic natural foraging, and taking patch depletion into consideration, we have decided to use a progressive ratio schedule to mimic an increase in motivated behaviors required to earn each successive reinforcement. That is, as the “patch” is depleted with each commodity received, it should be more difficult to obtain the following reinforcement. Aim of Study In order for us to address the issues of patch depletion in foraging and more specifically overconsumption with sodium appetite, we have decided to devise a protocol

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8 that can attend to both of these issues. The aim of our study is, by using a progressive ratio schedule, to assess the problem of overconsumption of sodium in sodium depleted rats, and thus suggest a behavioral method of sodium appetite satiation. By imposing an increasing cost on the sodium solution reinforcement, we hope to stop the rats from overconsuming their deficit, and to reach their “break point” when they are physiologically replete. In the present experiments, using rats chronically sodium depleted with furosemide combined with a sodium deficient diet, we examined satiation of sodium appetite using progressive ratio (PR) operant sessions. Additionally, we examined the physiological profile of rats at the onset and termination of PR responding in the same operant protocols.

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CHAPTER 2 METHODS Experiment 1: Simulated Work for 0.45M NaCl in Daily Progressive Ratio Sessions Animals and Housing The subjects were 16 male Long Evans rats (Harlan, Indianapolis, IN) weighing ~500 g at the start of the experiment. The animals were housed individually in hanging polycarbonate tub cages (48x27x20 cm) containing ~2cm depth of Sani-Chips bedding (Teklad, Madison, WI). The vivarium was maintained at 22 2 C with lights on from 2200 to 1000 h. Chronic Sodium Depletion Protocol The animals were maintained on a chronic sodium depletion protocol used in our lab previously (Rowland et al., 2004). Distilled water and a natural ingredient low sodium diet (TD 90228; Teklad, Madison, WI, manufacturer’s stated sodium range: 0.008-0.02%) were available in the home cage ad libitum. The animals were injected once daily (~1500 h) with furosemide (5 mg/kg; American Regent, Shirley, NY). Testing Chambers and Procedures The operant testing was conducted in standard rat operant chambers (34x24x29 cm; Med Associates, St. Albans VT) with a floor of stainless steel rods above a tray with absorbent Sani-Chips bedding. One wall of each chamber was fitted with one response lever on the right side of the cage, with a three-color cue light fitted above it. During testing the animals received 0.45M NaCl solution after completing their current required ratio of presses on the response lever. The fluid reinforcers were contained in bottles 9

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10 with spouts that could be moved on a motor driven platform to be accessible through a vertical slot (1x1.5 cm) above each lever. When in the reinforcement position, the tip of the spout was ~2 mm outside of the cage. There were no levers, cue lights or bottles on the left side of the cage. The animals were trained to lever press for the salt solution for about 3 weeks before testing, initially with free access to the solution, working up to a fixed ratio (FR) 1 schedule. The animals were tested in 60-minute sessions every day for two weeks. Two independent variables were imposed. The first variable was the step of the progressive ratio (PR), either low (1.25 fold) or high (1.50 fold) (see Figure 2-1). The second variable was the length of time the salt solution was available when the current ratio run was completed, either short (7.5 sec) or long (15 sec). The animals were randomly placed into four groups (n=4): 7.5 sec salt access/1.25x PR rate (Short/Low), 7.5 sec salt access/1.50x PR rate (Short/High), 15 sec salt access/1.25x PR rate (Long/Low) and 15 sec salt access/1.50x PR rate (Long/High). The initial progressive ratio requirement for all groups was 3 bar presses. Once the current ratio requirement was completed, the animal had their given access time to the NaCl reinforcement, followed by a delay time (22.5 s for the short access group, 45 s for the long access group) in which they could not continue on to the next ratio run. Once this delay was finished, the animals were alerted by the cue light to signal the start of the next ratio run. If the animal did not respond for 15 minutes, the program was shut off and the session was completed for that day (i.e., the break-point was reached). These conditions were run during the first week of testing. During the second week, the access time for each rat was switched to the alternate value but at the same PR step. Each reinforcement episode

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11 was recorded per session, as well as total intake of salt solution (measured gravimetrically before and after the session). Data Analysis Data were analyzed using SPSS (SPSS, Chicago, IL) one-way ANOVAs and post hoc comparison between group means using Newman-Keuls tests with a significance of p < 0.05 for number of reinforcements earned and for last completed number or bar presses (i.e., breakpoint) and two-way ANOVAs with subsequent t tests with a significance of p < 0.05 for mean intakes of the NaCl solution. Experiment 2: Simulated Work for 0.30M NaCl in Daily Progressive Ratio Sessions Animals and Housing The subjects were 16 male Long Evans rats (Harlan, Indianapolis, IN) weighing ~300 g at the start of the experiment and housed as described in Experiment 1. Chronic Sodium Depletion Protocol and Test Procedures The animals were maintained on the chronic sodium depletion protocol as described in Experiment 1. The operant testing was conducted as described in Experiment 1. During testing the animals received 0.30M NaCl solution after completing their current PR schedule on the response lever. Effects of Losartan on Operant Behavior The animals were again maintained on the chronic sodium depletion protocol as described in Experiment 1. The operant testing was conducted as described in Experiment 1, with the animals receiving 0.30M NaCl solution as a reinforcement. Additionally, for one testing day the rats were injected with either a saline/distilled water solution, 10mg/kg losartan potassium (a gift from Dr. R Smith of the DuPont-Merck

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12 Research and Development Division) or 20mg/kg losartan potassium 30 minutes prior to their session. Operant Behavior of Non-depleted Animals At a later date, the animals were placed back on regular chow and water and operant testing was conducted as described in Experiment 1. Data Analysis Data were analyzed as in Experiment 1. Experiment 3: Physiological changes at satiation of operant responding for NaCl Animals and Housing The subjects were those used in Experiment 2, but after ~1 month free access to standard chow (Purina 5001) and water, and now weighing ~500g. They were housed as described in Experiment 1. Testing Chambers and Protocol Due to their previous training, the animals were placed in the operant chambers three days before testing. During these three days, animals were allowed to adapt to establish previous performance. They were then tested at the conditions of the Short/High group (7.5 sec salt access/1.50 fold PR rate) for three days before blood collection. Blood Sampling and Assay Blood was collected from each rat on two occasions, about 3 weeks apart. On the first occasion, half of the rats were fed standard chow and so were in sodium balance (Control) while the other half were acutely depleted of sodium by injection of furosemide (5 mg/kg) and access to the low sodium diet and distilled water for ~24 hr (Acute). The rats were sedated using ~15 s inhalation of isoflurane (Aerrane; Schein, Melville, NY)

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13 and ~1 ml blood taken via heart puncture with a 25 gauge needle. An initial small volume was placed in a chilled, EDTA-coated tube for later analysis of plasma renin activity (PRA, described below). The major part of the sample was collected in a plain tube for later aldosterone radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA). A small volume of this latter sample was drawn into a capillary tube to determine hematocrit ratio and plasma protein concentration (Atago refractometer). On the second occasion, after the rats had been re-adapted to the chronic sodium depletion protocol and operant responding for NaCl described above, blood was collected from half of the subjects immediately before their sessions were started (the chronic depletion condition; Pre), and from the other half of the subjects when they ceased responding on the PR ratio and were thus behaviorally satiated (Post). PRA Assay Assay of PRA relies on generation and subsequent radioimmunoassay of Ang I. A kit that contains reagents for both parts of this assay was available for many years from Perkin-Elmer (NEN), but this has now been discontinued. Instead, a radioimmunoassay kit for human Ang I is now available from Phoenix pharmaceuticals (Belmont, CA; kit # RK-002-01) but does not contain reagents for the incubation phase. Thus, we performed a preliminary validation of the incubation procedure. First, based on Osmond et al (1974) we used 0.1M sodium phosphate buffer adjusted to pH 6.0 for the incubation. Second, we compared the results using 10, 100 or 1000M of the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical So, St. Louis MO). Each of these concentrations had comparable effects, and yielded higher values than tubes run in the absence of inhibitor. Thus, the final procedure was as follows. Duplicate polystyrene tubes (12x75mm) on ice contained 90l phosphate buffer to which 1l of a

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14 10mM solution of PMSF in absolute ethanol was added (final concentration approximately 100M). Finally, plasma sample (10l) was added and the tubes vortexed. One set of tubes was then incubated at 37 o C for 1h, while the other set was kept in ice water. The warmed tubes were then replaced on ice and the remainder of the assay including handling of standards was as described in the Phoenix kit instructions. Data Analysis Data were analyzed using SPSS one-way ANOVAs and post hoc comparison between group means using Newman-Keuls tests with a significance of P < 0.05.

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15 # of reinforcements 024681012# of bar presses 020406080100120140160180200 1.25x PR rate 1.50x PR rate Figure 2-1. Differences between PR rates. A graphical representation of the two PR rates used during operant testing (1.25 and 1.50 fold). As the number of reinforcements earned increases along the x-axis, the number of bar presses required to receive that reinforcement increases along the y-axis according to the two different rates.

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CHAPTER 3 RESULTS Experiment 1: Simulated Work for 0.45M NaCl in Daily Progressive Ratio Sessions The results for number of reinforcements and average intake of the salt solution are shown in Figure 3-1 and Table 3-1. During the first week of testing (Figure 3-1, left), the Short/Low group earned significantly more reinforcements than the Long/High group, F (3,12) = 25.88, p < .01. Groups that were reinforced with short access to the salt solution drank significantly less (p < .05) than the groups that had long access (Table 3-1). For the second week of testing, there were no significant group differences in the number of reinforcements earned (Figure 3-1, right). Groups that were reinforced with short access to the salt solution drank significantly less (p < .05) than the groups that had long access (Table 3-1). The results for the number of final completed ratio schedule (breakpoint) are shown in Figure 3-2. During the first week of testing (Figure 3-2, left), the Short/High group completed significantly more bar presses in the last ratio schedule than did the Long/Low group, F (3,12) = 7.157, p < .05. For the second week of testing (Figure 3-2, right), the Short/High group completed significantly more bar presses in the last ratio schedule than did the Long/Low group, F (3,12) = 4.364, p < .05. The overall average intake of the 0.45M NaCl solution was approximately 4ml or 1.8 mEq. Experiment 2: Simulated Work for 0.30M NaCl in Daily Progressive Ratio Sessions The results for number of reinforcements and average intake of the salt solution are shown in Figure 3-3 and Table 3-1. For the first week of testing (Figure 3-3, left), the Short/Low group earned significantly more reinforcements than the Long/High group, F 16

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17 (3,12) = 3.39, p < .05. There was no significant difference in intakes between the two access times (Table 3-1). For the second week of testing (Figure 3-3, right), the Short/Low group earned significantly more reinforcements than the Long/High group, F (3,12) = 3.57, p < .05. Groups that were reinforced with short access to the salt solution drank significantly less (p < .05) than the groups that had long access (Table 3-1). The results for the number of final completed ratio schedule (breakpoint) are shown in Figure 3-4. During the first week of testing (Figure 3-4, left), the Short/High group completed significantly more bar presses in the last ratio schedule than did the Long/Low group, F (3,12) = 5.123, p < .05. For the second week of testing there were no significant differences in the number of bar presses in the last completed ratio schedule (Figure 3-4, right). The overall average intake of the 0.30M NaCl solution throughout the experiment was approximately 6ml or 1.8 mEq. There was no significant difference found in number of reinforcements for groups given either dose of losartan when compared with controls. Also, there was no significant operant behavior observed when the animals were non-depleted. Experiment 3: Physiological Changes at Satiation of Operant Responding for NaCl The results for hematocrit ratio, plasma protein and aldosterone concentrations, and PRA are shown in Figure 3-5. Compared with controls, plasma volume was decreased in rats treated either acutely or chronically (Pre) with furosemide, and was reversed to control level by the end of the PR session (Post). Thus, hematocrit ratio of the Pre group was significantly higher than those of Acute, Post and Control groups, and hematocrit ratio of the Acute group was also significantly higher than that of either Control or Post groups, F (3,28) = 9.815, p < .05. Plasma protein concentrations were significantly

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18 higher in both the Pre and Acute groups compared with those of Control and Post groups, F (3,28) = 6.468, p < .05. Plasma hormone concentrations were likewise elevated by salt depletion and reduced by the end of the PR session. Thus, plasma aldosterone level in the Pre group was significantly higher than in the Acute, Post and Control groups, and plasma aldosterone of the Acute group was significantly higher than that of either Control or Post groups, F (3,28) = 26.704, p < .05. PRA in the Pre, Acute and Post groups was significantly higher that of the Control group, and PRA of the Pre group was significantly higher than those of Acute, Post and Control groups, F (3,27) = 29.81, p < .001.

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19 Table 3-1. Intake of NaCl solution as a function of access time per reinforcement in progressive ratio experiments NaCl solution Short (7.5 sec) reinforcement Long (15 sec) reinforcement Experiment 1 week 1 0.45 M 3.22 .24 * 4.39 .21 Experiment 1 week 2 0.45 M 3.38 .28 * 4.73 .27 Experiment 2 week 1 0.30 M 4.73 .44 6.64 .69 Experiment 2 week 2 0.30 M 4.66 .47 * 6.38 .35 Note: Shown are MSE intakes in ml (N=8 per cell) * P < 0.05 versus corresponding long access groups

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20 Group Short/LowShort/HighLong/LowLong/High# of NaCl reinforcements 05101520 *Week 1 Week 2Group Short/LowShort/HighLong/LowLong/High# of NaCl reinforcements 05101520 Figure 3-1. Number of earned reinforcements in Experiment 1. Mean (SE) number of 0.45M NaCl solution reinforcements earned in chronically sodium depleted rats. Week 1: 7.5 sec salt access/1.25x PR rate (Short/Low), 7.5 sec salt access/1.50x PR rate (Short/High), 15 sec salt access/1.25x PR rate (Long/Low) and 15 sec salt access/1.50x PR rate (Long/High). During week 2, the access time for each rat was switched to the alternate value but at the same PR step. *P<0.05 vs each group.

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21 Week 1Group Short/LowShort/HighLong/LowLong/HighBreakpoint (# of bar presses) 010203040506070 *** Week 2Group Short/LowShort/HighLong/LowLong/HighBreakpoint (# of bar presses) 020406080100120 * Figure 3-2. Number of bar presses in last completed ratio schedule in Experiment 1. Mean (SE) number of bar presses completed during last ratio schedule (i.e., breakpoint) in chronically sodium depleted rats. *P<0.05 vs each unmarked group.

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22 Group Short/LowShort/HighLong/LowLong/High# of NaCl reinforcements 05101520 *Week 1 Group Short/LowShort/HighLong/LowLong/High# of NaCl reinforcements 05101520 * Week 2 Figure 3-3. Number of earned reinforcements in Experiment 2. Mean (SE) number of 0.30M NaCl solution reinforcements earned in chronically sodium depleted rats. Week 1: 7.5 sec salt access/1.25x PR rate (Short/Low), 7.5 sec salt access/1.50x PR rate (Short/High), 15 sec salt access/1.25x PR rate (Long/Low) and 15 sec salt access/1.50x PR rate (Long/High). During week 2, the access time for each rat was switched to the alternate value but at the same PR step. *P<0.05 vs each group.

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23 Week 1Group Short/LowShort/HighLong/LowLong/HighBreakpoint (# of bar presses) 020406080100120140 * Week 2Group Short/LowShort/HighLong/LowLong/HighBreakpoint (# of bar presses) 020406080100120140 Figure 3-4. Number of bar presses in last completed ratio schedule in Experiment 2. Mean (SE) number of bar presses completed during last ratio schedule (i.e., breakpoint) in chronically sodium depleted rats. *P<0.05 vs each unmarked group.

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24 Group ControlAcutePrePostHematocrit (%) 404244464850 *** Group ControlAcutePrePostPRA (ng Ang l/ml/hr @ 37C) 010203040 ***+ Group ControlAcutePrePostPlasma protein (g/dL) 7.07.58.08.59.09.5 ** Group ControlAcutePrePostPlasma aldosterone (pg/mL) 050010001500200025003000 *** Figure 3-5. Physiological measures of chronically depleted rats. Mean (SE) hematocrit ratio, plasma protein, plasma aldosterone and plasma renin activity (PRA) in rats. Control group received no diet, pharmacological or behavioral manipulations; Acute group acutely depleted of sodium by injection of furosemide (5 mg/kg) and access to the low sodium diet and distilled water for ~24 hr; Pre group were chronically sodium depleted and run through operant testing as described in Experiments 1 and 2 with blood assayed before their daily operant session; Post group were chronically sodium depleted and run through operant testing as described in Experiments 1 and 2 with blood assayed immediately after their daily operant session. *P<0.05 vs all other groups. **P<0.05 vs unmarked groups. +P<0.05 vs. all other groups.

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CHAPTER 4 DISCUSSION Rats tested under progressive ratios to obtain salt solutions were found to satiate need-related sodium appetite. For the first and second experiments, we took control of the temporal access to NaCl (once per day) in chronically depleted rats, and simulated effort during salt presentation in the form of a progressive ratio that simulates either resource (patch) depletion and/or lengthened duration of the consummatory episode that would correlate with increased likelihood of predation. Under these conditions, and in dramatic contrast to common free consumption tests (Jalowiec, 1974; Sakai et al., 1987; Stricker et al., 1991; Rowland & Fregly, 1992) rats were found to satiate depletion-related sodium appetite both reliably and accurately. Although not measured in these experiments, the average sodium loss seen in other sodium depletion protocols (Rowland & Fregly, 1992) with similar dose injections of furosemide is approximately 2.0 mEq. For both progressive ratio experiments the average sodium intake was ~1.8 mEq, which was consumed on average during the first 20 minutes of the session. These rats did not overconsume their deficit. In Jaloweic’s paper (1974), rats given furosemide (10 mg/kg) were placed on either a sodium replete (NaR) diet or a sodium deficient (NaD) diet prior to injection. Even though sodium loss was greater for the NaR rats than the NaD rats, The NaD rats were the only ones to overconsume their sodium deficit when given 0.51M NaCl. This overconsumption occurred rapidly, during the first 30 minutes of access. Jalowiec proposed that a “rapid and precise” satiety mechanism may not be required since overconsumption of sodium is not harmful to the animal. Also he postulated that 25

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26 the overconsumption seen in the NaD rats may have been caused by an increase in mineralocorticoid secretion, which has been demonstrated in rats maintained on a sodium deficient diet, and when in combination with furosemide elicits a strong sodium appetite. The urinary Na+/K+ ratio of the NaR group never gets as low as that of the NaD group; this implies that their aldosterone is not very high, and could explain why those animals did not overconsume. Additionally, the NaD animals have lower plasma Na+, higher plasma K+ and higher plasma protein at the time of furosemide injection. Jalowiec thus suggested that aldosterone may sensitize the intake circuits. Rowland & Fregly (1992) suggested that passage of time is needed for the satiation of sodium appetite. McCutcheon & Levy (1972) showed similar results to our study, in which acutely depleted (2.5 ml of 0.6% formaldehyde, regular chow and distilled water) rats stopped bar-pressing (variable interval 20s schedule) for 0.4M NaCl solution once they were physiologically repleted (note that the authors used sodium content of the urine as their only physiological measure of sodium deficiency). They suggest that the rats stopped bar-pressing and did not continue to consume the NaCl solution due to the more effortful behavior demanded by their procedure. We believe that the protocols used in this current paper, namely reliable and non-painful depletion procedure and direct measures of deficiency build significantly on the prior work of McCutcheon & Levy. Does that behavioral satiation correspond to physiological need? In the long term, because these were chronic experiments and the rats maintained weight throughout, the answer must be affirmative. Within the duration of the operant sessions (~40 min), we showed that substantial physiological repletion of the sodium deficit was achieved and that hormonal signals correlating with deficiency were greatly reduced. Rowland,

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27 Rozelle, Riley, and Fregly (1992) found that central administration of losartan, an AT1 receptor antagonist with a t 1/2 of ~5 hr (Timmermans et al., 1993), partially inhibited salt intake in sodium depleted rats. Surprisingly, in our experiment, treatment with losartan did not attenuate operant salt consumption. This suggests that in animals experienced with depletion and the operant situation that Ang II is not a crucial motivating signal. However, because the animals did not press for sodium when they were not depleted, there must be some physiological trigger. Aldosterone is a possible candidate; indeed, Jalowiec (1974) suggested that the overconsumption in his free access protocol might be related to aldosterone secretion. Sakai, Nicolaidis, and Epstein (1986) found that central mineralocorticoid blockade combined with captopril (an ACE inhibitor) completely suppressed salt appetite in sodium depleted rats. Additionally, aldosterone and Ang II have been shown to work synergistically in the brain to affect salt appetite (for review see Daniels & Fluharty, 2004). Thus, the decline of aldosterone levels as our PR session progresses could diminish the appetitive or motivational aspects of the situation. Indeed, Tordoff and McCaughey (2001) found a drop in aldosterone levels within 1h in Na-depleted rats either ingesting or intubated with NaCl, raising the possibility that the signals that blunt aldosterone secretion may also inhibit NaCl intake. Aldosterone could then be thought of as an “on” switch for sodium appetite, and perhaps the decline in aldosterone levels could be thought of as part of an “off” switch for sodium appetite, although other factors may contribute to that (e.g., increasing behavioral costs). Further study will be needed to identify whether the role of aldosterone is critical. Thus, our specific results refute Jaloweic’s (1974) suggestion that a sodium satiation mechanism is neither present nor necessary, and suggest instead that overconsumption (of salt) is a

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28 product of testing in an environment devoid of organismically important adversity. Rowland and Fregly (1992) found evidence of need-matched satiation in rats in which repletion of sodium was slowed by providing a hypotonic solution to drink. Thus, passage of time could facilitate the satiation of sodium appetite and may have contributed to the present results because intake in PR sessions occurred more slowly than in typical free drinking protocols. A direct test of the contribution of timing is needed to resolve this issue. Sodium appetite is an excellent model system for study of motivated behavior: it is driven by definable need state, can be physiologically measured easily, and the behavior is taste guided toward a very small range of commodities. From this perspective, we learn from the present study that the amount of a commodity consumed is related to the ease or safety with which it can be procured. This will perhaps come as no surprise to students of motivation, but it points to the fact that overconsumption of a commodity is a natural consequence of removing environmental adversity. One of the principal health problems of our modern society involves overconsumption. Oveconsumption of sodium over a lifetime, especially paired with low potassium, is a high risk factor for hypertension and stroke, now one of the major killers in post-industrial societies. Overconsumption of food, especially if paired with lack of exercise, leads to “diabesity” and hypertension. To combat these threats to health and its associated costs, it is imperative that we better understand the conditions under which natural satiety factors can operate.

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31 Rowland, N. E. (2002). Thirst and sodium appetite. In H. Pashler & C.R. Gallistel (Eds.), Stevens' Handbook of Experimental Psychology, 3rd edition, vol. 3: Learning, Motivation and Emotion, (pp. 669-707). Wiley, New York. Rowland, N. E. & Colbert, C. L. (2003). Sodium appetite induced in rats by chronic administration of a thiazide diuretic. Physiology and Behavior, 79, 613-619. Rowland, N. E., Farnbauch, L. J. & Crews, E. C. (2004). Sodium Deficiency and salt appetite in ICR:CD1 mice. Physiology and Behavior, 80, 629-635. Rowland, N. E. & Fregly, M. J. (1988). Sodium appetite: Species and strain differences and role of renin-angiotensin-aldosterone system. Appetite, 11, 143-178. Rowland, N. E. & Fregly, M. J. (1992). Repletion of acute sodium deficit in rats drinking either low or high concentrations of sodium chloride solution. American Journal of Physiology (Reg. Int. Comp. Physio.), 31, R419-R425. Rowland, N. E., Goldstein, B. E., & Robertson, K. L. (2003). Role of angiotensin in body fluid homeostasis of mice: fluid intake, plasma hormones, and brain Fos. American Journal of Physiology (Reg. Int. Comp. Physio.), 284, R1586-R1594. Rowland, N. E., Morien A., & Fregly, M. J. (1996). Losartan inhibition of angiotensin-related drinking and Fos immunoreactivity in hypertensive and hypotensive contexts. Brain Research, 742, 253-259. Sakai, R. R., Fine W. B., & Epstein, A. N. (1987). Salt appetite is enhanced by one prior episode of sodium depletion in the rat. Behavioral Neuroscience, 101, 724-731. Sclafani, A. & Ackroff, K. (2003). Reinforcement value of sucrose measured by progressive ratio operant licking in the rat. Physiology and Behavior, 79, 663-670. Stafford, D., LeSage, M. G., & Glowa, J. R. (1998). Progressive-ratio schedules of drug delivery in the analysis of drug self-administration. Psychopharmacology, 139, 169-184. Stricker, E. M., Thiels, E., & Verbalis, J. G. (1991). Sodium appetite after prolonged dietary sodium deprivation: a sexually dimorphic phenomenon. American Journal of Physiology (Reg. Int. Comp. Physio.), 29, R1082-R1088. Timmermans, P. B. M. W. M., Wong, P. C., Chiu, A. T., Herblin, W. F., Benfield, P., Carini, D. J., Lee, R. J., et al. (1993). Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacological Reviews, 45, 205-251. Thunhorst, R. L., Xu, Z., Cicha, M. Z., Zaredetto-Smith, A. M., & Johnson, A. K. (1998). Fos expression in rat brain during depletion-induced thirst and salt appetite. American Journal of Physiology (Reg. Int. Comp. Physio.), 274, R1807-R1814.

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32 Tordoff, M. G., Fluharty, S. J., & Schulkin, J. (1991). Physiological consequences of NaCl ingestion by Na + -depleted rats. American Journal of Physiology (Reg. Int. Comp. Physio.), 261, R289-R295. Tordoff, M. G., & McCaughey, S. A. (2001). Influence of oral and gastric NaCl preloads on NaCl intake and gastric emptying of sodium-deficient rats. American Journal of Physiology (Reg. Int. Comp. Physio.), 281, R1152-R1160.

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BIOGRAPHICAL SKETCH Laura Jennifer Starr was born in West Palm Beach, FL, in 1981. She completed high school at Wellington High School in Wellington, FL. Laura received her B.S. in psychology from the University of Florida in May of 2003. She attended the University of Florida for her M.S. in psychology. 33