LLRLAE DSMOL‘A’LBIY AND . ‘ LEGTRBLYTE RESPONSES 0F ‘jsi' jig? ALLXLETL’ LAEALED MAL-E RATS ~.;<£i;;j::£7:»? 1:15: 1:1- m; :XERCISE 'DissertALLOA for the Degree of PA D; MECALGAN SFAFE UALVERSL"? . MLCHAEL C GREEMLSEM ‘- ~1974_ .Mqunit... ”at...“ 3': L I F R. A K Y I1”! ‘vi:1(;dn Univer j, «acme-rm .- . This is to certify that the thesis entitled ‘l Urine Osmolality and Electrolyte Responseswof Anxiety Treated Male Rats to Exercise w presented by Michael C. Greenisen has been accepted towards fulfillment of the requirements for Ph.D. Physical Education degree in 1/ . (”g/27944 21/ A4 (WW! ‘1”. Major professor Date November 15, 1974 . «'I 021639 - alumna BY "OAS & SONS" 300K BINDERY - LIBRARY BINDERS 35mm". Lg " ‘ ABSTRACT URINE OSMOLALITY AND ELECTROLYTE RESPONSES OF ANHETY TREATED MALE RATS TO EXERCISE By Michael C. Greenisen This investigation was undertaken to determine the effects of exercise treatments on responses to experimentally induced anxiety by examining the urine osmolality and the Na+ and K+ electrolyte concentrations of male rats during a twelve-hour home-cage environment. Twenty rats (Sprague-Dawley) were observed for 21 days. Four groups of five animals each were used. Group I: ArE was exposed to anxiety conditions by random faradic stimulation through the floor of individual anxiety chambers for three hours daily. A forced exercise treatment, consisting of interval running, immediately followed the anxiety exposure. Group II: SA-E received a sham anxiety exposure followed by the exercise treatment. Group III: ArNE was exposed to the anxiety treatment but did not receive the forced exercise treatment. Group IV: SA-NE was exposed to sham anxiety conditions and did not exercise. Three-day control data, similar to experimental data, were collected from the animals in each of the experimental treatments but in the absence of any faradic stimulation. Michael C. Greenisen With the inception of the eighteen—day experimental treatment phase, total urine volume‘was collected during: anxiety or sham anxiety exposure, the exercise treatment or a holding period of the same duration, 3 recovery period, and the twelve-hour period of sedentary home-cage environment. Body weight and the food and water ingested also were recorded during the recovery and home—cage environment periods. During the home—cage period, the urine osmolality of the SArNE group returned to normal values. However, they did demonstrate elevated total urinary Na+ retention (non—significant) and K+ excretion patterns (p 2 .05). Animals exposed to the A—NE and SA—E treatments exhibited increased and similar urine osmolality (p é .05). Both groups showed enhanced urine Na+ retention (p E .05), but neither group demonstrated a convincing K+ imbalance. The ArE group responded with urine osmolalities significantly higher (p é .05) than the A—NE and SAeE groups, apparently indicating the additive nature of exercise and anxiety during this study. In addition, the A—E group had increased urinary Na+ retention and K+ excretion (p é .05). These findings indicate that exercise does not alleviate experimentally induced anxiety but rather seems to act as a second stressor. URINE OSMOLALITY AND ELECTROLYTE RESPONSES OF ANXIETY TREATED MALE RATS TO EXERCISE By -.L’ \‘r Michael Ct Greenisen A DISSERTATION Smeitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physical Education College of Education 1974 Dedicated to Virginia Gail Greenisen my wife ii ACKNOWLEDGMENTS The challenges of a mountain and a Ph.D. dissertation are more similar to one another than they may appear to the uninitiated. Both require rigorous training in specialized skills. Both offer the personal challenge for man to compete against himself. Both present the uncertainty of accomplishment, for if victory were certain, why bother? A mountain challenges by its altitude, which makes progress, mentally and physically, more taxing as the summit is in View. It punishes the body with its moraine, scree, jagged rocks, ice and snow. The brain and extremities must work in concert to select the route, pitch by logical pitch, with coordinated reach and step to safely navigate the route, each with its own unexpected moments. A dissertation challenges by producing a myriad of unexpected data which make progress more laborious as the final two pitches are at hand. It punishes the body over sleepless nights demanding nicotine and caffeine. The brain and extremities must work in concert as they select table, figure, and appendix to navigate a logical route through five hazardous pitches. In the end, even Everest and McKinley are mere mountain walks during the final steps to the summit. No sudh pleasures are extended the writeru .A dissertation presents its most dangerous terrain at the iii summit, where the tired or the unprepared may flounder and be lost forever. Ultimately the challenge of either is taken up essentially because it is there and blocks our view. Both appear much more overwhelming and hazardous at the base than at the summit. To challenge a mountain and defeat it provides a few moments of personal victory but, in and of itself, it is an end. Accepting the challenge of a dissertation and writing it pro- vides fleeting, if any, victory for, in and of itself, it is a beginning. The mountaineer and the researcher are one and the same-- explorers who seek a better view. I acknowledge the dissertation which, like the mountain, taught me new things about myself and considerably more regarding my profession. In particular, I appreciate the help of: Dr. Glenn Hatton, who provided the technical training necessary to select this particular route; Dr. Wayne Van Huss, who was consistent in his encouragement, interest and his insistence that his students probe new areas in the interest of physical education; Professor William Heusner, who was for four years teacher, counsellor, and scathing critic. As a student this author learned more from.his courses and over midnight coffee than all the other combined graduate school input. His expectations would be unreasonable were it not for his own involvement; Professor Lloyd Bohn, retired chairmen of the Physics Depart- ment, Temple University, Researcher, Teacher, Coach, Friend: a iv rare kind of man, the kind of man we would all like to be. His influence was paramount in the author's decision to complete this dissertation; Charles Beach, for his friendship, and for always standing ready to help--AIRBORNE - ALL THE WAY; Michael Marshall, for his encouragement, friendship and the countless hours spent together as we learned from each other the techniques of analyzing human movement. DEDICATION. . TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . LISTOFTABLE8:................... LIST OF FIGURES . . . . . . . . . . . . . . . . . . . LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . Chapter I. INTRODUCTION . . . . . . . . . . . . . . . . . Statement of the Prdblem. . . . . . Rationale . . . . . . . . . . . . . . Limitations of the Study. . . . . . . II. REVIEW OF THE LITERATURE . . . . . . . . . . . ADH Relationships to Urine Osmolality . Extracellular Fluid Volume Shifts vs Exercise or Anxiety ADH Secretion . . . . . . Exercise or Anxiety Stress Effects on Urine Osmolality . . . . . . . . . . Urinary Sodium and Potassium Concentration Reactions to Stress. . . . . . . . III. EXPERIMENTAL PROCEDURE . . . . . . . . . . . . Perspective of the Experimental Design. Receipt and Assignments of Subjects . . Animal Care . . . . . . . . . . . . . Pretreatment Protocol . . . . . . . . . Urine Collection Periods. . . . . . . . Urine Collection Technique. . . . . . . Experimental Protocols. . . . . . . . . Anxiety. . . . . . . . . . . . . Sham Anxiety . . . . . . . . . . Exercise . . . . . . . . . . . vi Page ii iii xiii ix xi WNN 12 13 16 16 l8 l8 19 20 21 21 21 22 22 Chapter Holding. . . Recovery . . . . . . Zero-Phase . . . . . . Home-Cage Environment. Sacrifice Protocol. . . . . . Urine Osmolality Analysis . . Page 0 O 0 23 . . 23 O O 24 . . . . . . 24 . . . . . . 24 Urine Na+ and K+ Electrolyte Concentrations . . 25 Statistical Analysis. . . . . IV. RESULTS. . . . . . . . . . . . . . . Response to the Running Exercise. Body weight . . . . . . . . . Urine Osmolality Responses. . Urine Na+ Concentration in mEq/l. Urine K+ Concentration in mEq/l. Total Urinary Na+ . . . . . . Total Urinary K+. . . . . . . Na+/K+ Ratio. . . . . . . . . Relative Adrenal Weight . . . Food Ingestion Comparisons. . water Consumption Comparisons Urine Volume Excretion Patterns Recovery Period Water Consumption Recovery Period Urine Excretion . Discussion. . . . . . . . . . V. SUMMARY, CONCLUSIONS, RECOMMENDATIONS. Conclusions . . . . . . . . . Recommendations . . . . . . . LIST OF REFERENCES. . . . . . . . . . . . “PENDICES. O O O O O 0 O O O O O O O O O 0 vii . . . 25 . . 28 . . . . 28 . . . 29 . . . . . . 29 . . . . . . 35 . . . . . . 39 . . . . . . 39 . . . . 46 . . . . . 53 . . . . . 57 . . . . . . 64 . . . . 64 . . 67 Table 10 11 LIST OF TABLES Page Results of Statistical Tests on the Investigated Variables to Determine Their Meeting the Assump- tions of Equal Variance and Compound Symmetry. . . . . 27 Analysis of Variance Summary and Scheffé Post-Hoe Test on Home-Cage Urine Osmolality . . . . . . . . . . 33 Analysis of Variance Summary and Scheffé Post-Hoc Test on Home-Cage Urine Na+ Patterns . . . . . . . . . 37 Analysis of Variance Summary and Scheffé Post-Hoe Test on Home-Cage Urine K+ Patterns. . . . . . . . . . 41 Analysis of Variance Summary and Scheffé Post—Hoc Test on Home-Cage Urine Total Na+ Patterns . . . . . . 44 Analysis of Variance Summary and Scheffé Post-Hoc Test on Home-Cage Urine Total K+ Patterns. . . . . . . 48 Analysis of Variance Summary and Scheffé Post-Hoe Test on Home-Cage Urine Na+1K+’Ratios. . . . . . . . . 51 Means for Relative Adrenal Weight. . . . . . . . . . . 53 Analysis of Variance Summary and Scheffé Post-Hoe Test on Home-Cage Food Ingestion . . . . . . . . . . . 55 Analysis of Variance Summary and Scheffe Post-Hoe Test on Home-Cage water Consumption. . . . . . . . . . 59 Analysis of Variance Summary and SCheffé Post-Hoc Test on Home-Cage Urine Excretion Volume . . . . . . . 62 viii Figure 10 LIST OF FIGURES Perspective of the experiment by groups and treatment protOCOI O O O O O O O O O O O O O O O O O 0 Average percent of expected revolutions (PER) and total number of revolutions run (TRR) of the exer- cise treated rats over the three experimental Observations . . . . . . . . . . . . . . . . . . . . . Mean body'weight comparisons by groups across the Study 0 O O O O O O O O O C C O O O O I O C O O O 0 Mean home-cage urine osmolality in mOSm/L/gram body weight during control and experimental observations . . . . . . . . . . . . . . . . . . . . . Mean home-cage urine Na+ concentration in mEq/ liter/gram body weight x 100 for the control and experimental observations. . . . . . . . . . . . . . . Mean home-cage urine K+ concentration in mEq/ liter/gram body weight x 100 for the control and experimental observations. . . . . . . . . . . . Mean home-cage total urine Na+ (Na+ mEq x urine volume) in mEq/liter/gram body weight x 100 during the control and experimental observations. . . . . . Mean home—cage total urine K+ (K+ mEq x urine volume) in mEq/liter/gram body weight x 100 for the control and experimental observations. .1. . . . . Mean home-cage total urine Na+7K+ ratios in mqu liter/gram body weight x 100 over the control and experimental observations. . . . . . . . . . . . . Mean home-cage food ingestion levels in grams/gram bodwaeight x 100 over the control and experimental observations . . . . . . . . . . . . . . . . . . . ix Page 17 31 32 36 40 43 47 50 54 Figure 12 l3 14 Mean home-cage water consumption in ml/gram body weight x 100 during the control and experimental observations . . . . . . . Mean home-cage urine excretion patterns in ml/gram body weight x 100 for the control and experimental observations . . . . . . . Mean recovery period.water consumption in ml/gram body weight x 100 over the control and experimental observations . . . . . . . Mean recovery period urine excretion volume in ml/gram body weight x 100 over the control and experimental observations. Page 58 61 65 66 ACTH ADH ArNE CS LIST OF ABBREVIATIONS adrenocorticotrophic hormone antidiuretic hormone anxiety-exercise anxiety-no exercise compound symmetry equal variance potass ium sodium percent of expected revolutions sham anxiety-exercise sham anxiety-no exercise percent of expected revolutions total number of revolutions run CHAPTER I INTRODUCTION An area of investigation relatively unexplored by physical educators is the role of exercise in mammalian psychophysiological adaptation to the environment. 0f specific interest is the effective- ness of exercise as a therapeutic measure intended to alleviate concurrent emotional trauma (anxiety). The stress of exercise or anxiety stimulates increased mammalian pituitary-adrenocortical activity. This increased activity by the pituitary and adrenal glands results in measurable quantitative and qualitative changes in the excreted urine. Considerable data have been assembled regarding posterior pituitary antidiuretic hormone (ADH) responses in urine and/or blood as a result of emotional or exercise stress (7,42,56). ADH acting at the kidney is a direct regulator of urine osmolality (14,55,69). Emotional anxiety and environmental stress have been reported to be reflected in increased urine sodium (Na+) retention and potassium (K+) excretion (17,49,89). ADE is further implicated in Naf resorption occurring in the distal tubule of the kidney (69). This electrolyte imbalance also implies a.hyperfunction of the adrenal cortex, specifically its secretion of aldosterone or corticosterone (55). 2 These findings lead logically to the conclusion that physio- logical reactions regarding the effects of exercise on anxiety may be analyzed by urinalysis examining urine osmolality, and the Na+ andK+ electrolyte concentrations. One study which essentially dealt with the prevention of heart disease and/or the reduction of cardiac sympathetic tone seemed to show that physically active and placid individuals respond less to mental and sensory stress than do sedentary and emotionally irritable persons (64). However, the available literature demon— strates a lack of convincing evidence regarding the interrelation— ships of exercise and anxiety-prone subjects. A need exists to expand human knowledge regarding the direct effects of exercise on those parameters that have been identified as being sensitive to anxiety. Statement of the Prdblem The purpose of this study was to investigate the effects of daily periods of a selected interval running program on male rats following daily exposure to induced anxiety conditions. Home cage urinary Na+ and K+ electrolyte concentrations and urine osmolality were the parameters of the investigation. Rationale Pioneered by Steinhaus' (82) classical publication, "The Chronic Effects of Exercise", selected positive rewards of exercising on the physiological function of the mammalian organism have become well documented. Specifically, the cardiovascular, respiratory, 3 and skeleto—muscular systems have been studied in detail. The rationale for this study is to bring another part of the organism into focus by obtaining relevant data regarding the effects of exercise on mammalian emotional adjustment. The albino laboratory rat was selected as the experimental model for this study, in part to utilize the controlled setting of the laboratory. More dramatically, this author knew of no method by which he could convince any of the animals that exercise would be "beneficial" for them. Therefore, it was reasoned that the physiological responses elicited in the subjects would be uncon— taminated by their expectations. The argument for this investigative approach is based on the following assumption: shifts from control data in the osmolality and the Na+ and K+ concentrations of the animals' urine, collected in their home-cage environment, were results of the experimental treatments . Limitations of the Study 1. Available metabolism cages dictated that a maximum of 20 subjects could be used at one time. 2. The effect of faradic stimulation (foot shock) as the moti- vator utilized by the electronically controlled running wheel is an unknown, and uncontrolled for, variable. 3. The inability to maintain twenty—four hour home-cage control subjects for the duration of the study is traceable to the availa- bility of metabolism cages. Evaluation of such a control group is thus lacking. 4 4. Important data may have been lost when urine samples and other measures were not taken during the zero time period eaCh day. CHAPTER II REVIEW OF THE LITERATURE The following literature review cites previous studies linking ADH secretory changes, urine osmolality, and experimental stress. The available research regarding urine osmolality and urinary Na+ and K+ mEq/l concentrations with respect to experimentally induced stress will conclude the review. ADH Relationshipg to Urine Osmolality Bernard (93) in 1859 was the first experimenter to observe that emotional stress in dogs and rabbits may suppress the rate of urine secretion even to the degree of total inhibition. Rydin and Verney (67) found that emotional stress, induced by mild faradic stimula- tion or a frightening noise, results in the inhibition of a water diuresis. To support their theory that the agent responsible for this emotional antidiuresis was a humorally transported antidiuretic srbstance of posterior pituitary extract, Rydin and Verney performed extensive neural sectioning. They denervated the renal, suprarenal, and splanchnic nerves as well as the entire abdominal symathetic system of test dogs without finding a reduction in emotional-s tress antidiuresis. From these data they concluded that emotional inhibi- tion is mediated by posterior pituitary antidiuretic hormone. In 6 the same‘work, they discovered that mild exercise (running 4 to 5 umh for 4 min.) also results in the inhibition of water diuresis. They theorized that the inhibition produced by exercise is due to an emotional factor associated with exercise, for after repeated training sessions this antidiuresis disappeared. After extinction, however, if the dogs were exposed to a loud frightening noise while exercising, an antidiuretic response was elicited once again. O'Connor and Verney (57) further supported this earlier research by showing that removal of the posterior lobe of the pituitary diminishes the antidiuretic response to only about 52 of its pre- operative inhibition on urine flow induced by emotional stress. Kozlowski et al. (42) reinforced O'Connor and Verney's conclu- sions using human subjects. He and his co~workers found no increase in blood antidiuretic activity during mild exercise (450 kpm/min. on a cyclometer). This research did show that heavy exercise (1,200 kpm/min.) produces a rise in plasma antidiuretic activity. They hypothesized that the rise was due more to a homeostatic reflex mechanism than to emotional inhibition. Fendler at al. (23) forced female rats to swim in 18°C water until exhausted daily. On days 12, 18 and 29, animals were sacrificed and posterior pituitary extracts from these experimental animals and from control animals were injected into dogs. Extracts from the exercise-treated animals suppressed urine flow in the dogs by an average of 3.5 ml. per five minutes. Extracts from control animals suppressed urine flow an average of only 1.5 ml. per five minutes. Extracts from rats treated for 18 days were found to be the most 7 dynamic, suppressing urine flow an average of 5.0 ml. per five udnutes. Dempster and Joekes (l8) autotransplanted the kidneys of dogs to the dogs' necks in order to more precisely observe the effect of emotional stress on water diuresis inhibition. USing faradic stimulation as the emotional stress, they demonstrated that the kidneys of the animals exhibited the prolonged inhibition of a water diuresis representing ADH involvement. Corson (7) experimented.with chronic conditioned and uncondi- tioned responses. Using Pavlovian type experiments, he demonstrated the participation of ADH in the responses of dogs to psychologic stress. Some dogs produced a marked and persistent release of ADH in response to repeated psychologic stress. Corson's treatment combined auditory tones with faradic stimulation. The secretions of ADH in response to this treatment were indicated by consistently high urine osmolality values (up to 1,500 MOS/1.). Urine concen- tration remained high, and occasionally increased, even though the dogs received a large water load prior to treatment. Fendler et al. (24) demonstrated that electroshocks (AC,12 ma) administered daily for 16 days, until a state of lethargy had been developed in male rats, resulted in a significant increase in pituitary ADH. (No mention was made of the number of repetitions of electroshocks per day.) The evidence seems to be clear that emotional stress activates the supraoptic nucleus, hypothalamus, and posterior pituitary system, thus stimulating the secretion of antidiuretic hormone (ADH) into 8 the circulating blood. Noble and Taylor (56), in an experiment on venisection using human subjects, demonstrated that all those who fainted excreted ADH in their urine; ADH was not found in their presyncopal urine. ADH was not found in the urine of those subjects who did not faint. Once again, the emotional implication of ADH involvement is clear. Extracellular Fluid Volume Shifts vs Exercise or Anxiegy ADH Secretion 0f major interest to the interpretation of the data presented in this study is the elucidation of the physiological mechanisms which influence the secretion of ADH. Specifically there is concern regarding extracellular fluid volume shifts affecting the normal secretion of ADH during exercise and training induced by a condi- tioned emotional response. The acute expansion.by endogenous infusing of the extracellular fluid volume in dogs, rats, and man results in an increased urine flow, sodium excretion and osmolar clearance (l6,25,28,3l,43,50,55,57,60). The inunediacy of this response rules out the slow aldosterone mechanism affecting natriuresis or antinatriuresis (8). Stahl at al. (81) demon- strated that the diuretic and natriuretic substances elicited during extracellular fluid volume expansion and stimulation of the intrar thoracic volume receptors by immersion in.water originated in the liver. Interestingly, ADH destruction occurs in the liver and kidneys (50). This natriuretic substance released by the liver may be the substance Xfiwhich Homer Smith describes (94). 9 Expansion of the intrathoracic blood volume by extracellular fluid volume expansion, negative pressure breathing, assuming a supine posture, or immersion in water, activates volume receptors, more specifically those of the left atrium, resulting in a diuretic and natriuretic response (5,38,50,56,6l). A diuresis also is elicited by expanding a balloon in the left atrium (30,50). Gauer et a1. (23) demonstrated that the degree of distention of the left atrium controls water excretion by the antidiuretic mechanism of the posterior pituitary, and that the degree of distention of the right atrium governs the excretion of sodium. This is a complementary mechanism for, unless accompanied by the excretion of sodium, loss of water is limited by the increasing osmolality of the body fluids. If the stimulation of the intrathoracic volume receptors (specifically in the left atrium) is reduced by a reversal of the maneuvers described (negative to positive pressure breathing, supine position to erect standing, etc.) an antidiuresis ensues accompanied by the return to normal values of sodium and osmolar clearances. Activation of the intrathoracic volume receptors, by the pooling of central and intrathoracic blood volume, triggers an anti-antidiuresis. This response in turn maintains the homeostatic balance of the living organism in response to these expansion maneuvers. However, this central and intrathoracic blood-pooling is not seen during exercise. This is the first evidence to refute the possibility of an anti-antidiuresis response to exercise. There is an increase in arterial blood pressure during exercise. Among the first mechanisms to sense this pressure rise are the 10 aortic and carotid sinuses, which are baroreceptors. Cort (8,9,10,11) found that bilateral occlusion of the common carotid arteries produces a diuresis and natriuresis. This is readily explanable in.that lack of the carotid sinus pressor response leads again to central and intrathoracic blood volume pooling, distention of the left and right atria, etc. It is noteworthy that after approximately ten minutes of occlusion, blood pressure begins to return to normal; however, it does not reach pre—occlusion levels. The intact carotid sinus and aortic sinus, when sensing increased blood pressure, demonstrate two responses which interact to reinforce each other: (a) there is a release of vasoconstrictor tone in the periphery of the body which opens vascular beds lowering systemic.blood pressure, and (b) there is a decrease in cardiac output due to both bradycardia and a decrease in stroke volume. The latter need not always be present (8). This second point has no relevance to the investiga- tion of exercise. As exercise builds in intensity, blood volume shifts from the thoracic and abdominal regions to the working muscles. As discussed above, mmscle vasoconstrictor tone decreases, and muscle capillary beds open to accept this blood volume shift. This situation now permits cardiac output to adjust to the demands of the intensity of the exercise. The baroreceptors and the sympathetic domination of the autonomic nervous system during exercise reinforce this maneuver. As intrathoracic blood pooling declines, so does its effectiveness to impose an anti-antidiuretic situation. Segar and Moore (73), employing exposure to a hot environment, demonstrated a situation somewhat similar to exercise. As exposure 11 to heat increases, vascular beds open and increased ADH secretion ensues as a result of decreased distention of intrathoracic stretch receptors. The marked levels of ADH secretion could have resulted from.a loss of extracellular fluid as well as a redistribution of blood due to an increased flow to peripheral beds. However, since no measurable Changes in plasma sodium or total solute concentration occurred, it is not likely that an osmoreceptor could have mediated the ADH release. The abrupt drop in blood ADH concentration that occurred within 15 minutes after return to normal temperature pro- vides evidence that the changes in ADH concentration.were the result of redistribution of blood. This exposure to heat resulted in an antidiuresis and decreased sodium and total solute excretion. If a similar situation did not occur in exercise, there would be a constant urge to void as a result of the previously mentioned anti- antidiuresis response. The carotid chemoreceptors are sensitive to changes in P002: increased P002 results in enhanced ADH secretion (8,76,77). Share and Levy (77) concluded that under situations in which there is a simultaneous activation of the chemoreceptors which stimulate ADH release and receptors which inhibit ADH release (left atrial recep- tors and possibly some arterial baroreceptors), the net result will either be no change or a slight reduction in ADH release. This situation is difficult to envision in the intact animal. It is apparent that a variety of sitmuli excite and inhibit the secretion of ADH. Intact vagi are necessary to transmit afferent volume receptor information to the central nervous system (8,10,77,78). 12 Presumably, the CNS integrates neural and hormonal responses to maintain body fluid homeostasis in response to this variety of stimuli. This review has not produced a basis to suspect that exercise or conditioned emotional response treatment will effect an anti- antidiuresis. The immediacy of the response to the various stimuli described.would seem to rule out any long-term effect which was not specifically associated with homeostasis. Therefore, any treatment effects should not be normally masked when interpreting the animals' home-cage data which are collected some ten hours after exposure to the treatments. The effect of anxiety exposure and/or exercise treatments on urine osmolality in the home-cage environment should prove to be meaningful research in the elucidation of the physiological mechanisms regarding the effects of exercise on responses to anxiety. Exercise or Anxiety Stress Effects on Urine Osmolality Corson (7), utilizing a combination of auditory tones and electrical stimulation to create anxiety conditions in dogs, found that this experimental treatment resulted in consistently high urine osmolality. These findings remained valid even when the dogs were water loaded prior to the treatment. Changes in urine osmolality concentrations resulting from exercise seem to be related to the intensity of the exercise. Kachadorian (39) found that urine excreted after moderate exercise reflected a higher osmolality than urine excreted following mdld exercise. But, in this same study, urine after heavy exercise was 13 found to have a lower osmolality than urine after either mild or moderate exercise. Surprisingly, these findings occurred even though the volume of excreted urine was less following heavy exercise than following moderate exercise. Kachadorian's results support earlier investigations by Raisz at al. (65) and Schrier et a1. (71), who found that heavy exercise produced diluted urine osmolality levels. These investigators speculate that an impairment of the renal concentrating mechanism.during heavy exercise may account for the low post-exercise urine osmolality concentrations. These find- ings should stimulate interest among exercise physiologists as they are contrary to the popular view that exercise, in general, not only reduces excreted urine volume but also increases urine osmolality. Urinary Sodium.and.Potassium.Concentration Reactions to Stress Dauphinée (17) reports the active retention of Na+ andK+ excre- tion to be a characteristic mammalian response to physical, emotional, or surgical stress. Moore (49) reports elevated Na+ retention and K+ excretion by humans as a result of surgical stress. Thorn at al. (84), although being nonspecific about the stressors, directly relates Naf retention and K+ excretion to environmental stress. Intense Na+ retention and K+ excretion also are reported as a result of infusion of ACTH, hydrocortisone, or corticosterone. In an actual environmental stress situation, these researchers postulate that abnormal K+ excretion would begin three to four hours following the activation of the adrenal cortex to meet the stress. 14 Hoagland at al. (21), employing pursuit meter tests to induce anxiety, examined 20 subjects. These investigators found increases in both urinary Na+ and K+ concentrations for those subjects over 20 years of age. Subjects under 20 years of age also had increased Naf concentrations. However, the subjects under 20 years of age demonstrated a K+ retention of 132. - Gann and wright (28), collecting urine samples from five dogs after exposure to induced trauma, clearly demonstrated elevated Na+ retention to be a result of the experimental variable. Spigel and Ramsay (80) housed thirty-nine S-year-old male turtles in individual anxiety chambers. The turtles received one second of electrical shock, delivered randomly on the average of one shock every fifty-nine seconds, twelve hours a day for four days. All turtles responded with elevated urinary Na+ and K+ excretion. This research further demonstrated a conditioned emotional response in some of the turtles. Those turtles maintained in the anxiety chambers after the conclusion of the experiment continued to reflect elevated urinary Na+ andK+ concentrations even though they were no longer receiving shocks. Turtles previously shocked but removed from the anxiety chambers returned to control-level values. On the other hand, studies have been performed.which suggest that Na+ and K+ excretion levels do not respond significantly to imposed anxiety. Frost (19), utilizing nine subjects involved in the 1950 annual Indianapolis 500 Automobile Race, found no consistent Na+ or K+ responses in the post-race urine samples of the drivers. a». a l 'L 10!" HI. *N ”I - r‘: 15 Paré and McCarthy (61), utilizing forty-eight 90-day-old male rats divided into four groups of twelve, conducted a 22-day, 20-hour- a—day stress experiment. The animals were placed in identical individual anxiety chaubers and designated as: l - tone-shock, 2 . no tone-shock, 3 - no tone - no shock, 4 - home-cage environment controls. Electrical shocks of 3.5 ma intensity were used with one shock occurring on the average of every four minutes, 20 hours a day. This study yielded no significant Na+ or K+ concentration changes . CHAPTER III EXP ERIMENTAL PROCEDURE The present study was undertaken to determine the effects of daily interval running as an immediate post-treatment for male rats exposed to daily anxiety conditions. Effectiveness of the treatment was determined by measuring the osmlality, the Na+ and K+ mEq/l. concentrations of urine excreted in the home cage. Perspective of the Experimental Desigi An overview of the experiment is shown in Figure l. Twenty subjects were randomly assigned to four groups: 1) Anxiety - Exercise (A-E) 2) Sham Anxiety - Exercise (SA-E) 3) Anxiety - No Exercise (A-NE) 4) Sham Anxiety - No Exercise (SA-NE) The anxiety treatments (including sham) were administered via indi- vidual grid-box snidety chambers. The exercise groups were placed on a moderate-duration, medium-intensity, interval-running program (Appendix A). This type of program is intended to simulate the training program for middle-distance running training (880—yard or one-mile run) in man. Urine samples were collected daily and frozen for later analysis. 16 Individual Grid Boxes Anxiety Exposure With tones and electrical foot pad shocking Anxiety Exposure With tones but no electrical foot Iflid shocking FIGURE 1. l7 I I I l I I I I I I Forced I I I I No I I Interval Running I I I ' Exercise I I Exercise I I I I Treatment I I Treatment I I I I I l. ArE 3. A—NE N - 5 N = 5 2. SArE 4. SA-NE N = 5 N = 5 Perspective of the experiment by groups and tl‘eatment protocol . 355* act: Eli? 18 The purpose of this design was to determine the effects of running exercise as a therapeutic treatment intended to alleviate concurrent emotional trauma. Receipt and Asflgnments of Subjects TwentyL-six male Sprague-Dawley rats, 80 days of age, were purchased from Hormone Assay Laboratories in Chicago, Illinois. Upon arrival, each animal was placed in an individual wire-mesh cage, 20.3 x 20.3 x 25.4 cm., with free access to an adjacent activity wheel. The ensuing nine days were reserved for general environmental adaptation and foot-pad conditioning. Mechanical revolution counters attached to each activity wheel allowed indi- vidual activity records to be kept daily for each animal. The three most active and the three least active rats were excluded from the study. This procedure was designed to eliminate chronically hyper- aetive or hypoactive animals. On the tenth day, the subjects were transferred to individual sedentary Acme Metabolism Cages located in the same quarters. At this time, five subjects were randomly assigned to each of the four t1‘eatment groups. Thereafter, all groups received the same general Care with the exception of the experimental variables. Animal Care During all phases of the experiment, all of the animls were halulled at least once daily. The animals were directly exposed to 01'lly three researchers during the experiment. All other laboratory 19 personnel were asked to avoid entering the animal quarters where the experimental home-cage environment was being maintained. The animals were fed ground Wayne Food blocks ad Zibitwn. Food containers were used which permitted the amount of food con— sLmned in any particular phase of the study to be determined. These food containers also prevented food from falling into the stainless steel urine collection funnels of the metabolism cages. Water was provided ad Zibitum from 100 ml. graduated cylinders in order to calculate the amount of water ingested. Cylinder corks were main- tained via Dow high vacuum silicone grease. Daily body weights were taken and recorded to the nearest gram. Room temperature was maintained at 70°F and 502 relative humidity by air conditioning. To minimize diurnal variability, the quarters were maintained on 24 hours per day of continuous light With the initial receipt of the animals. Pretreatment Protocol The stbjects were maintained under experimental conditions for Seven days without receiving the actual treatments. During the first f(bur days, the animals in all groups received their initial exposure to three hours in the individual anxiety chambers. This phase was followed by movement to holding cages for the A—NE and SA-NE groups. The A—E and SA—E groups were placed in individual running wheels but were not allowed to exercise as the wheels were locked. The looked running wheels and holding cages were adjacent to each other and in the same room as the anxiety chambers. During this 20 pretreatment period the groups remained in their respective positions for the same length of time as that for the first day of the interval running program. These treatments were followed by the recovery period and then the twelveehour home-cage environment. For the last three days, the same conditions were imposed except that the running wheels were unlocked and could be operated by the animals. This permitted the work-rest cycles for the first day of the exercise program to be presented to the animals. In this manner, they became familiar with the freely moving*wheel (work cycle) and its braking action (rest period). At no time during this seven-day period were the animals subjected to any electrical stimulus. The last three days of this pretreatment phase also were used to collect control data in each of the treatment conditions. In addition, measurements were taken on body weight, food and water ingested and urine volume excreted.when appropriate. Urine Collection Periods Control urine was collected for three days immediately prior to the treatment period. Excreted urine volume was measured and samples‘were frozen in 20 m1. plastic vials, with snap caps, for later analysis. Samples were taken when the subjects were in the anxicety Chambers (6:00 to 9:00 a.m.), during the holding period and/or exercise treatment (9:10 to 10:00 a.m.), during the recovery period (10:15 a;m. to 12:15 p.m.) and during a twelve-hour home-cage environment period (5:45 p.m. - 5:45 a.m.). Urine collection times during the eighteen-day experimental period were identical to those used to obtain the control data. 21 Urine Collection Technique Stainless steel funnels were placed under the sedentary home metabolism cages, the anxiety chambers, the holding cages, and the interval training wheels. These funnels caught and collected the excreted urine. The spout of the funnel was protected from feces by a catch tray and roof apparatus. Spun glass was placed in the spout to filter the urine. A glass 50-ml. graduated centrifuge tube was attached to the funnel spout by means of a two-hole rubber stopper. After eaCh sample collection period, the collecting apparatus was washed, rinsed with distilled water and thoroughly dried. The spun glass was replaced each time the apparatus was cleaned. The same glass centrifuge tube was used for each rat in each treatment period. Experimental Protocols Anxiety (6:00 to 9:00 a.m.) Approximately at 5:45 a.m. each morning, all of the animals in each group were transferred from their home-cage quarters to the adjacent but separate animal training room. Body weights to the nearest gramnwere determined during the transfer process. The APE and ArNE groups received the following treatment protocol: EaCh animal was placed in a 20.3 x 20.3 x 10.16 cm. anxiety chamber for three hours daily. Each chamber had a stainless steel rod floor, plastic walls, and a securable plywood door for a roof. Stainless steel metabolismrtray funnels were placed under each Chamber to collect the excreted urine. Anxiety was induced by faradic 22 stimulation via the stainless steel rod floor. Each electrical shock (60 volts, 15 ma) was preceded by a 1700 Hz tone 0.5 seconds before the shock was administered. Each shock lasted 0.5 seconds and was administered randomly on a lOO-second time base. Ninety-five percent of the electrical shocks were from 5 to 20 seconds apart; five per- cent of the shocks were from 21 to 100 seconds apart. The average number of randomly presented shocks was four per minute over the three-hour period. Sham Anxiety (6:00 to 9:00 a.m.) The two sham anxiety groups (SA-E, SA-NE) were placed in identi- cal anxiety chambers as those of the A—E and A-NE groups. These groups were in their chambers during the same time period and in the same room as the A—E and A-NE groups. The two sham anxiety groups, however, received only the auditory tones with no electrical shocks. Movement from the living quarters, with body weight determina- tions, occurred at the same time as that for the two anxiety groups. Exercise (9:10 to 10:00 a.m.) 'Itvo of the experimental groups (A—E and SA—E) received a forced exercise treatment immediately after exposure to anxiety or sham- anxiety conditions. This procedure consisted of a moderate—intensity, medium-duration interval running program (Appendix A), in electroni- cally controlled running wheels. By its description, this program is aerobic in nature and within the physiological work capacity of the rat. Another attractive feature of this training method is that it employs running which is a physical skill common to the rat. 23 The detailed description of the interval training program found in Appendix A was modified for this study. The purpose of this study was to exercise the animals, not necessarily to train them. A decision was made to follow the training program up to day eight, and then to maintain the exercise level at that intensity through day 18, the final day of the experiment. Appendix B contains a comprehensive explanation of the electroni- cally controlled interval running wheels. This appendix also describes the motivation stimulus which prompted the animals to run (95)- Body weig'its before and after the exercise treatment were determined to the nearest gram for each rat. Holding (9:10 to 10:00 a.m.) The holding protocol consisted of an intermediate holding area for the A-NE and SA-NE groups. Upon termination of the anxiety or sham anxiety protocols, these groups were immediately transferred to 20.3 x 20.3 x 25.4 cm. wire mesh holding cages within the same room that housed the anxiety chambers and the electronic running wheels. During the holding period food and water were withheld. Urine samples were collected as previously described. Body weights before and after holding were measured. Recovegz (10:15 a.m. to 12:15 p.m.) At 10:00 a.m. each morning, the two exercise treatment groups and the two non-exercised groups were transferred back to their home cages in the living quarters. The following two hours were 24 designated as a recovery period. Body weight was determined before and after recovery. Food and.water were available ad libitum, ingestion levels were measured, as well as the amount of urine excreted. Zero Phase (12:15 p.m. to 5:30 p.m.) During this time, the animals remained in their home cages. Food and water were available ad Zibitum. No urine samples were collected. Home-Cage Environment (5:45 p.m. to 5:45 a.m.) The twelve-hour home-cage environment period was the focus at which the impact of this current research was aimed. Food and water were available ad'Zibitum, the amounts of each that were ingested were measured. Total urine volume excreted was measured and samples were frozen in 20 ml. plastic vials for later analysis. Sacrifice Protocol Approximately one hour following the final treatment period on the last experimental day, the animals were sacrificed by decapi- tation. Their adrenal glands were removed and immediately weighed on a Mettler analytical balance. Urine OsmolalitygAnalysis A Precision Systems Osmometer was used to determine urine osmolalities. This is an uncomplicated procedure‘whereby a.2-ml. sample is placed in the osmometer and its freezing point is determined. At the freezing point, the apparatus produces a digital readout, in 25 milliosmols per kilogram, of the osmolality of the fluid analyzed. The step by step procedure that was used for analysis was the standard method provided by the manufacturer of the osmometer. Urine Na+ and K+ Electrolyte Concentrations Na+ and K+ electrolyte concentration gradients (mEq/l.) were measured using a Coleman Flamephotometer. The standard procedure described by the manufacturer was employed in the operation of the apparatus for these analyses. The procedure is straightforward; however, care must be taken to recalibrate the flamephotometer every ten to fifteen samples in order to insure accurate measures. Statistical Analysis In order to standardize the data so that meaningful comparisons could be made, the raw data for each animal were divided by the animal's body weight and then multiplied by 100 (osmolality values were not multiplied by 100). This procedure converts all data to values which are relative. As a result of this procedure, the data for each anima1.were expressed in terms of units per gramflbody weight; i.e., a reported mean osmolality of 6.212 was the amount of osmolality per gram of body weight. After compiling the results for each rat, mean values for each group in each observation period were obtained. It was decided that the collected data lent itself to a repeated measures analysis of variance. This analysis requires that there must be more subjects per group than there are observations on eaCh subject (97). There were five animals in eaCh group; therefore the 21 days of control and experimental data collection were divided 26 into four observation intervals. Observation interval 1 was the three-day control data interval. The remaining three observation intervals consisted of six days each. That is, observation interval 2 consisted of treatment days l-6. Based on research by Danford at al. (15), concerning the analysis of repeated measurements experiments, the data were tested for equal variances (EV) and compound symmetry (CS). If the data met the assumption of equality of variance and compound symmetry, a onedway repeated measurements univariate analysis of variance was performed. If the data had equal variances, but failed the test for compound symmetry, the same analysis was performed using the "conservative F—test." Table 1 shows the variables investigated and the results of these tests of assumptions. The probability of making a type I statistical error was set at the .05 level. A SCheffé Post-Hoe procedure was performed on the data in order to identify the observation periods where significant changes had occurred. 27 TABLE 1 Results of Statistical Tests on the Investigated Variables to Determine Their Meeting the Assumptions of Equal Variance and Compound Symmetry variable EV DS 1. Home-cage urine osmolality yes no 2. Home-cage urine Na+ in mEq/l. yes no 3. Home-cage urine K+ in mEq/l. yes no 4. Home-cage food digested yes yes 5. Home-cage HOH digested yes yes 6. Home-cage urine excreted yes no 7. Home-cage body weight yes no 8. Recowery HOH digested yes yes 9. Recovery urine excreted yes yes 10. Home-cage total urine Na+ yes no 11. Home-cage total urine K+ yes yes 12. Home-cage urine Nah/K+ ratios yes no CHAPTER IV RESULTS The results presented here were based upon the repeated measure- ments univariate analysis of variance, further clarified by a Scheffé Post-Hoe test, as described in the experimental methods section. The probability of chance occurrence was set at the .05 level of significance. unless otherwise indicated, all of these results reflect the animals' home-cage environment. When the overall treatment and duration-of-treatment effects were statistically significant (p § .05) for the variables investi- gated, in the rats' home-cage environment, specific treatment and duration effects tests were run. Therefore, the specific treatment results between groups within observation periods and the duration of treatment effects between observation periods within groups are tabulated for the appropriate variables as they appear in the results section. Response to the Runninngxercise Two of the four groups (APE, SAFE) were exercised daily by a program.of interval running conducted in electrically controlled interval running*wheels. These data demonstrate that the animals reacted favorably to the imposed interval running exercise. The 28 29 first six days of running‘was, essentially, when the rats learned to operate the wheels and also represented their initial, programmed, physical conditioning. During this initial sixrday period (second observation) the ArE group accumulated an average of 479 total revo- lutions run per day (TRR = 479), which was 107 percent of the revo- lutions run (PER - 107). These results are shown in Figure 2. During this same period the SAeE group had TRR.= 462 for PER.- 103. During the third observation interval, the APE group averaged 850 TRR, which was 112 PER, while the SA-E group averaged 784 TRR for 105 PER. For the fourth observation, the A-E group compiled 830 TRR and 110 PER, while the SA-E group had 783 TRR, which was 105 PER. The differences between the groups were not statistically significant. These findings lend further validity to the effectiveness of the electronically controlled interval running wheel as a reliable method of exercising laboratory rats. Body Weight Body weight comparisons between the groups (Figure 3) showed that all groups except the SA—NE group lost weight during the course of the study. However, this weight loss was statistically significant for only the SAFE group. A general weight loss by young postpubertal animals was surprising. Possibly this was the initial evidence of the imposed stress the animals were experiencing. Urine Osmolality Responses All of the groups showed statistically significant and similar increases in urine osmolality (Figure 4, Table 2) by the second £0225 cozoiomoo 62087.33 «85 9: .26 29. no.3... $8.98 9.: so Ext :2 30:226.. so .363: .22 can Emmy 323.96. 2:898 23.3 39.23 .N 959... 30 ./.A ./AA ./A _.,A .e ../A ./..A .AA A w... I / / / l L HM , L e L , y / / / i A. wr W ”L -wA m m L 1:. i / .102 A. W W “A. wmm L L H ung suoIInIonaa Io JequInN IoIol 31 30. "3 3:20 3.36:. Eat E9636 EECoEcEm .mode 28 .828 58. 222.6 reocceem + * $035 2: 39.8 33.5 .5 meow...ooEoo £22.. .63 zoos. .m 832... 2-». 2.8 «I. :8 0-. as. .228 v m>mo m m>mo N m>mo _ m>mo 7 . . _ Ho a 40.» I»: + {oi In.» a a a An _+ a Low» szqm sz< m-..o»oo BEoEtooxo can .923 05.3 26 onmeAOE 5 2:20:30 9...: 33 @602 5.02 .v «52“. a-m. 26.. «I. :8 o; 2.8 .228 w m>mo m m>mo N m>mo _ m>mo a U _ J o E o E < a. d 2. 9000.0 m n a 000.» 00m.n 000.? 0006 0006 009m 000.0 00nd 000K M8 wD/‘I/wsow IIIIIoIowso x 33 TABLE 2 Analysis of Variance Summary and Scheffé Post-Hoe Test on Home-Cage Urine Osmolality Treatment Observation period group 1 2 3 4 A—E Mean 3.634 4.960 6.596 6.987 S.D. .506 .868 1.038 .461 SArE Mean 3.608 4.904 5.244 5.234 S.D. .234 .798 .788 .615 AeNE Mean 3.270 4.883 4.933 4.841 S.D. .400 1.173 .931 .307 SA-NE Mean 3.296 4.649 3.625 3.549 S.D. .234 .465 .378 .331 Duration of treatment effects, between observations, within groups F P A-E 75.886 § .01 SA-E 12.938 g .05 APNE 23.058 § .01 Treatment effects,gbetween groups, within observations Observationjperiod 1 2 3 4 F .902 .388 9.322 21.427 P NS NS § .05 E .01 34 TABLE 2 (continued) Scheffé Post-Hoe Test Observations Treatment Treat- Periods Mean Obser- Groups Mean ment cone dif- vation con- dif- groups treated ferences period trasted ference A-E 2-1 1.326* 3 A-E - SA-E 1.352* 3—1 2.962* A-E - A-NE 1.563* 4-1 3.353* A—E - SA-NE 2.171* SA-E - ArNE .311 SA-E 2-1 1.296* SA-E - SA-NE 1.619* 3—1 l.686* ArNE - SA-NE . 1.308* 4—1 1.626* 4 A-E - SA-E l.753* A—NE 2-1 1.6l3* A-E - A-NE 2.146* 3—1 1.663* A—E - SA—NE 3.338* 4—1 l.57l* SArE - A-NE .393 SA-E - SA-NE 1.685* SA—NE 2-1 l.353* A-NE - SA-NE 1.292* 3—1 0.329 4-1 0.253 *Mean difference - 1.277 required *Mean difference - 1.277 for significance at p § .05. required for significance at p § .05. 35 observation interval. The ArE treatment group continued to reflect increasing urine osmolality over the next two observation intervals, nearly doubling their control urine osmolality. They also reflected significantly higher home-cage urine osmolalities than either of the other groups at observation intervals three and four. The SAFE and A-NE groups remained at about the same elevated urine osmolali- ties over the third and fourth observation intervals; these osmolali- ties were nearly fifty percent greater than their control urine osmolalities. These two groups were not significantly different from each other, but their home-cage treatment urine osmolalities at observations three and four were significantly different from those of APE and SAeNE groups. The SArNE group returned to control level urine osmolality by the third observation interval. Of par- ticular interest is that the increased osmolality at the second observation interval was accompanied by a failure to gain weight (:eee Figure 3). These associated findings warrant further investigation. Urine Na+ Concentration in mEq/l. All groups demonstrated a statistically significant Na+ reten- tion (Figure 5, Table 3) which was evident by the second observation interval. There were no significant differences between the groups with respect to Na+ retention based on concentration gradients alone. The Na+ electrolyte retention factor will be brought more into focus 4. when the total Na retention patterns are examined. 36 A00.» 3 Son .938 Eat Eocotfi 2285.65 .I. * 68.8388 6.8:...an 08 .9380 9: .8 00. x 222... .68 6333:. 5 8.8.2828 +02 2...: 88 oEo... :82 .o 053... 9.». 2.8 «I. :8 m.-. :8 .228 v m>mo m m>mo N m>mo . m>mo . _ _ _ .H I Com. m a, * [/0' 1 I. 00m. * 4 [<‘L‘L . I Own. I 00¢. I one. I 000. mz-mo ~ m>mo _ m>mo . _ _ _ a #000 I 00w. 0 N ._ + . . I 08. 0 m . I . .u. .m. < 00m A I one a a a m J 000. 3.8 u o E mz-< u o .8 MIA I q a. A I 08. wI< u ‘ a: me wb/‘I/baw 4.x 5 41 TABLE 4 Analysis of Variance Summary and Scheffé Post-Hoe Test on Home-Cage Urine K+ Patterns Treatment Observation_Qeriod group 1 2 3 4 A-E Mean .425 .549 .610 .560 S.D. .032 .091 .063 .104 SA-E Mean .450 .538 .642 .580 S.D. .036 .080 .028 .109 A-NE Mean .405 .565 .598 .535 S.D. .033 .080 .041 .139 SA-NE Mean .420 .526 .550 .427 S.D. .036 .087 .062 .072 Duration of treatment effects, between observations. withinfigroups F P SA-E A-NE SA-NE 9.129 9.801 9.364 4.283 IIA .05 “A .05 IIA .05 NS Treatment effectsi between groups, within Observations Observation period 1 2 3 4 F .226 .247 1.435 1.859 P NS NS NS NS 42 TABLE 4 (continued) Scheffé Post-Hoc Test ‘Observations Treatment Treat- Periods Mean Obserb Groups Mean ment con— dif- vation con- dif- groups trasted ferences period trasted ference A-E 2—1 .124 4 A-E - SA-E .020 3—1 .185* A-E - A-NE .025 4-1 .135* A-E - SArNE .133* SA-E - A-NE .045 SArE 2-1 .188* SArE - SA-NE .153* 3-1 .192* A-NE - SA-NE .108 4-1 .130* A-NE 2-1 .100* 3-1 .198* 4-1 .130* SA—NE 2-1 .106 3—1 .130* 4-1 .007 *Mean difference - .128 required for significance at p 2 .05. 43 3063 23 .828 29.. 298:6 >..coo:.co_m u * 68.8238 2282.83 98 .828 2.. 9.....6 00. x 20.2.. .63 Bax—own. c. .25.? 8...... x Axum... 2. 3.2.2828 +02. +3 2.... .22 88 2.9. :85. K 83E 0...... 2.8 «I. :8 0-. 16.. .9....8 w m>mo m 98 N m>mo . m>mo T . . . 0.08.0 1 8...” 1 8m.» 1 8nd 1 08...... 1 con.» 1 com.» .900...» 1:80... 1 oood 1 80¢ mz-< u o .m. 1 coo... ”.73 u q a. 93 a.m.o 1 Good mind 2. N2... . 2.... 180m 9-4m ... o .e. +°N W404 & Analysis of Variance Summary and Scheffé Post-Hoc Test 44 TABLE 5 on Home-Cage Urine Total Na+ Patterns Treatment Observationgperiod group 1 2 3 4 AeE Mean 9.314 3.100 3.688 3.632 S.D. 1.817 .373 .255 .284 SA-E Mean 8.452 3.052 3.258 3.376 S.D. .872 .791 .345 .159 A-NE Mean 8.212 3.160 3.460 3.352 S.D. 3.628 .259 .752 .426 SA-NE Mean 7.340 3.686 3.622 3.698 S.D. 1.782 .261 .235 .260 Duration of treatment effects, between observations,¥yithin groups F P A-E 33.084 3 .01 SA-E 26.313 é .01 A-NE 23.042 3 .01 SA-NE 12.969 3 .05 Treatment effects, between groups, within observations Observationgperiod 1 2 3 4 F 2.347 .309 .131 .110 P NS NS NS NS 45 TABLE 5 (continued) Scheffé Post-Hoc Test Observations Treatment Periods Mean groups contrasted difference 2-1 -6.214* A—E 3-1 -5.626* 4-1 -5.682* 2"]. -5 0400* SAFE 3—1 -5.l94* 4-1 -5.076* 2-1 -5.052* A-NE 3-1 -4.752* 4—1 -4.860* SA-NE 3-1 -3.718 4-1 -3.642 *Mean difference = 3.859 required for significance at < p g .05. 46 Total Urinary K+ Regarding tota1 urine K+ (Figure 8, Table 6), the AFE and SA-NE groups showed a statistically significant urinary excretion of total K+ at Observation intervals three and four. The AeNE group demonstrated a similar significant rise in total K+ excretion during observation period three. The SAFE group was the only group not to reflect a statistically significant rise in K+ excretion. The F ratios (7.429) were not as high as those for urine osmolality (71.409) or Na+ mEq/l. concentration (29.304); however, the total K+ data produced thought-provoking information. Interest is aroused as to why the SAFE group responded at observation interval two with a Ki retention (not significant) and otherwise had an essentially stable K+ excretion pattern. The possibility of K+ excretion being sensitive essentially only to emotional stress should be examined. This is emphasized further by the fact that the SArNE group did not demonstrate a total Naf retention but clearly showed a K+ wasting effect when their tota1 excreted quantity of the K+ electrolyte was examined.’ Na:[K+ Ratio Na+/K+ ratios were depressed for all of the groups across all three experimental observation intervals (Figure 9, Table 7). The depressed concentration ratios were significantly different from the control Na.+/K+ ratios. All groups responded similarly. 47 .85... 28 .828 8.. 2.83.... 3.82.86 u 4 68.2388 228.890 oco .228 o... .8 00. x 20.03 .62. Exam... 2. 2829.. 2...: x Axum... 2. 8.2.2828 +5 +x 2...: .22 88 9.6.. :82 .m 8:0... a-m.-.8 «I. :8 8-. 2.8 .228 c 98 n 98 m 98 . m>mo . A . a _ IHOOOO gfi < o 1 000.0 / l 000.0 < 5V 4 l 0006 O X. 4 O 1 8nd * mz-m u o 2.. 108... w21< n 0 Am. * m-mo n m>mo N m>mo . m>m0 . a 2 . _ IPOOO * m L 000. 1 mNm. 1 x 1 one * O m 1 nhm. l 000. 973 u o .3 wz-q u o .n. 1%8 m-I/+DN owmon 00an 1010; 5 50 50."... 0.00 .228 22. 20.022 328.285 u * 6.8.2.808 2202.898 .20 2280 05 8.6 00. x 20.03 .63 20329.. c. 8.2. +v.\+oz 0...... .22 008 08; 80s. .0 0.30.... 2.928 0.... :8 0... :8 .228 w 550 m m>mo N m>mo . m>mo . . _ _ 1 .U-OOO 1. .u * .x .m I 005 \1: .x .1 mum. * 1 *o l 005 * O * 4 1 0.5. 1. 000. 02.8 n o .4. i 000. wZI< u C Amy M.’ r 0.4.... u 4 .0. 18»... 0-4 u 4 2. 82 4 out; JCNV. «04.. 1 05.4.. o 0...... L 00?; 90130.1 +>ll+DN owmon augm logo; 5 51 TABLE 7 Analysis of Variance Summary and Scheffé Post-Hoe Test on Home-Cage Urine Na+YK+'Ratios Treatment Observation period group 1 2 3 4 A-E Mean 1.452 .502 .496 .490 S.D. .062 .047 .059 .043 SA-E Mean 1.426 .574 .516 .556 S.D. .047 .053 .036 .036 A-NE Mean 1.398 .628 .514 .566 S.D. .062 .065 .059 .038 SA—NE Mean 1.470 .624 .550 .568 S.D. .052 .034 .019 .020 Duration of treatment effects, between observation§,,within groups F P A-E 504.146 2 .01 SAFE 504.397 5 .01 A—NE 454.295 5 .01 SA-NE 519.307 § .01 Treatment effects, between gropps, within observations Observation period 1 2 3 4 F 2.157 3.549 1.112 3.004 P NS NS NS NS 52 TABLE 7 (continued) Scheffé Post-Hoc Test Observations Treatment Periods Mean groups contrasted differences 2-1 -.950* A-E 3-1 -.956* 4-1 -.962* 2—1 -.852* SA-E 3-1 -.910* 4-1 -.870* 2-1 -.770* A-NE 3—1 -.884* 4-1 -.832* 2-1 -1.146* SA-NE 3—1 -.920* 4-1 -.920* *Mean difference - .146 required for significance at p § .05. 53 Relative Adrenal Weight This comparison (Table 8) revealed that the relative adrenal weight for the APE group was significantly greater than were those of the SA-NE and A-NE groups, but similar to that of the SAFE group. In turn, the SA-E and ArNE groups had similar relative adrenal weights. Both were significantly greater than that of the SArNE group (p . .05). TABLE 8 Means for Relative Adrenal Weight Groups ArNE SArNE SArE ArE Mean 1.784 1.402 1.987 2.150 Food Ingestion Comparisons All groups had similar and statistically significant drops in food ingestion which were evident at the second observation interval (Figure 10, Table 9). By the third and fourth Observation intervals, food ingestion had returned to the control-period intake'(adjusted for body weight changes, i.e., gram food per gram body weight). The A—E group demonstrated a significantly elevated food ingestion at the fourth observation interval. 54 50."... 0000.0 0020.0... 8.. 20.2.2 328.....05 .00."... 0.00 2.200 8... 20.0.2.0 3200:...05 1T * 88.220000 2202.898 08 2.28 0.... 8.6 00. x 20.0... >000 0.08.8.0 c. 0.08. 8.800... 002 008 0.8... 80.... .0. 0.00.“. 070. :8 0.-.. 8 0-. 8 .228 0 080 m 080 m 080 . m>0o . . . u. . 10.000 0 1:000. 02.8 n o .4. m 02-4 u o 2. 0-40 u G .0. m 1000. m-q u 4 .3 m 0.. + . 1 08. n; +0 1 08. 1 .00. 1 000. L I {Quin .00 ' p005 ,0 swo19 55 TABLE 9 Analysis of Variance Summary and Scheffé Post-Hoc.Testi on Home-Cage Food Ingestion Treatment Observation period group 1 2 3 4 A-E Mean .043 .033 .043 .048 S.D. .006 .005 .002 .002 SAFE Mean .039 .029 .037 .039 S.D. .004 .005 .005 .004 A-NE Mean .042 .032 .040 .041 S.D. .006 .002 .005 .005 S.D. .006 .001 .005 .007 Duration of treatment effectspibetween observations, within groups F P APE 13.464 3 .01 SA-E 8.980 f. .01 A—NE 6.808 .‘i .01 SA-NE 4.000 E. .05 Treatment effects, between groups, within observations Observation period 1 2 3 4 F 2.877 1.020 1.083 5.259 IIA P NS NS NS .05 56 TABLE 9 (continued) Scheffé Post—Hoe Test Observations . Treatment Treat- Periods Mean Obser~ Groups Mean ment con- dif— vation con— dif- groups trasted ferences period trasted ference A-E 2-1 -.010* 3-1 .000 4 A-E - SA-E -.009* 4-1 -.005* A-E - A-NE -.007* A—E - SA-NE -.012* SA-E 2-1 -.010* SA-E - A—NE -.002 3—1 -.002 SA-E - SA-NE -.003 4-1 .000 A-NE - SA—NE -.OOS* A-NE 2—1 -.010* 3-1 -.002 4-1 -.001 SArNE 2-1 -.006* 3—1 .001 4-1 .001 *Mean difference - .005 *Mean difference - .005 required for significance required for significance at p i .05. at p 5 .05. 57 Water Consumption Comparisons All groups had a significant and similar decrease in water ingestion at the second observation interval (Figure 11, Table 10). Water ingestion was only partially restored'by the third observa- tion interval. During the fourth observation interval, all groups returned to control levels of water ingestion, with the exception of the AFE group, which continued to exhibit depressed.water intake. The water consumption of the ArE group‘was significantly different from.that of the other groups in the fourth observation interval. This is consistent with the fact that they were also statistically different from.the other three groups during the control period. Urine Volume Excretion Patterns The urine excretion pattern for all of the groups followed a trend similar to that of their water consumption (Figure 12, Table 11). All groups had significantly depressed urine volume excretions which were evident by the second observation interval and.which con- tinued through the third Observation interval (except for the SAHNE group). During the fourth observation interval, the AFE and ArNE groups returned to control urine excretion volumes. However, the SAFE group urine excretion rates remained depressed while the SArNE group demonstrated a significantly increased urine volume during the fourth interval. The urine excretion patterns verify the validity of utilizing tota1 electrolyte patterns. If only a mEq/l. concentration gradient analysis had been employed, it would have mistakenly appeared that .00."... 0000.0 0200.0... 0.0.. E0830 3200.....05 u + .00."... 28 .928 22. 228.... 2.82.220 u m 68.83030 .28....808 0:0 .0....00 0.: 00.80 00. x 20.03 >000 E0)... 0. 00.880003 883 0000 060; 000.2 .: 0.50.“. 58 07». :8 0.-.. :8 0-. 2.8 .0....8 w 050 m m>00 N m>mo _ m>mo . _ . . . 1._.000 1.- 02-40 - o .3 4 1 9.0. 02 .. < m - «m a 000. m 1 4 1 000. 1 000. O 1 000. 1 000. 0.0.0 + .0. . 1 08. L 2.0. l 000. 1810M ,0 1w 59 TABLE 10 Analysis of Variance Summary and Scheffé Post-Hoe Test on Home-Cage Water Consumption Treatment ' Observation period group 1 2 3 4 A-E Mean .076 .051 .068 .069 S.D. .009 .010 .007 .017 SArE Mean .065 .046 .056 .064 S.D. .011 .013 .010 .006 A-NE Mean .065 .040 .059 .062 S.D. .016 .008 .020 .014 SA-NE Mean .058 .043 .049 .056 S.D. .008 .004 .008 .006 Duration of treatment effects,,between Observations, within groups F P SAFE 7.831 g .01 AFNE 13.485 § .01 SAFNE 4.938 5 .05 Treatment effects,4between groups, within observations Observationgperiod 1 2 3 4 F 4.103 .927 1.199 3.247 60 TABLE 10 (continued) Scheffé Post-Hoe Test Observations Treatment Treat- Periods Mean Obser- Groups Mean ment con— dif- vation con- dif- groups trasted ferences period trasted ference A-E 2-1 -.025* 1 A-E - SAFE -.011* 3—1 -.006* A-E - A-NE -.011* 4—1 -.OO7* ArE - SA-NE -.018* SA—E - A-NE .000 SA-E 2-1 -.019* SA-E - SArNE -.001 3-1 -.009* AeNE - SA-NE -.003 4—1 -.001 3 A-E - SA—E -.012* A-NE 2-1 -.025* A-E - AeNE -.009* 3-1 -.006* A-E - SA-NE -.019* 4-1 -.003 SArE - ArNE -.003 SA-E - SA-NE -.007* SA-NE 2-1 -.015* A—NE - SA-NE —.010* 3-1 -.OO9* 4-1 -.002 4 A-E - SArE -.005* A-E - AeNE -.007* A-E - SArNE -.013* SA-E - A-NE -.002 SA-E - SAeNE -.003 A-NE - SArNE -.002 *Mean difference - .004 required for significance at p 5 .05. *Mean difference . .004 required for significance at p S .05. .00.»... 0000.0 00.00.00. E0... .008...0 >_...00.....0.m u + 30.00. 0.00 .9000 0.0.. 008.20 >_.000...00.m u * .000..0>8m00 .0.00E..00x0 000 .9200 0... .0. 00. x 20.0... >000 60:0. 0. 008.80 00:08.8 0...... 0000 00.0.. 0002 .m. 0.00.... 61 0.1». 0>00 NT.- 0>00 01. 0>00 .0....00 0 «>00 . 0 «>00 0 900 _ m>mo . 0 0 q . 10.000 1.0.00. 02-4010 .0. 4.2.... 02-4 u 0 .8 1 000. 0.40 n 4 .0. 0-4-4... 1000. 1 000. 1 «no. 1 08. 1 000. 1. mno. 1. 000. L 000. 00an ,0 H11 62 TABLE 11 Analysis of Variance Summary and Scheffé Post-Hoc Test on Home—Cage Urine Excretion Volume Treatment Observationgperiod group 1 2 3 4 ArE Mean .041 .031 .036 .040 S.D. .008 .008 .008 .006 SA-E Mean .038 .029 .029 .033 S.D. .004 .008 .004 .004 A—NE Mean .037 .025 .034 .036 S.D. .016 .007 .014 .011 SArNE Mean .033 .031 .033 .038 S.D. .007 .004 .005 .006 Duration of treatment effects, between Observations, within groups F P A-E 5.478 f. .05 SA-E 4.722 :- .05 A-NE 7.690 :- .05 SA-NE 2.487 S .10 Treatment effects,pbetweenggroups,_within Observations Observationgperiod l 2 3 4 F .852 .469 .655 .454 63 TABLE 11 (continued) Scheffé Post-Hoc Test Observations Treatment Treat- Periods Mean Obser- Groups Mean ment COD? dif- vation con— dif- groups trasted ferences period trasted ference APE 2-1 —.010* 1 A—E - SA-E -.003 3-1 -.005* A—E - A—NE -.004 4-1 -.001 A-E - SArNE -.008* SArE - A-NE -.001 SAFE 2-1 -.009* SA—E - SA-NE -.005* 3-1 -.009* A-NE - SA—NE -.005* 4-1 -.005* 2 A—E - SA-E -.002 A-NE 2—1 -.012* A-E - A-NE -.006* 3—1 -.005* A-E - SArNE -.003 4-1 -.001 SA-E - A-NE -.001 SArE - SAeNE -.006* SArNE 2-1 -.002 A-NE - SA-NE -.003 3—1 .000 4-1 .005* 4 APE - SA-E -.007* A—E — A-NE -.004 A—E - SA-NE -.002 SArE - A-NE -.OO4 SA—E - SA-NE -.005* A-NE - SA-NE -.003 *Mean difference - .005 *Mean difference - .005 required for significance at required for significance at p 5 .05. p 3 .05. 64 the SArNE group had returned to a normal urine K+ electrolyte balance. It was not until examining this group's K+ mEq/l. with respect to their total excreted urine volume that an accurate picture of their K+ excretion levels was apparent. The same situation occurred for the SA-NE group regarding their retention of Na+. It is the total amount of electrolytes which is important, not just the concentration gradient. Examining electrolyte concentration gradients alone may be misleading. For example, if two rat urine samples were being compared, one sample at 400 mEq/l. of K+ and the other at 200 mEq/l. of K+, a quick estimate would be that the rat with the 400 mEq/l. concentration.was excreting the most K+. However, if the 400 mEq/l. concentration was from a 10 m1. sample and the 200 mEq/l. concen- tration was from a 25 ml. sample, the rat with the lower concentrae tion gradient actually would be excreting the most K+. Recovery Period Water Consumption All of the groups except the SA—NE group showed a significant increase in.water consumption by the second observation interval (Figure 13). Only the ArE group retained this increase across all three experimental observation intervals. Recovery Period Urine Excretion The decrease in urine excretion for all groups was pronounced during the recovery period (Figure 14). The groups were not sta- tistically different from each other across the three experimental observation intervals, but all had values which were significantly different from their control excretion levels. 65 30.00. 0000.0 00.00.00. 0.0.. .008...0 >_...00...00.m .005-.0. 0.00 .2080 0.0.... .008...0 >_.000...00.m .+. fl. 002.00.00.00 3.006.808 000 .9008 0... 88 00. x 20.0.... >000 0.0:... 0. 02.00.0003 8.02. 00.80 >.0>000. 000.2 .m. 0.00.... 0.-.... 2.8 0.-.. 2.8 0-. 2.8 .228 0 m>00 m m>mo . N m>mo _ m>mo . _ . 0 T . . O .+ * .0 u 0.n.~+ .0 0 02-40 u o .0. 02-4 u o .0.. 0-40 u d 8. 0-4 u 4 ... 0+... 11.1000. L 4000. 000. 000. 000. 000. 000. 000. «no. 000. 000. 80. Nvo. 19,01“ ,0 11.1.1 66 60.00. 0.00 .0..000 0.0.. .008...0 >....00.....0.m u * .000..0>8000 .0.00E.80..0 08 .0..000 0... .000 00. x 0.0.03 >000 .5)... 0. 00.0.0) 00.8.9.0 00.8 00.80 >838. 000.... .0.. 0.00.... 0.-.". :8 0.-.. 2.0.. 0-. 0...... .828 v m>mo m m>m0 N m>mo _ m>mo . . 0 . 1. *0 */ l I. 9 .fl . * I * J 02-40 1 o .0. 0214 u 0 .3 L UIQm u q RNV o m1< u ‘ A: l L N00. #00. woo. 000. 0.0. N_O. v.0. 0.0. 0.0. ON 0. 80an ,0 In: 67 Recovery urine excretion and water ingestion were included to support and clarify the corresponding home-cage parameters. Once more the SAeNE group presented thought-provoking data. These animals appeared to be demonstrating an antidiuretic effect similar to those exhibited by the groups that received the experimental treatments . Discussion An evaluation of the exercise data shows that the animals were able to successfully complete the imposed interval-running program. These data provide supporting evidence for the effectiveness of the Controlled Running Wheel (37,95) as a reliable method of exercising and/or training research rats. The results of the body weight comparisons of these animals‘were puzzling. Generally, exercised animals weigh less than sedentary animals as was the case in this study. However, this investigator was unable to uncover any other studies where young postpubertal rats lost weight as a result of similar experimental treatments. The loss of weight by the animals in this study‘was the first indi- cation of the possible pronounced reaction they may have had to the induced stress. Urine osmolality data in this study directly support Corson's (7) findings in dogs. It is interesting that the ArNE and SA—E groups, which theoretically were exposed to one stress each (shocking and exercising, respectively), had approximately 50% increases in urine osmolality. The A—E group which was exposed to two stresses 68 (shocks and exercise) had approximately a 100% increase in urine osmolality by the fourth observation interval. The implication that can be drawn from these results seems to be that increases in urine osmolality reflect increases in imposed stress--at least in that part of the osmolality continuum Observed in this investigation. The Na+ and K+ electrolyte data of this study are in agreement with earlier data published by Dauphinée (17), Moore (50), Thorn et al. (89) and Gann and Wright (28). However, the findings of this study contradict those of Spigel and Ramsay (80), who found signifi- cant 00* as well as K+ excretion. Hoagland et al. (21) also found elevated Na+ and K+ excretion in human subjects over 20 years of age whereas, for those under 20 years, they reported elevated K+ retention accompanied by increased 05* excretion. Paré and McCarthy (61), in a somewhat similar study, reported no disturbance of the urinary Na+-K+ electrolyte balance. This may be attributable to the fact that they used only 3.5 ms of shock in their study. It is important to note the Na+ and K+ electrolyte changes which occurred in the current investigation. All of the groups except the SArNE group had increased total Na+ retention-I Only the ArE and SArNE groups had consistently elevated total K+ excretions, but the ArNE group also had an elevated K+ excretion during the third observation period. The SA—E group was unique in that they did not respond with elevated K+ excretion and even showed a non- significant retention in the second Observation period. 69 One could speculate that urine K+ responds only to severe stress were it not for the data of the SA-NE group. Taking this group's responses into consideration, it becomes entirely plausible that Rf electrolyte excretion changes may be more associated with emotional stress than physical stress (exercise). This assumes that SA involves a significant amount of emotional stress. This theory becomes more interesting as it is noted that the SA-NE group was the only group which did not consistently retain 05+ during the study. The mechanism directly responsible for these Na+ and K+ electro- lyte shifts seems to be associated with mineralocorticoid aldosterone rather than corticosterone. While it is known that aldosterone may be secreted independently of pituitary function(.55,69), there is a rapid increase in aldosterone secretion following anxiety stress or physical stress (45). ACTH, which is secreted by the anterior pituitary, stimulates the adrenal cortex to secrete elevated levels of aldosterone in a stress situation (55). This evidence leads to the conclusion that, in the current investigation, aldosterone was stimulated by ACTH to respond to the induced stress situations andwwas the agent responsible for the electrolyte imbalances. Enhanced aldosterone secretion also is reported to result in a depressed Na+ to K+ ratio (55). Figure 11 shows a significant Na+ to Rf ratio depression for all of the groups, with that of APE group being the furthest depressed. This writer chooses to disagree with those investigators (16,61,78) who have reported that aldosterone is not implicated in the stress reaction. 70 Relative adrenal weight data reveal adrenal hypertrophy fOr the A-E, SA—E, and A—NE groups. These data, along with the urinary osmolality and Na+ and K+ imbalances, suggest significant hyper- function of the pituitary and the adrenal cortex as responses to the induced experimental stress situations. The relative adrenal weights support the findings of Paré and McCarthy (61) for male Sprague—Dawley rats of the same age. It is clear that the A-E group responded dramatically to the experimental protocol as evidenced by statistically significant increases in urinary osmolality, Na+ retention and K+ excretion. The SArNE group, however, yielded equally dramatic data. During the first six treatment days, their home-cage urine osmolality increased in a manner similar to those of the three treatment groups, but it did return to control level by the end of the study. However, their recovery urine data showed a marked anti- diuretic effect equivalent to that found in the other groups across all experimental periods. While they did not demonstrate signifi- cant hommrcage total Na+ retention, their total K+ excretion pattern paralleled that of the ArE group. Significant alterations occurred in both groups over time. This SA-NE group provides further supporting evidence concerning the role of aldosterone in stress. ADH secretions probably are not responsible for their Naf retention pattern since their home-cage urine osmolality returned to normal while their Na+ retention remained consistently elevated. Aldosterone is approximately 30 times more + potent than corticosterone in influencing Na retention and 5 times 71 greater in its influence on K+ excretion (55). It seems reasonable, therefore, since ADH responses may be elicited only by some physical source which involves a direct treatment such as physical activity, loud noises, pain or fear of pain, hemorrhage, etc., that the marked antidiuretic effect in the SArNE group recovery period was mediated by A111. This enhanced ADH secretion probably was a result of direct stress in response to the loud tones in combination with the squeals of the shocked animals during the experimental treatment immediately prior to the recovery period. The Na+ and K+ imbalances detected in their home-cage urine were not the results of directly applied stressors but rather may have reflected stress by association. The fact that the SA-NE animals were in the same home-cage environment as the stressed animals may have produced an association stress reaction in them. This stress could.have been via olfactory stimuli given off by the treated animals. If this hypothesis is tenable one would have to assume that over the days of the study the other treatment groups learned that removal from the treatment environment merely signaled a delay before the onset of the next treatment cycle. Thus, for the APE and ArNE animals the fear of pain and/or a general environmental threat may have remained with them in the home-cage environment. This could explain their significantly increased home-cage urine osmolalities. An extension of this thinking is that general, undefined anxiety‘which is detected by olfaction may stimulate changes in the 00+ and K+ electrolyte levels. In addition, this observation may show that it is possible 72 to produce anxiety in the absence of a direct anxiety stressor- Laboratory rats may be susceptible to emotional anxiety merely by being directly associated with other animals who are living under high anxiety conditions. These olfactory stimuli, possibly detected by the SA—NE group, could have triggered elevated ACTH secretions which, in turn, may have maintained enhanced aldosterone secretions. This hypothesis is not contradicted by Ganong et al. (48), who showed that lesion of the median emminence of the hypo— thalamus results in markedly decreased ACTH and aldosterone secre- tion plus adrenal cortical atrophy. Aldosterone is further implicated here as a result of the SAFNE group's significantly depressed Na+/K+ ratio. The ArNE and SArE groups responded.with thought-provoking data. Their urine osmolalities were significantly elevated and similar; their Na+ retention patterns were similar; their K+ excretions were similar except that the third observation period the ArNE group had a significantly elevated total K+ excretion. The ArNE group's elevated K+ excretion at the third Observation period supports the possibility of K+ excretion reacting as a response to emotional stress since only the SA-E group did not alter its K+ excretion. The ArNE and SAFE groups seemed to clearly demonstrate similar responses to a single stress even though the stressors were different (anxiety and exercise, respectively). This supports the opinion of those investigators who claim that response to stress is nonspecific. In this study, however, differences in K+ electrolyte response must be ignored in order to categorically accept that hypothesis. 73 In conclusion, the urine data gathered in the home-cage environment and the body weight comparisons indicate that both experimental treatments produced pronounced stress reactions in the experimental animals. Urine osmolality seems to represent a reliable and sensitive measure of physical and emotional stress. Posterior pituitary secreted ADH appears to be the logical mechanism controlling this response. The fact remains, however, that APE, SArE, and A-NE treatment groups all may have been responding to physical stress. The electrical shocking protocol may not have elicited an emotional stress situation but rather another form of physical stress. This would mean that the SArNE group might have been the only actual anxiety treatment group. The existing litera- ture and the data available from this investigation make this a difficult position to support. The Na+ retention by all of the groups and the K+ excretion by the A-E, A-NE and SA-NE groups make it reasonable to infer that the mineralocorticoids do play a role in the physiological response to stress. ACTH stimulated aldosterone is probably the controlling factor mediating this electrolyte response. These electrolyte imbalances, supported by adrenal hypertrophy and increased urine osmolality, also imply a hyperfunction of the hypothalamus, anterior pituitary and adrenal cortex as responses to anxiety and/or exercise stress. The data resulting from this study provided physiological evidence that the stresses of exercise and anxiety are additive in nature and elicit an apparent double stress syndrome. This evidence 74 is contrary to the popular view that exercise alleviates emotional anxiety. The.implications these findings may have regarding cardiac rehabilitation alone warrant continuing research with respect to the relationships between exercise and anxiety. For example, the hearts of the animals used in this investigation were examined in a come panion study of the possibility that myocardial damage might have resulted from the experimental treatments the animals received. After completion of the current investigation, the animals were immediately sacrificed by decapitation. Fbllowing quick freezing of the hearts, five sets of four transverse sections (eight microns thick) were taken from the heart muscles at equal intervals from the lower half of the ventricles. These sections then were stained and rated for possible myocardial damage. The hearts of the SA-NE group were used as the control heart tissue. The difference in heart damage ratings between the SA—NE and the A-E groups was found to be significant (p § .10). Slight damage also was recorded in the SAeE and A—NE groups, but this damage was not statistically significant (86). These findings provide supporting evidence regarding the severe stress the ArE animals were confronting. CHAPTER V SUMMARY, CONCLUSIONS, RECOMMENDATIONS The purpose of this study was to determine the effectiveness of exercise as a therapeutic measure for alleviating emotional trauma (anxiety). Home-cage excreted urine osmolality, sodium (00') and potassium.(K+) electrolyte concentrations were examined as the primary variables. Twenty male albino rats (Sprague—Dawley) were randomly assigned to four experimental groups and studied for 21 days. The four treatment groups consisting of 5 rats each were as follows: "A—E" Group: Anxiety (A) treatments were mediated via random faradic stimulation through the stainless steel grid floor of individual anxiety chambers for three hours daily. Exercise (E) was by forced interval running (Appendix-A) immediately following the anxiety treatments. "SA-E" Group: Sham Anxiety (SA) was presented at the same time as the anxiety treatments. These animals were placed in similar individual anxiety chambers located in the same room as,'and adjacent to, the treated animals' chambers. However, faradic stimulation was not administered to the sham anxiety group. Exer- cise (E) was by forced interval running immediately following the sham anxiety treatments. "ArNE" Group: The Anxiety (A) treatment was the same as for the ArE group. No exercise (NE) followed this group's exposure to anxiety conditions. 75 76 "SA-NE" Group: Sham Anxiety (SA) was imposed on this group in the same manner as for the SA—E group. No exercise (NE) followed this group's exposure to the sham anxiety treatment. The daily experimental treatment protocol began with anxiety or sham anxiety treatments from 6:00 to 9:00 a.m. All four treat- ment groups were placed in individual anxiety chambers during this time period and all were in adjacent chambers in the same room. Anxiety was induced by random electrical shocks (60 volts, 15 ma) preceded by a 1700 Hz tone 0.5 secs. before the shock was administered. The two sham anxiety groups, however, received only the auditory tones with no electrical shocks. Imediately after anxiety or sham anxiety, 9:10 to 10:00 a.m. , the mo groups scheduled for exercise (A-E, SA-E) followed an interval running program of moderate intensity, medium duration in electroni- cally controlled running wheels. Since the purpose of this study was to exercise the animals, not necessarily to train them, the description of the interval training program found in Appendix A was modified. It was decided to follow the training program up to day 8 and then to maintain the exercise level at that intensity through the final day of the experiment. All of the exercise rats ran simultaneously in individual wheels. These electronically controlled interval running wheels employ operant conditioning utilizing a light (60 volts, 120 watts) as the conditioned stimulus and faradic stimulation (.2 ma) through the running surface as the unconditioned stimulus. During this same time those animals who were to receive no exercise (A-NE, SA-NE) were transferred to wire mesh holding cages. This holding cage protocol was conducted in the 77 same room and at the same time as the exercise animals were running. From 10:15 a.m. to 12:15 p.m., all four groups were trans- ferred back to their home cages in the living quarters. These two hours were designated as a recovery period. The zero phase was from 12:15 p.m. to 5:30 p.m. During this time period the animals remained in their home cages and no data were collected. The period of home-cage environment in the living quarters was from 5:45 p.m. to 5:45 a.m.; this was the time interval on which the current research was focused. Body weights were taken before and after: 1) anxiety or sham-anxiety treatments, 2) exercise or holding protocol, 3) recovery and 4) home-cage environment. Urine samples (total volume) were collected during all of the treatment periods using stainless steel metabolism trays and frozen for later analysis. Food and water were available ad Zibitum during the recovery period and in the home-cage environment. Daily ingestion levels of both food and water were recorded for each animal. Daily records of running performance also were maintained for the two exercise groups. The animals were exposed initially to the experimental protocol for four days prior to the onset of control data collection. Con- trol data were then collected for three days immediately prior to the 18—day treatment period. The technique used for control data collectionuwas exactly the same as that used to collect data during the treatment periods with one exception: at no time during the 78 control data collection period.were the animals subjected to any electrical stimuli. All animals were decapitated approximately one hour following their final treatments. The adrenal glands of each rat were removed and weighed. Urine osmolality was determined utilizing an osmometer. Milli- equivalents of Na+ and K+ were measured by flame photometer. The data.were statistically analyzed.by a onedway repeated measurements univariate analysis of variance. The results were further analyzed, whenever appropriate, by the Scheffé Post-Hoe procedure. Conclusions The conclusions that can be drawn from this study are as follows: 1. Exercise, as administered in this study, does not reduce coinciding emotional trauma (anxiety). 2. Exercise is an additive stress on an animal which is also exposed to anxiety. 3. Urine osmolality is a reliable and sensitive indicator of emotional or physical stress. 4. Urine sodium electrolyte imbalance (retention) is a sensitive indicator of emotional or physical stress. 5. Urine potassium electrolyte imbalance (excretion) may be a reliable measure of emotional stress. 79 Recommendations 1. Further studies should utilize an additional control group which is continuously located in the home-cage environment. 2. A similar study on previously trained rats, employing a larger sample size, should be undertaken. 3. Rats previously exposed to an anxiety treatment should be exercised with the anxiety regimen continuing during the conduct of the daily exercise program. 4. Rats exposed to an anxiety treatment should be exercised with the anxiety regimen terminating at the start of the daily exercise program. 5. The ordering of the treatments in items 3 and 4 should be reversed, i.e., exercise followed by anxiety. 6. 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APPENDICES 89 00NH 00NH 00.4w 0.4 N.H 0.m 4 ON mH mmH m.H ON 0 00NH 00NH 00.4w 0.4 N.H 0.m 4 0N mH mmH m.H 0H 8 00NH 00NH 00.4w 0.4 N.H 0.n 4 ON mH mnH n.H 0H 3 00NH 00NH 00.4w 0.4 N.H 0.m 4 0N mH an m.H 0H H 0m0H 00NH 0Nu4m m.m N.H 0.m 4 0m 0H 00H m.H 0H 2 4 0m0H 00NH 0N.4m m.m N.H 0.m 4 0m 0H 00H 0.N mH m 0m0H 00NH 0Nu4m m.m N.H 0.m 4 0m 0H 00H 0.N 4H 9 0m0H 00NH 0Nu4m m.m N.H 0.m 4 0m 0H 00H 0.N MH 3 0m0H 00NH 0Nu4m m.m N.H 0.m 4 0m 0H .00H 0.N NH H 000 00NH omnm4 0.m N.H 0.m m 04 0H 00H 0.N HH z m 000 00NH 0m.04 0.m N.H 0.m m 04 0H 00H m.N 0H m 000 00NH 0m.04 0.m N.H 0.m m 04 0H 00H m.N 0 H 0m> 00NH 00.04 m.N N.H 0.m m 04 0H 00H m.N m 3 000 00NH 0m.04 0.N N.H 0.m m 04 0H 00H m.N n H 0m4 00NH m4u0m m.H N.H 0.m m 04 m 00H m.N 0 2 N 0m4 00NH m4umm m.H N.H 0.m m 04 m 00H 0.m m m 0m4 00NH m4umm m.H N.H o-m m 04 m 00H 0.m 4 H 0m4 00NH m4.0m m.H N.H 0.m m 04 m 00H 0.m m 3 0n4 00NH m4umm n.H N.H 0.m m 04 m 00H 0.m N H 0m4 00NH m4umm m.H N.H 0.m m 04 m 00H 0.m H 2 H mas Hoomv A000 A000 A08. A048. 00:00 0:00 Aommv AaHav Avon. .0H .33. .x3 mooHu $00 .aHav \00. #0000 00000 no 000 0eHu no 05H» 00 mo 10Ho>mu 08H» .woum 00000 .000 .oz maOHu 000m Aommv 00Hum 000 000 .0x0 3003 mo 00m mEHH 1H0 oaHu lumHoo H0009 H0009 oeHu 1000M #003 10¢ H0009 mHmmmB UZHZZDMIQMHHOMHZOD zH mH00 0800 .0000 00000 .000 .03 00000 0003 A0000 00000 000 000 .000 3003 00 000 0a0H I00 0000 I00H00 H000H H000H 0000 I000m 3003 :00 H000H 30300080 4 00020004 APPENDIX B DESCRIPTION OF ELECTRONICALLY CONTROLLED INTERVAL RUNNING WHEEL FOR SMALL ANIMALS Motivation to run is provided by a low-intensity, controlled shock current which is terminated when the animal reaches a speci- fied speed. A light always precedes the shock so the animal may avoid shock entirely by responding promptly. At the beginning of each running period, a light is turned on above the wheel and remains on for a predetermined acceleration period. If a specified speed has not been reached by the end of the acceleration period, the light is turned off and a controlled shock current is applied to the animal through the running surface of the wheel. As soon as the animal reaches the specified speed, the shock is discontinued. If this speed is attained within the acceleration period, the light is turned off and no shock is applied. As long as the animal con- tinues to run at or faster than the specified speed, he avoids being shocked; if he slows down below the specified speed during the running period, the light and shock sequence is repeated. Initially, the animals run in response to the shock. By the end of the third 40-min learning period, most animals learn that the light always precedes the shock and will run in response to the light. 91 92 A typical running program consists of alternate periods of work and rest. During work periods, the wheel is free to turn; during rest periods, it is braked automatically to prevent spontaneous activity. (A specified number of work and rest periods (repetitions) constitutes 1 bout of exercise. A single training period may include several such bouts separated by relatively long periods of inactivity (time between bouts), during which the wheel remains braked. A buzzer signals when the running program has been completed. A bank of controlled-running wheels consists of 1 master control unit and up to 12 wheels. The master control unit . . . can be programmed for a given training period as follows: 1) Acceleration time can'be set for 0.5-5.0 sec at intervals of 0.5 sec. These times are accurate to 1:0.02 sec. 2) work time can be set for 5 sec-60 min in intervals of 5 sec. These times are accurate to i 0.02 sec. 3) Rest time can be set for 5—60 sec in intervals of 5 sec. These times are accurate to 1:0.02 sec. 4) The number of repetitions (of work and rest periods) per bout can be set for 1-399. 5) The number of bouts can be set for 1-10. 6) Time between bouts can be set for 1.0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 20.0, 25.0 and 30.0 min. These times are accurate to 110.02 sec. 7) Shock level can be set for 0.0-1.2 ma at intervals of 0.2 ma. Shock levels are accurate from + O to ~10% regardless of whether the animal is wet or dry. 8) Running speed can be set for l.0—6.0 ft/sec at intervals of 0.5 ft/sec. Speed settings are accurate to i 2%. The controlled-running wheel. . . is made of lightrweight plastic and has aluminum rods as the running surface to minimize 93 angular inertia. The circumference of the running surface is 122 cm (4 ft); the width is 11 cm. The wheel is supported by bearings in a steel frame. Fecal droppings are caught in a paper-covered metal tray below. Light baffles are provided to avoid confusion between adjacent animals due to nonsynchronous light signals. The top light baffle can be raised to permit easy removal of the wheel for washing. A simulus-control cage is provided in which a matched simulus-control animal receives light signals and shock simul- taneously with the animal in the wheel. A result unit. . . is attached to the frame of each controlled- running wheel. The following data are collected and displayed in digital fornnby the result unit: a) the total number of revolu- tions run (TRR) by the experimental animal, and b) the cumulative duration of shock (CDS) received by both the experimental and control animals. Two calculated variables have been derived to allow a comparison to be made between animals on different training programs: a) the percent of expected revolutions (PER), which is a measure of work performed relative to work expected, and.b) the percent shockrfree time (PSF), which is the percent of the total work time during which the animal avoided shock by running at or faster than the speed specified on the master control unit. The master control unit provides electrical power to itself and to a maximum of 12 wheels. It also provides work and shock information, shock current, and coded signals representing speed and acceleration time to each wheel. . . . 94 If an animal's speed drops below the set speed during a work period, the light must turn on for a period equal to the accelera- tion time set on the master control unit. Since no 2 wheels will decelerate simultaneously, timing of the light must be accomplished independently within each result unit. Thus, a signal representing acceleration time must be continuously available to each result unit. Providing this signal is the function of the acceleration time control circuit. A synchronizing pulse generator develops pulses at a rate of 2 per second. These pulses are derived from the 60-Hz power line and serve as a timing reference for the entire system. Pulses from the synchronizing pulse generator are counted by the timing unit composed of 4 silicon controlled-rectifier (SCR) ring counters. The timing unit generates intervals of .5 sec—60 min for accelera- tion time, work time, rest time, and time between bouts. Five distinct modes of operation are recognized in the master control unit: work, shock, rest, time between bouts, and program complete. The mode logic unit selects the appropriate mode in the proper sequence at intervals determined by the timing unit. The repetition counter consists of 3 silicon controlled- rectifier ring counters which count work periods to a number preset on the repetitions switches. The bout counter consists of 1 SCR ring counter which counts bouts to a number preset on the bouts switch. An output is provided on the rear of the master control unit for connection to an external alarm speaker. A second output 95 permits interconnection of a master control unit for synchronized oepration of as many as 24 wheels. Light from a small bulb passes through one side of the plastic wheel and illuminates 2 photocells. Light to one cell is inter- rupted by a black stripe on the wheel during half of each revolution. The output from this cell is used in counting revolutions. Light to the other cell is interrupted 8 times per linear foot of running surface (32 times per revolution). The output of this cell repre- sents wheel speed. . . . The tachometer section of the result unit monitors the output of the second photocell (wheel speed) and compares it with the speed set on the master control unit. The tachometer output is a DC signal: 0 volts if set speed exceeds wheel speed and -12 volts if wheel speed exceeds set speed. The logic section determines whether light, shock, or neither shall be applied. Assuming the master control unit is in either the work or shock modes, the logic section will turn on the light whenever wheel speed falls below set speed. Whenever the light comes on, the acceleration time generator begins timing the acceleration period according to instructions from the acceleration time control circuit in the master control unit. If wheel speed reaches set speed during the acceleration period, the logic section turns the light off. Otherwise, at the end of the acceleration period, the logic section switches the light off and the shock on. As soon as wheel speed reaches set speed, the logic section switches the shock off. 96 An alarm circuit in the result unit is activated whenever shock current is applied continuously for more than 3 sec. A buzzer in the master control unit signals that 1 or more animals is in diffi- culty. A red light on each result unit directs the operator to the appropriate animal. The usual cause of difficulty is fecal matter short-circuiting the bars of the wheel so that little or no current flows through the animal. In rare cases, an animal may be sick or injured and require attention. The alarm also is activated by failure of the photocell driver bulb. In this case, light and shock are inhibited. If this were not done, the animal would be unable to avoid shock by running. The alarm reset switch serves a dual purpose. In the center position, the result unit functions normally. Pressing the switch down resets the alarm circuit. In its upper position, the switch prevents application of either light or shock stimulus. In this mode, voluntary exercise may be observed since the wheel is free- running during all work periods and the TRR is recorded. Shock stimulus arrives at the result unit as a voltage (0-600 volts AC). It was found in preliminary work with the CRW that shock must be applied as a controlled current rather than a controlled voltage. An animal's skin resistance varies greatly according to whether it is wet or dry, causing corresponding variations in shock current when controlled current assures uniform shock stimulus to all animals at all times. In the result unit, shock current is switched by the shock relay. When the relay is activated by the logic section, current 97 passes through the relay contacts, a neon light, and the animal. The neon light indicates that current actually is passing through the animal (95).