ABSTRACT EFFECTS OF LESIONS IN THE POSTERIOR HEDIAL HYPOTHALAHUS 0N SALINE SOLUTION PREFERENCE AND BODY HATER REGULATION 3:! John Hilliam Wright The maintenance of extracellular fluid volume and tonicity is primarily the responsibility of the antidiuretic hormone and aldo- sterone systems. which have aided the transition of animals from an aquatic to a terristrial habitat. The more sodium retained in the extracellular fluid the greater is the volume of this fluid space. Therefore the phenomenon of an exaggerated sodium appetite. i.e.. saline solution consumption in excess of that needed for normal body maintenance is a paradox.not readily explained. The present series of experiments utilised albino rats housed in unit metabolism cages which allowed for a rather intense monitoring of food and water consuaption. urine volume output and urinary const- ituents. Periodic blood sampling was also employed. The first of five experiments was of a methodological nature and established a recommended delay tine of ten.days between blood samples or a blood sample and surgery. performed on the same aniaal. This suggestion is primarily based on the observation that hematocrit and serum sodium values were depressed following blood loss and did not recover to the levels of the initial sample until post-sample days seven and nine. respectively. A second blood sample prior to this delay duration may. therefore, result inra misinterpretation of the effects of an experimental manipulation. John Hilliam Hright Bilateral electrolytic or radio frequency lesions of the posterior aedial hypothalamus were considered in the remaining four experiaents and were shown to result in a substantial increase in isotonic saline solution intake that persisted throughout the several post-operative observational weeks in both normal and previously adrenalectoaised animals. Considering the intake and excretion of total sodium it was established that a balance existed.i.e.. there was an increased output of total sodium but only in response to the heightened sodium intake. The isotonic saline polydipsia did not appear to be due to an inability of the kidney to conserve water for the administration of exogenous pdtressin tannate resulted in a decreased fluid intake and increased urinary specific gravity and electrolyte concentrations (sodium and potassium were measured). Hater'deprivation differentially affected the experiaental and shaa lesioned groups. With the initiation of water’deprivation all animals evidenced slight and inconsistent decreases in food intake and urine volume output. There were urinary constituent differences between the control and experimental groups. In general the experi- mentally lesioned animals revealed an inhibited ability to concentrate their urinary electrolytes to a degree equivelant with their corresponding control groups. Urinary specific gravity also reflected this deficit. It is not known whether these differences were directly due to the experimental brain lesions or possibly reflected kidney function alterations due to the polyuria accompanying the heightened isotonic saline solution intake. an indirect effect of the lesions. John um.- Bright These data are interpretted to support a neural aodel proposed for the control of body water regulatory processes. Experimentally induced dange to medially located brain structures lave been noted in the literature to often result in increased fluid intake while lateral brain structure damage has often led to an adipsic ani/or aphagic coalition. Bilateral posterior medial hypothalaaic lesions represent daaage near the brain midline ani isotonic saline polydipia results. Bilateral lesions in what the author has chosen to call the posterior lateral hypothalams. the area Just lateral to the region ablated in this investigation. have yielded adipsia and aphagia. There are neural pathways that link the medial structures and comparable neural systems that connect the lateral brain structures. Fiber tracts appear to arise from the ventroaedial. anteriomedial an! posteriomedial hypothalaaic nuclei and contribute to the periventricular system at least a portion of which flows into the stria terminalis which courses around the anterior commissure terminating primarily in the central nucleus am medial portion of the basal nucleus of the mgiala. There is also a branching from the stria terminalis in the fore of the stria aedularis which establishes connections with the medial habenular nucleus. On the other hard, fibers originating free the posterior and anterior lateral hypothalaaus pass through the aedial forebrain bundle foraing the ventral anygdalofugal pathway which appears to terminate in and about the lateral aaygdaloid nucleus. Reciprocating fibers have been identified between the posterior lateral hypothalaaus an! the lateral septal nucleus and between the lateral septal nucleus aid the lateral habenular nucleus . EFFECTS OF LESIOIB IN THE KBTERIOR HEDIAL HYPOTHALAHUS ON SALINE SOLUTION PREFERENCE AND BODY HATER REGULATION By John William Wright ATHESIS Submitted to Michigan State University in partial fulfill-cut of the requirements for the degree of DOCTOR OP PHIWOPNY Department of Psychology 1971 PLEASE NOTE: Some Pages have indistinct print. Filmed as received. UNIVERSITY MICROFILMS DEDICATION To my Family. Donna. Tin and Shanna Ann For they alone realize the true cost that these pages represent In effort expended over the last several years. And they alone remember the Hellos and Good-byes Separated by wuch too little tine. ii ACKNOWLEDGMENTS A thesis chairman's guidance and assistance is appropriately acknowledged by his students at this point in the writing of a dissertation. however. in the case of Dr. L. I. O'Kelly the relationship was more than simply Professor and student. In spite of his extremely demanding duties as Chairman of the Department Dr. O'Kelly always had time for my problems. concerns and general welfare. His positive attitude and cheerfulness were certainly appreciated. I am going to sincerely attempt to develop future student-faculty relationships along the lines of those that I have enjoyed with Dr. O'Kelly. Dr. Glenn I. Hatton also deserves a very special thanks for he was always available and willing to listen and then supply very sound advice. His time expenditure on my behalf was considerable and will not be forgotten. Appreciation is also extended to Drs. J. I. Johnson and Robert Raisler for pertinent comments on this manuscript. Their influence and guidance as committee members was valuable. Thanks are also due Miss Dalene DeGraaf and Mrs. Emmy Height for technical assistance rendered. iii TABLE OF’ CONTENTS Ptse LISTOF‘TABLES...................... vi LISTOF'FIGURES..................... vii LISTOFAPPENDIX.................... xv INTRODUCTION....................... 1 EXERIHEM 1 O O O O O O O O O O O 0 I O O O O O O O O O O 18 Repeated Blood Samples Subjects . 18 Appr‘tus O O O O O O O O O O O O O O O O O O O O O 1 8 Roofing I O C O O O O O O O O O O O O O O O O O 0 1 9 Ros“ 1“ C O O O O O O O O O O O O O O O O O O O O O 20 D1. cuss 10 n O O O O C O O O O O O O O O O O O O O O C “5 EXERIHENT 2 O O O O O O O O O O O O O O O O O O O 0 O O O [‘7 Posterior Medial Hypothalamic lesions sabjeda O O O O O O O O O O O O O O O O O O O O O O “7 Nut. 0 C C O O O O O O O O O O O O O O O O O 0 % Raulta O O O O O O O O O O O O O O 0 O O O O O I O “9 AmtomicalFindings............... 70 Discussion . . . . . . . . . . . . . . . . . . . . . 72 EXERIHEM 3 O O I O O O O O O O O O O O O O O O 0 O O O O 7“ Replication Lesions Subjects . 7a Placed”. e e e e e e e e e e e e e e e e e e e e e 75 Results e e e e e e e e e e e e e e e e e e e e e e 75 Anton“). Filflim e e e e e e e e e e e e e e e 1 08 Dumaion e e e e e e e e e e e e e e e e e e e e e 109 mlmmuOOOOOOOOOOOO0000......O 113 Pitressin Influence upon the Experimental Lesion SUbJCCts e 1 1 3 medur. O O O O O O O O O O O O O O O O O O O C O 1 1 3 Results 0 O O O O O O O O O O O O O O O O O O O O O 1 1 5 anaaion O I O O O O I O O O O C O O O O O O O O 0 1“ iv EmeIHENTSeeeeeeeeeee The Effect of Hater Deprivation Subjects . . Procedure . Basal“ e 0 Discussion . General Discussion LIST 0? REFERENCES . APENDIX O O O O O O O O 1&6 1h? 1“? 147 170 172 181 190 Table 3. LIST OF TABLES M Volume and extent of lesion damage inflicted upon the experimental and control animals of ExperimentZ.................. 71 Mean serum sodium. potassium. protein levels and hematocrit values for pre- (A) and post-lesion (B) blood samples taken from the sham and experimentally lesioned groups of Experiment 3 . 106 Volume and extent of lesion damage inflicted upon the experimental animals of Experiment 3 . . 107 vi Figure 3. 5. 7. 9. 10. LIST 01" FIGURE Mean water consumption in 12 hour units for the intersample interval 1 day and 3 day groups during eight days prior to blood sampling and eleven days following the initial sample. . . . Mean water consumption in 12 hour units for the intersample 5 day and 7 day groups during eight days prior to blood sampling ani eleven days following the initial sample . . . . . . . Mean water consumption in 12 hour units for the intersample interval 9 day group during the eight days prior to blood sampling arr! eleven days following the initial sample. . . . Mean food consumption in 12 hour units for the intersample interval 1 day and 3 day groups before and during blood sampling . . . . Mean food consumption in 12 hour units for the intersample interval 5 day and 7 day groups before and during blood sampling . . . . Mean food consumption in 12 hour units for the intersample interval 9 day group before andduringbloodsampling. . . . . . . . . . . Mean urine volume output in 12 hour units for intersample interval 1 day and 3 day “on” before ‘m during 1310“ ”.pling e e e 0 Mean urine volume excretion in 12 hour units for the intersample interval 5 day and 7 day groups before and during blood sampling . . . . Mean urine volume excretion in 12 hour units for the intersample interval 9 day group before and during blood sampling. . . . . . . . Mean urine sodium concentration for the intersample interval 1 day and 3 day groups before and during blood sampling. . . . . . . . vii 23 23 25 25 28 28 30 33 33 Figure 11. 12. 13. 11+. 15. 16. 17. 18. 19. 20. Mean urine sodium concentration for the intersample interval 5 day and 7 day groups before and during blood sampling . . . . . . . Mean urine sodium concentration for the intersample interval 9 day groups before andduringbloodsampling. . . . . . . . . . . Mean urine specific gravity for the inter- sample interval 1 day and 3 day groups before and during blood sampling . . . . . . . Mean urine specific gravity for the inter- sample interval 5 day and 7 day groups before and during blood sampling . . . . . . . Mean urine specific gravity for the inter- sample interval 9 day groups before and duringbloodsaspling. . . . . . . . . . . . . The mean serum sodium concentrations for each 181 group on its first (a) and second (b) blood samples. The mean serum sodium concentration of the grouped first blood samples (a) is also provided. labelled MEAN . The mean serum potassium concentration for each 181 group on its first (a) and second (b) blood samples. The mean serum potassium concentration of the ground first blood samples (a) is also provided. labelled MEAN . The mean serum protein concentration for each 181 group on its first (a) and second (b) blood samples. The mean serum protein concentration of the grouped first blood samples (a) is also provided. labelled MEAN . The mean hematocrit for each 131 youp on its first (a) and second (b) blood samples. The mean hematocrit of the groupd first blood samples (a) is also provided. hull“ mu 0 O O I O O O O O O O O O O O 0 Mean tap water an! isotonic saline solution consumption exhibited by the control lesioned group for the last ten days of a twenty day pre-lesion period and twenty days post-op. . . viii 35 35 3? 37 b2 ’62 Figure 21. 22. 23. 2h. 25. 26. 27. 28. 29. 30. 31. 32. Mean tap water and isotonic saline solution consumption exhibited by the posterior medial hypothalamic radio frequency lesioned group pre- and post-operatively. . . . . . . . . . . Mean tap water and isotonic saline solution consumption exhibited by the posterior medial hypothalamic electrolytically lesioned group FTC- ‘nd FOBt-Oper‘tivelye e e e e e e e e e a Mean food intake in 2“ hour units for the three lesioned groups pre- and post- 0p‘r‘tivoly e e e e e e e e e e e e e e e e 0 Mean urine volume output in 2“ hour units for the three lesioned groups pre- and DOBt-Oporltivaly e e e e e e e e e e e e e e e Mean urine sodium concentration for the three lesioned groups pre- and post- operatively . . . . . . . . . . . . . . . . . Mean total urinary sodium excreted per 2“ hour units for the three lesioned groups ”0- It‘d ”at-opnt1'°1ye . o o o o e O o O 0 Mean urine potassium cone. for the three lesioned groups pre- and post- operatively . . Mean urine specific gravity for the three lesioned groups pre- and post-operatively . . Mean body weights exhibited by the three lesioned groups pre- and post-operatively . . Mean serum sodium and potassium concentration for the control lesioned group on one pro-op and two post-operative blood samples . . . . . Mean serum sodium and potassium concentrations for the posterior medial radio frequency lesioned group on one pro-op and two post- operativebloodsamples ........... Mean serum sodium and potassium concentrations for the posterior medial hypothalamic lesioned electrolytic group on one pro-op and two DOBt-Oporttlve bIOOd BCIPIOB e e e e e e e e e ix 5? 57 63 63 65 65 67 35. 36. 37. 38. 39. “1. #2. #3. Mean serum protein and osmolality for the control lesioned group on one pro-op and two post-operative blood samples . . . . . . Mean serum protein and osmolality for the posterior medial hypothalamic radio frequency lesioned group on one pro-op and two post- operativebloodsamples ........... Mean serum protein arrl osmolality for the posterior medial hypothalamic electrolytically lesioned group on one pro-op and two post- operative blood samples . . . . . . . . . . Mean body weights for posterior medial hypothalamic and sham lesioned groups maintained on tap water. pre- ard post- Opntivolyeeeeeeeeeeeeeeee Mean body weights for posterior medial hypothalamic and sham lesioned youps maintained on tap water and isotonic saline solution pre- and post-operatively . . . . . Mean body weights for the adrenalectomised posterior medial and sham lesioned groups maintained on isotonic and 2.00% saline solutions. pre- and post-operatively . . . . Mean fluid intake corrected for body weight differences by the groups maintained on tap water pre- and post-operatively . . . . . . Mean fluid consumption corrected for body weight differences by the groups maintained on tap water and isotonic saline solution pr.- ‘11,. post-opetlthIOIY. e e e e e e e e 0 Mean fluid intakes corrected for body weight differences of the adrenalectomised groups maintained on isotonic and 2.00% saline solutions pre- and post-operatively . . . . Mean food intake corrected for body weight differences of the groups maintained on tap water pre- and post-operatively. . . . . Mean food intakes corrected for body weight differences of the groups maintained on tap water and isotonic saline solutions. pre- andpost-cperatively. . . . . . . . . . . . X 67 77 79 82 82 85 85 #5. 47. “9. 50. 51. 53. Mean food intake corrected for body weight differences of the adrenalectomised groups maintained on isotonic ani 2.00% saline solutions. pre- and post-operatively . . . . . Mean urine volume output corrected for differences in body weight for the groups maintained on tap water pre- ani post- operativoly . . . . . . . . . . . . . . . . . Mean urine volume output corrected for body weight differences for the groups maintained on tap water and isotonic saline solutions. prs- I’m Mt-Opntively e e e e e e e e e e Mean urine volume output corrected for body weight differences for the adrenalectomised groups maintained onLisotonic and 2.00% saline solutions. pre- and post-operatively . Mean urine sodium concentrations for the poups maintained on tap water pre- and mat‘opntivelyeeeeeeeeeeeeeee Mean urine sodium concentrations for the groups maintained on tap water and isotonic saline solution. pre- arrl post-operatively . . Mean urinary sodium concentrations of the adrenalectomised groups maintained on isotonic and 2.00% saline solutions pre- tndPOSt-Opentidy............. Mean total urinary sodium excreted in 21+ hour units by the groups: maintained on tap , water pre- and post-operatively . . . . . . . Mean total urinary sodium excreted in 24 hour urits by the groups maintained on tap water and isotonic saline solution pre- ‘mmt’opnt1VQIYQeeeeeeeeeeee Mean total urinary sodium excreted in 24 hour units by the adrenalectomised groups maintained on isotonic and 2.00% saline solutions pre- and post-operatively . . . . . . Mean urinary potassium concentration of the groups maintained on tap water pre- an! ”tropntivolyeeeeeeeeeeeeeee xi 87 87 92 95 95 100 55. 56. 57. 59. 65. 66. Mean urinary potassium concentrations of the groups maintained on tap water and isotonic saline solution pre- an! post- opsntivsly ................ Mean urinary potassium concentration of the adrenalectomised groups maintained on isotonic and 2.00% saline solutions pre- sndpost-operatively.. . . .. .... .. Mean urinary specific gravity for the groups maintained on tap water pre- and post- operativsly . . . . . . . . . . . . . . . . Mean urinary specific gravity for the groups maintained on tap water and isotonic saline solution pre- and post-operatively . . . . . Mean urinary specific gravity for the adrenalectomised groups maintained on isotonic and 2.00% saline solutions pre- ‘mmt“°”nt1VOIYOeee e eeee eee Mean water consumption in It hour units for the groups maintained on tap water. pre- ”ripest-injection. eee eee e e eeee Mean food consumption in 1+ hour units for the groups maintained on tap water. pre- and Wt-inJOCtioneeeeeeeeeeeesee Mean urine volume excretion in 1+ hour units for the groups maintained on tap water. pre- and post-injection . . . . . . . . . . . . . Mean urinary sodium concentration in h hour units for the groups maintained on tap water m- ‘M pat-1nJ.Ct1°n . o g Q o Q Q Q g 0 Mean urinary potassium concentration in 15 hour units for the groups maintained on tap water. pre- and post-injection . . . . . . . . . . Mean urinary specific gravity in it hour units for the groups maintained on tap water. pre- .mmt'injectioneeeeeeeeeeeee Mean water consumption in lo hour units for the groups provided tap water. no saline. m- ‘1“ wt-inJCCtion e e e e e e e e e e xii 100 102 102 1% 101+ 11? 117 120 120 123 123 126 Figure 67. 69. 70. 71. 73. 7“. 75. 76. 78. 79. Mean food intake in a hour units for the groups maintained on tap water. no saline. pre- and post-injection . . . . . . . . . . . . Mean urine volume output in h hour units for the groups maintained on tap water. no saline. pre- and post-injection . . . . . . . . Mean urinary sodium concentration in h hour units for the groups maintained on tap water no saline. pre- and post-injection . . . . . . . Mean urinary potassium concentration in h hour units for the groups maintained on tap water. no saline. pre- and post-injection . . . Mean urinary specific gravity in # hour units for the groups maintained on tap water. no saline. pre- and post-injection . . . . . . . . Mean isotonic saline consumption in a hour units for the adrenalectomised groups maintained on 0.87% saline solution. pre- ‘mwst-inJOCtioneeeeeeeeeeeeeee Mean food intake in h hour units for the adrenalectomised groups maintained on 0.87% saline solution pre- and post-injection . . . . Mean urine volume excretion in a hour units for the adrenalectomised groups maintained on 0.87% saline solution. pre- and post- injoction O O O O O O O O O O O O O O O O O 0 0 Mean urinary sodium concentration in 0 hour units for the adrenalectomised groups maintained on 0.87% saline solution. pre- ‘mmt‘inj¢ct1°neeeeeeeeeeeeeee Mean urinary potassium concentration in 4 hour units for the adrenalectomised groups maintained on 0.87% saline solution. pre- ‘M mt-1njoct1°n O O O O O O O O O O O O O O 0 Mean urinary specific gravity in a hour units for the adrenalectomized groups provided 0.87% saline solution. pre- and post-injection . . . . Mean food consumption in a hour units for the groups maintained on tap water. pre- and duringdem'ivation............... Mean food consumption in # hour units for the groups provided tap water. no saline. pre- ‘Mdunnsdaw1V‘t10n e e e e e e e e e e e e e xiii 126 129 129 132 132 135 135 139 139 1u2 1u2 150 150 80. Mean food consumption in A» hour units for the adremlectomised groups maintained on 0.87% saline solution. pre- and during deprivation . . 152 81. Mean urine volume output in 4 hour units for the groups provided tap. pre- and during dep . . 152 82. Mean urine volume output in 1+ hour units for the groups maintained on tap water. no saline. pre- and during deprivation . . . . . . . . . . 156 83. Mean urine volume output in it hour units for the adrenalectomised groups maintained on 0.87% saline solution. pre- and during deprivation . . 156 81+. Mean urinary sodium cone. in 1+ hour units for the groups maintained on tap pre- and during dep 158 85. Mean urinary sodium concentration in ‘5 hour units for the groups maintained on tap water no saline pre- ani during deprivation . . . . . 158 86. Mean urinary sodium concentration in ‘0 hour units for the adrenalectomised groups provided 0.87% saline solution. pre- and during dep. . 161 87. Mean urinary potassium concentration in a hour units for the groups maintained on tap water pre- and during deprivation . . . . . . . . . . 161 88. Mean urinary potassium concentration in M» hour units for the groups maintained on tap water. no saline. pre- and during dep. . . . . . 161+ 89. Mean urinary potassium concentration in 1+ hour units for the sdrenalectomised groups provided 0.87% saline solution. pre- and during dep. . . 16“ 90. Mean urinary specific gravity in 1+ hour units for the tap water groups. pre- and during dep . 167 91. Mean urimry specific gravity in 0 hour units for the tap water. no saline. groups pre- and during dOMV‘tion. e e e e e e e e e' e e e e e 167 92. Mean urinary specific gravity in ‘1 hour units for the adrenalectomised groups maintained on 0.87% saline solution. pre- and during deprivation . 169 LIST OF APPENDIX Page Photomicrographs for Experiment 2 Animals . . . . . . . . . 190 Photomicrographs for Experiment 3 Animals . . . . . . . . . 199 Total sodium intake-output figures for four animals fromExperiaentB..................... 205 INTRODUCTTON The existence of mechanisms for maintaining body fluid constancy has been appreciated for many years (hters. 1935. Starling. 1896). The development of such homeostasis has depended upon two major mechanisms aiding in the transition of animals from an aquatic to a terrestrial habitat. Both of these systems are primarily concerned with the extracellular fluid (ECF) volume. for water is thought to freely diffuse across the majority of cell membranes equating extra- cellular and intracellular concentrations.1 Antidiuretic Hormone The first fluid regulation mechanism. concerned with body water conservation. involves the antidiuretic hormone (ADR) which is sensitive to changes in the effective osmolality of the extracellular fluid (Sims and Solomon. 1963). It was Verney (1947) who first pointed out the relationship between the ADM effect and possible osmoreceptors in the forebrain. Verney infused equal osmolsrity hypertonic solutions of heel. glucose and urea into the carotid artery of dogs during the apex of'water diuresis. The order of effectiveness of these solutions in reducing the diuresis was sodium. glucose then urea. Since this order is the reverse of their ability to penetrate cell membranes from the extracellular fluid he proposed that osmotic removal of water from ”osmoreceptor“ cells located in the supraoptic nucleus of the hypoth- alasus initiated neural impulses from that nucleus for the release of 1There is some debate on this issue with the suggestion that intracellular concentration may be slightly hypertonic to that of the extracellular fluid (Conway and Geoghehan. 1955: Robinson. 1950). 1 2 ADM from the posterior pituitary. Thus Verney envisioned a negative feedback system. i.e. . a rise in blood osmotic pressure results in cellular dehydration causing an increased release of ADM which in turn facilitates water reabsorption by the kidney. This added reabsorption of water. together with excretion of ions decreases plasma osmotic pressure arxi ADM output decreases. Histological evidence (Jewell. 1953) coupled with the results of intracranial vascular ligations of the cerebral vascular bed (Jewell ani Verney. 1957) and recording studies um. revealed ”osmopotentials" (changes in firing rate in preoptic ani supraoptic areas resulting from hypertonic saline injections (Brooks. Ushiyama and lange. 1962; Cross ani Green. 1959: Nakayama. 19553 Sawyer arrl Gernandt. 1956; Von Euler. 1953) have identified the anterior region of the hypothalamus with ADM secretion. Antidiuretic hormone is synthesized in the supraoptic nucleus. It then migrates down the supraoptic-hypophyseal tract to the prs nervosa of the pituitary glam! where the ADM is stored until release is signalled (Leveque and Sharrer. 1953; Sawyer ani Mills. 1966). Cort (1963b) has suggested that ADM may be secreted directly from the hypothalamus. however this has not been substantiated (Raisman. i966). Hith the advent of new ADM analysis techniques (Gilmore and Vane. 19703 Meller arrl Stule. 1959) this possibility may be explored further. The method by which ADM acts upon the kidney nelsiron is not clear. It may be that the characteristics of the vasopressin chemical bond alters cell membrane water permeability (KoepoedeJohnsen and Ussing. 1954) thus allowing more water to pass through the membrane. Mater reabsorption is also aided by the osmotic gradient created by the ”counter cment multiplier system” in the ppilla of the loop of Menle (lira. 1956. Gottschalk. 1960). ADM does not seem to act on the proximal tubule or upon the descerxling or ascending loop of Meals. The action is specific to the distal tubule and collecting duct (Pitts. 1968). Under normal conditions the adequate stimulus for ADM release is hyperosmolarity. or cortical influence upon the hypothalamus resulting from fear or pain (Moran and Zimmermann. 1967). A decrease in ADM secretion accompanies mechanical stretch of the left atrium an! stretch of carotid sinus receptors (Pitts. 1968). The control of water excretion by ADM in addition to regulating osmotic pressure of the ECF. has some affect on the volume of the ECF. Infmion of isotonic saline causes changes in EC? volume which results in a water diuresis (Strauss et al.. 1951) suggesting that ADM may be controlled by EC? volume changes alone (Cart. 1955: Leaf "I! Hit-by. 1952). Aldosterone Primary input into the secorrl fluid regulation mechanism. the aldosterone system. comes from stretch receptors in the carotid sinus. right atrium and juxtaglomerular apparatus. Hith a decrease in blood volume the afferent glomerulus becomes passively constricted art! there is a reduction in EC? volume. An enzyme. renin. released from the kidney into the blood. acts on the glycoprotein of the plasma resulting in the reduction of a decapeptide to angiotensin I which is converted via a plasma ensyme to an octapeptide. angiotensin II. Angiotemin II functions as 1) a vasoconstrictor within the blood and 2) acts on its target organ. the cortex of the adrenal gland. stimulating secretion of aldosterone into the blood resulting in a greater reabsorption of sodium by the nephron. This appears to be accomplished by a change in the protein synthesis. i.e.. aldosterone stimulates sodium transport by influencing a nuclear receptor to increase the DNA directed synthesis of RNA wich codes a particular protein. This protein may function in one of three ways: 1) to control entry of sodium into the mucosal epithelium from the urine side: 2) to act as the ion carrier of the sodium pump; or 3) to regulate the production of ATP enzymatically. and thus control the energy supply on which transport depends (Crabbi arr! DeHeer. 1961M Edleman et al.. 1963; Fimognari et al.. 1967: Forte and Landon. 1968: Porter et al.. 196a; Sharp‘and Leaf. 1966). Because protein synthesis has a minimum time lag of one or more hours after the introduction of aldosterone. changes in sodium reabsorption of the kidney are delayed for that time period. If the reabsorption of sodium is considered along the nephron. the proximal tubule accounts for the reabsorption of some 75% of the sodium. Since approximately 99% of the body sodium is reabsorbed. the descending limb of Menle accounts for some of the remaining 24%»by accepting sodium from the ascending limb via the ”counter current multiplier mechanism” (Gottschalk. 1960). Reabsorption by the distal tubule and collecting duct. as governed by aldosterone level. accounts for the remaining sodium reabsorption. and obviously. the more sodium retained in the ECF the greater is ECF volume (Pitts. 1968). Sodium Appetite Because animals are capable of closely monitoring their body fluid concentrations in a homeostatic manner. the phenomenon of exaggerated sodium appetite (saline consumption in excess of that needed for normal body maintenance) is a paradox not readily explained. Rats offered a choice between hypotenic saline solution ani water evidence a heightened ingestion of the saline solution (Bare. 1918: Nelson. 191073 O'Kelly. 195M Stellar arrl McCleary. 1952). Young arr! Chaplin (1989) simultaneously offered normal rats eight concentrations of saline: 0.1. 0.2. 0A. 0.7. 1.5. 3.0. 6.0 ani 12.0 per cent. These animals drank more from the 0.7% than from all other solutions. net-e (191.9) foam! that as he increased the sodium chloride concentration. fluid consumption increased while tap water intake slightly decreased. Maximum solution intake occurred with a 0.9% saline solution ami fell off with concentrations above this level. At saline concentrations above 2.5% consumption was almost entirely of tap water. The £13233 M has also been employed in such preference tests. typically assuming the fen of the presentation of one test fluid for one hour. The animals are under a water deprivation schedule until fluid presentation. 81th this approach leiner and Stellar (1951) placed maximum saline solution consumption at a concentration of 0.8%. Stellar. Myman and Samet (1951;) also indicated maximum preference at between 0.7 and 0.8% using similar intake methods. In general investigations utilising two drinking tubes have established a preference for salt solutions in the normal rat beginning at concentrations of 0.05 to 0.06 per cent. Maximum differential consumption appears to take place with concentrations of 0.7 to 0.9 per cent. At a saline solution of appoximately 1.5% the normal rat drinks equal quantities of tap water ad the solution. Young end Falk (1956) and Falk ard Titlebaum (1963) have criticised earlier saline preference work for the implicit assumption that "the more an animal drinks of a fluid the better he likes it”. They have argued that a brief-exposure preference test relieves the experimenter from subscribing to this definition of preference. Under these conditions an animal is released fr0m a start box and is allowed to approach the two test fluids (either distilled water and a saline solution or two saline solutions; the saline solutions tested in the Young all! Falk study were 0.38. 0.75. 1.5. 3.0 and 6.0%). Once substantial contact was ads with one of the two fluids the cup containg the fluids were withdrawn out of reach. Urder these experimental conditions the preferred saline concentration of non-thirsty rats was between 0.75 ani 1.5 per cent. Thirsty rats often chose water over the saline solution and took the weaker of two salt solutions. Isotonic saline preference may be relatively indepenient of bodily sodium need in that the preference is not significantly affected by parenteral saline loading or by changed sodius food content (Freglymt al.. 1965). Also rats given prolonged free choice between water air! isotonic saline ingest saline to such excess that they may reveal signs of chronic sodium overloading (Nelson. 1907). This preference exhibited by the normal rat for saline solutions is of some interest for such solutions would appear to be less effective in reducing thirst than pure water. Hook (1963) he argued for the relative ineffectiveness of such solutions in relieving thirst on two bases: 1) ”isotonic saline solutions are absorbed less rapidly than water from the rat intestine (o'xeny. Falk end Flint. 1958)” 2)"sodium chloride is confined wimarily to the extracellular space after absorption (Gamble. 199+)“. Hook suggests that osmotic pressure equilibration holds the solutes of the ECF‘ at isotonicity thus ingestion of an isotonic solution will only add to the Bar volume with no water gain by the cells. A number of seemingly adequate theories have been postulated in an effort to explain this apparent discrepancy bottleen intake an! need. The Diluted-Hater hypothesis by Deutsch ard Jones (19 59; 1960) argues that the Central lervous System has receptors semitive to the taste of water. Saline solution. to some degree. mask the “water signal” resulting in the imbibition of more saline before a given level of satisfaction is reached. They cite neurophysiological work by Zotterman (1956) and Zotterman and Dissent (1959) as support for their hypothesis. These investigators found that nerve fibers carrying water and salt signals fire at high spontaneous rates art! are markedly inhibited when water is placed on the tongue. but inhibited to a lesser extent when hypotonic saline is applied. Thus hypotonic saline acts as a ”diluted“ water yielding a weaker water signal and consumption of a greater volume to reach satiation. Deutsch (1953) has reported that a choice between equal quantities of water and saline in a '1‘ mass situation should result in the selection of water which has a water water signal. His results confirm this prediction. These basic findings utilising a T man have been replicated by Chiang an‘l Wilson (1963) and by Brookshire (196%) who also found tint water reward resulted in faster running by water deprived rats than did a saline solution reward. However. if rats were raised from weaning on hypotonic saline they preferred the saline rather than water in the T maze situation (Brookshire. 1967b). Fisher (1965) held rats on a slight water deprivation schedule an reported that his animals would lick from a spout containing tap water in order to obtain access to a seconi tube containing 0.58% saline. In Iciner ani Deutsch's 1967 paper they criticised the work reported by Fisher (1965) and hit and Titlebaum (1963) in that animals used in these experiments may have been predisposed to saline solution due to imdvertent salt deficiency. Recently Myer and Hemmel (1969) confirmed the dilute water hypothesis in a lever pressing experiment. Rats kept at 80% body weight were placed on a 1-min. variable interval reinforcement schedule for one hour per day. A 0.025 ml. water or saline solution (0.10-2.01!) was offered as reinfu-cement. With higher concentrations of saline. response rates decreased; at no time did a saline solution prove more reinforcing than water. A second explanation emphasises gastrointestinal diultion of hypotonic saline rather than oral receptor influences in explaining saline solution preference. hook (1963) prepared each of his nts with both an esophageal fistula and a gastric fistula. In this way he could allow the animal to drink a particular fluid (water or saline) which flowed out the esophageal fistula and introduce the same quantity of that. or adifferent fluid into the stomich. Utilising this prepration Hook fouui that when isotonic saline reaches the stomach. water intake is elevated above normal ingestion levels. It would appear that isotonic saline is less effective in hydrating the animal when comps-ed with water and as a result the animal must drink more of the isotonic saline solution. It is interesting to note tint if the conequences of drinking water or of drinking isotonic saline are the same in terms of what is placed into the stoneh. the voluntary intake of these two fluids is nearly equal. Uith regard to this pestingestional osmotic effect ani mouth receptors Hook states: "This osmotic mectunism must influence intake in interaction with mouth factors: for when hypertonic saline enters the stench water drinking is enhanced ani the drinking of concentrated solution is depressed. even though the postingestioml events are precisely the same in both cases“. flatten (1965) placed rats on a 23} hour water depivation schedule ani once accustomed to this he stomach loaded the animals with tap water 2% Bl. tap water 4% Hal. 2%. 4% or 6% saline to 2% Bi. he then measured urine output for a 3 hour period following such loading. The curve of urine output beginning with a sham stomach load group and progressing through the saline loads. i.e.. 2%. 10% and 6% fuel. was negatively accelerated with a greater urine output as saline solution concentration increased. Upon reaching criterion performance (10 min. without a response) for two extinction periods under a lever pressing design for a water reward. flatten allowed animals water for a one hour period. The animals showed differential intakes of water that were depemient on the type of load earlier received. a W H tap water load group comummed a mean of 10 cc. 2% Bi tap water load group 16 cc. sham load group 21 cc. 2% ram load group 20 cc. 4% NaCl group 23 cc and the 6% heel group approximately 31 cc. If these intakes are accepted as a measure of the degree of dehydration suffered by the animals it appears that in general as the concentration of saline solution increases water intake ani therefore level of dehydration also increases. A third explanation takes a more general fern aid has been referred to as the "Sodium Reservoir“ hypothesis by Stricker airl lolf (i967. weir end Stricker. 1967). Sodium appetite is thought to be 10 elicited by hyponatremia. hypevolemia or increases in mineralcorticoid production. Bone sodium is the only reservoir that Stricker ani wolf have thus far identified (Bergstrom. 1955). The hypothesised reservoir is thought to release sodium as a respolue to hypevolemia. hyponatremia or increased mineralooroticeid levels. Sodium appetite thus appears in an animal when sodium loss from a reservoir reaches some threshold value (Stricker aui wolf. 1969). Stricker ani his colleagues have concerned themselves with manipulating this sodium reservoir by the use of several techniques. The injection of 5 ml of 10% polyethylene glycol (subcutaneous) results in a decreased blood volume with an unaltered senim sodium level approximately 8 hours later. About the same length of tine following the injection of 2.5 ml formalin both serum sodium and blood volume are decreased. And following a 5% body weight stomach load of distilled water blood volume is uncl'Ianged but the serum sodium level drape (Stricker and Self. 1966). In a recent study (Jalowiee and Stricker. 1970) rate were injected with formalin and then offered either water. 0.07M. 0.15! or 0.511! saline solutions to drink. Body fluid balance was restored most quickly by those animals given isotonic saline and less quickly by hypotonic and hypertonic solutions even though the solutions were ingested at comparable rates. Animals given subcutaneous injections of polyethylene glycol lose isosmotic intravascular fluid (Stricker. 1966). The animal's msponse to such treatment is similar to that following formalin injection. Fluid consumption. increases and a preference for hypotonic saline over water occurs with some ingestion of hypertonic saline 11 solutions (Stricker. 1966: Smith and Stricker. 1969). This hypothesis suggests that the ultiute responsibility for a sodium appetite lies with the sodium loss sufferred by some body reservoir which then affects body function (welt end Stricker. 1967: Stricker and Half. 1969). Nevin (1962) developed a method of measuring brain electrical conductivity in rats. With this technique he has measimed the impedance of the brain of thirsty rats (22 hour water deprivation schedule) given one of the following saline solutions: 0.0. 0.1. 0.2. 0.1!. 0.8 per cent (Nevin,et al.. 1966). It was found that measurable changes in conduct- ivity as a result of fluid intake occur within five minutes after the initiation of drinking. In Kevin's words. “As the concentration of odine decreases. the change in corductivity increases”. A saline solution of 0.8% results in very little impedance change. If it is accepted that changes in brain impedance prallel deviations in systemic electrolyte concentrations or effective osmolarity then Nevin would suggest that if effective osmotic pressure must reach a threshold change for the termination of thirst to occur the realisation of this threshold would require correspondingly larger volumes of solution ingested as the salinity concentration increased. The brain lesion technique has thus far offered limited insight into the identification of the underlying neural components of this saline preference in rats. Bilateral damage inflicted upon the ventro- medial hypothalamus (V10!) of the rat results in a substantial increase in a 1% sodium chloride solution intake (Xawamura et al.. 1970). Bilateral lesions just below the V101 which appear to damage the arcuate nucleus yield a heightened 2% saline intake (Covian and Antunes-Redrigues. 1963). Bilateral electrolytic lesions destroying the van. dersomedial 12‘ hypothalamus (Duh) or fornix are followed by a diabetes insipidio synirome with an unaltered 2% NaCl ingestion pattern (Antunes-Redrigues end Covian. 1965). A more recent piper (wolf end Quarternin. 1967) describes anterior lateral hypothalamic (LEA) lesioned animals that were maintained through adipsic air! aphagia by intragastric feeding until normal food aid water ingestion was again self-initiated. Such animals. once recovered. exhibited a preference for weak saline ami an aversion for hypertonic saline undistinguishable from a normal animal's preference. when adrenalectomised. however. these lesioned animals failed to satisfactorily regulate their saline intake and fell into a sodium deficiency. Holf (1967) has shown that normal regulation of saline alri water ingestion continues after the elimination of the lateral septal. lateral preeptic or ventral tegmental regions. Lesions in the thalamic gustatory nucleus an! in the reticular formation just cauial to the above nucleus decrease intake of a 0.5! (2.9%) saline solution instigated by a 2.5 ml injection of a sodium depleting 1.5% formalin solution (Rolf an! Steinbaum. 1965; Wolf. 1968). Large septal lesions involving the anterior ani posterior areas and exterrling from the medial to the lateral portions caused substantial increases in 1. 5% saline intake (Vilar et al.. 1967) arr! 0.87% saline intake (Donovick et al.. 1969) unier a self selection regime involving saline solution and water. Support for a neural circuit linking hypothalamus. septal ani amygialeid areas to bodily sodium balance is enhanced by the discovery that amygdaloid lesions result in altered saline intakes by rats. Bilateral dange to the corticosedial complex of the amygdala was 13 followed by increased 1. 5% NaCl intake: similar damage to the lateral nicleus brought decreases in 1. 5% saline intake (Gentil et al.. 1968). The posterior region of the hypothalamus has also been suggested as an.area involved in the regulation of saline ingestion. Gert (1963a) has claimed that this area is the site of production of a neuroeisiocrine hormone. a ”Substance X'. that serves as an anti-natriuretic agent at the kidney level. Bilateral.destruction of the posterior hypothalamic area appears to reflect an increase in 0.9% ilaCl intake with an accomp- anying urinary sodium loss of substantial magnitude. Electrolytic lesions of the posterior hypothalamus of rats and cats have been immediately (30 minute latency) followed by salt less in the urine (Cort and Keeler. 1951:; Cort end Lichesdue. 1963d: Cort. 1955: Lichardus end Jonec. 1961). There is no evidence of adrenal damage and storied therapy does not restore salt balance (Lichardue et al.. 1965) but availability of saline solutions (0.9 and 3.0%) for drinking. restores salt balance within about five days in rats (Cort. 1963a). Bats given a 2% saline solution to drink for ten days revealed significantly decreased cell nucleus also in the posterior hypothalamic nucleus as compared with the ventromedial. dersomedial an! arcuate nuclei. The supraoptic‘and paraventricular nuclei increased nucleus sise (Liehardus. Mitre and Cort. 1965). Cort's present position may be summarised as follows: In that salt loading results in decreased cell nucleus sise in the posterior hypothalamic nucleus. a neuroendocrine function may exist at this locus. The carotid occlusion work (Cort and Lichardus. 1963a: 1963c: Gert. 1965. p. 1105) suggests that during natriuretic "volume“ reflexes a natriuretic 1h substance is released frem the dienoepralon. This sub-tense is probebly not a steroid or a catecholamine. The rapidity of natriuretic onset and termination given the carotid occlusion stimulus indicates a peptide. Vasopresein arr! angietensin have been eliminated (Cort et al.. 1965: Cort aul Lichardus. 1963b). The substance may have a chemical structure similar to oxytocin. A question of major significance concerning Cort's work may be approached as follows: A natriuretic hormonal agent is secreted from a brain site during thoracic vascular bed expnsicn an! is not present. i.e.. a blood sample does not cause natriuresis in a recipient cat. after bilateral posterior hypothalamic lesioning. Bilateral posterior hypothalamic lesioning results in natriuresis an! diuresis in rats. cats. dogs an! human with sudden onset ani an extreme neptive medium balance. Hy question: How is it that bilateral posterior hypothalamic lesioning during carotid occlusion prevents mtriuresis ani diuresis ani in a normal animal causes acute natriuresis ani diuresis? It must be pointed out that the most recent of a series of attempts to replicate evidence of a blood-born natriuretic factor has offered an alternative explanation to this phenomenon. In cross-circulation experiments utilising rats. Bonjour ani Peters (1970) expanded the extracellular space of a donor animal by the slow infusion of isotonic saline (0.02 ml/min.). The donor animals demonstrated diuresis and natriuresis due to hemodilution (plasma potein dilution) ani/or expnmion of the extracellular space. The recipient animals which experiemced hemodilution via cross circul- ation but no expansion of the extracellular space. did not evidence any diuresis or natriuresis. Prevention of expmion of the extracellular space in the recipient animal was accomplished by placing the animal 15 on a sensitive balance (sensitivity 20 mg.) and adjusting the rate of blood flow in drops per minute so that the recipient animal was not allowed to gain weight in excess of the intravenous infusion which it received. i.e.. 0.075 slain. The cross circulation was achieved by cannulation of a femoral artery to a femoral vein. A blood-born mediator is thus ruled out unier these conditions. An alterntive explamticn for Cort's findings concerns the stimulation of intrarenal mechanisms by extracellular space expnsion. Thus the urinary patterns reported by Cort (Cort et al.. 1968. Cort asi Lichardus. 1968) and co-workers (Lichardus arr! Pearce. 1966) may be the result of extracellular fluid volume changes 25 22 rather than a “Substance X”. Lesion Location and Proposed Experiments For purposes of future reference the posterior medial area of the hypothalamus is tint region dorsal an! caudal to the dorsomedial nucleus in the post-tuberal region (Haymaker et al.. 1969) and medial to the fornix ani mamaillothalamic tract. The mesent set of experiments are an atteapt to more systematically ascertain the role of the area posterior hypothalami in body water regulation. with special emphasis on saline preference and ingestion. Experiment 1 is methodologically concerned with sampling techniques , i.e.. what time duration aunt pas following a 1% to 2 cc heart puncture blood sample before the body fluids return to pro-sample levels as determined by available iniices. Experiment 2 focuses attention upon the application of bilateral lesions in the area posterior medial hypothalami. Comparisons are made 16 with reference to the results obtained from electrolytic DC lesions and Radio Frequency (RF) lesions. The electrolytic lesion is referred to as an irritative focus in tlmt metal ions may slough off the electrode with the passage of current ani become deposited at the lesion site (Dahl and Ursin. 1969). The radio frequency technique has been said to be non-irritative for it relies upon high intemity heat which results in coagulation of tissue at the lesion point (Rolls. 1970). Experiment 3 is a preference experiment utilising three group of animals. Within each group the animals are equally divided into an experimentally lesioned segment with bilateral lesions of the area posterior medial hypethalami. ani a sham lesioned group with identical surgical prepration as the experimental group but with a sterile dura puncture rather than lesions. Group 1 received tap water for its fluid intake. Group 2 was provided both tap water and 0.87% saline to drink. Group 3 comisted of previously adrenalectoaised rats aui was allowed both 0.87 ani 2.0% saline. These same three groups of animals were utilised through the subsequent experiments . Experiment lb addresses the possibility of neural damage to the antidiuretic hormone system. Subcutaneous injections of pitressin tannate amd peanut oil were administered the experimental lesion and control groups in a counter-balanced design. Cort (1963b) has indicated that ADH may be secreted directly from the posterior hypothalamus in addition to its release by the pars nervosa. This experiment. therefore tests whether the heightened isotonic saline ingestion exhibited by experimentally lesioned animals can be reduced with exogenous pitressin. 1? Experiment 5 is concerned with the first day of a 231» hour water deprivation schedule. Such a schedule represents a rather stressful condition in that body water and electrolyte saving mechanisms are required to function well in order to satisfactorily nintain ECF volume and tonicity. Attention is given to comparisons between the experimental atxl control groups ' responses to this regimen. EXPERIMENT 1 Repeated Blood Samples Since it is necessary to use repeated blood samples in this set of studies an appreciation of the effects of a 1‘) to 2 cc heart puncture blood sample is advised. The literature offers no indication of urine arxi blood electrolyte changes following such a blood sample. flatton ani Thornton (1968) used ten days between heart puncture blood samples with good success. The following experiment was therefore designed to deternine whether the rat's body water balance recovers in less than ten days following a blood sample as measured by available indices of body water volume and constituencies. m Subjects Thirty male albino rats of the Holtsman strain. approximately 120 days of age were adapted to living in metabolisa cages under cormtsnt light for ten days before the initiation of experimental observations. The animals were maintained on _ad_ libitum tap water and powdered Wayne Breeder Blox. notabolism m The metabolism cages were of the Acme Metal Products design. The living dimension were 26.5 x 20.5 cm. as! 16.5 cm high. hob cage was supported by an accompanying base (also Acme Metal Products) that was 50 cm tall. There were 30 unit metabolism cages of this type 18 19 arranged on three tables . 10 per table. the surfaces of which were elevated 914 cm above the floor. All cages were kept in the same room which was maintained at approximately 23-25 c with a relative) humidity of 20-50%. Procedure Following ten days of adaptation to the metabolism cages the animals were placed into five group of six animals each. Experimental observation then began with eight pre-saaple days prior to the first blood sample. The groups were raniomly assigned to one of five treatment coalition. These coalitions were: first blood sample taken on day 0. second blood sample on day 1. this comprised the group henceforth referred to as Inter-Sample Interval (ISI) - 1. The next group was designated ISI - 3 with the first blood sample taken on day 0 the second sample on day 3. Other groups were treated in similar fashion with five days between blood samples, i.e. . group 181 - 5. seven days between samples group 131 - 7 and nine days between the two samples group 181 - 9. The ‘first blood saaple was taken from the animals in a random ordernon day 0. The second saaple taken mm an animal was determined by the group to which it had been peviously assigned. The second sasple for a given animal was taken within five minutes of the time of day the first sample had been taken. The 151 - 3 group, for example. represents animals that were sampled at a given time on day 0 and then sampledagain 72 hours later within five minutes of that given time for each animal. The samples were taken unier light ether anesthesia between 0800 and 1330 hours on all sasple days. The serum samples were taken via left ventricle heart puncture and were immediately 20 centrifuged at 3300 rpm for 3 minutes. Two hematocrit samples per animal were prepared and centrifuged in standard heparinised capillary tubes (1.5-2.0 mm 1.0.. length 75 mm. Scientific Products) at 10.000 rp for h mimtes. and subsequently read. A small portion of the retained serum was immediately analysed for protein in a refractoaoter (AmericanJOptical Company). The remaining serum was divided between two analysis methods. A 0.025 ml serum sample was taken for’eodium and potassium concentration determination by flame photometer (instru- mentation Laboratories. hodel 1&0). The remaining serum was quickly frosen for later’determination of osmolality by freesing point osmometer. Throughout the ten day adaptation period. the eight day pre-eaaple period amd the nine days of blood samples. urine samples were taken at the same times twice daily. 0800-0830 and 2000-2030 hours. Food and water consumption and urine volume output were recorded at the same time as the urine collection. Body weight was recorded at the 0800- 0830 reading time. Sodium and potassium concentrations were determined for each urine sample by flame photonetry and total solids readings (specific gravity) were obtained by the use of a refractometer. Results The data for water and food intake. urine output and electrolyte concentrations have been grouped across two day intervals for the eight days preceding the first blood sample ani nine or eleven days after the first sample depending upon the group being considered. Hhen a blood sample was taken the next reading period values are given following the sample. This is the only departure from the presentation of'data grouped across two days. The means and standard errors (S.E.) of the seam are plotted across days for each of the five groups. Points are 21 reliably different from one another at a minimum of the 0.05 probability level where 8.8. bars do not overlap. water intakes for the five groups are shown in Figures 1. 2 and 3. The points represent mean values of tap'water intake per 12 hour reading periods with reference to a given sample group. The eight days prior to the first sample represents a base rate period and in the case of water intake the five groups are significantly different from one another (Ft-5.28, df 40/15. p<0.01). Groups 131-3 end 131-9 deviated from the other three groups. The next reading period after the first blood sample for all groups revealed significant drops in water intake. as compared with the base rate periods for the ISL-1 group which dropped an average of 3.2 ml and the ISL-3 group which dropped 6.2 ml. The other three groups (131-5. -7. -9) were not different from the base rate water intakes. The effects of the second blood sample upon water intake were that groups 151-3. -5 and -9 significantly decreased water intake as compared with the groupod reading periods prior to the sample. with declines of “.87. 2.88 and 5.33 ml. respectively. Utilising the above criteria. four out of the five groups revealed decreased water intake following the first end/or the second blood sample. The exception. group 181-? also revealed a significant decrease in water intake following the initial blood sample if post-sample day 1 is used as a reference value to conpare with the base rate periods rather than the first reading period after the sample. Comparisons between groups concerning food intake disclose tendencies not unlike those evidenced for water intake. Figures h. 5 and 6 represent food intakes for the five groups for the 12 hour reading 22 Figure 1. Mean (t S.E.) water consunption in 12 hour units for the intersample interval (131) - 1 day and - 3 day groups during the eight days prior to blood sampling and eleven days following the initial sample. Figure 2. Mean (1‘ S.E.) water consunption in 12 hour units for the intersample interval (ISI) - 5 day - 7 day groups during the eight days prior to blood sanpling and eleven days following the initial sanple. ML.) WATER INTAKE (l2 hour units in WATERINTAKE UZhourumninML) 23 g L l l A L 4 s e l‘ 3A BLOOD SAMPLES (DAYS) N N 20 5 ;\ O A A A l l lShS e———e ISI-7 o~---o 1 A A A A L 6 8 I 3 BLOOD SAMPLES 5‘ 7A 9 u (DAYS) 2# Figure 3. Mean (t S.E.) water consumption in 12 hour units for the intersample interval (ISI)-9 day group during the eight days prior to blood sampling end eleven days following the initial sample. Figure 4. Mean (t S.E.) food consunption in 12 hour units fer the intersanple interval (ISI)-1 day and -3 day groups during the eight days prior to blood sampling and eleven days following the initial sanple. 25 3's. e. I... are: a: u21h8. £9.33 BLOOD SAMPLES (DAYS) T“..:.‘:‘.‘...‘..‘.‘~“Le . . g e e a I val s x \ I I 8 6 4 2 Amldco E 3:5 Laos N: u¥0.05). Food intakes dropped significantly for groups 131-3 and -5 after the initial blood saaple. i.e.. 6.87 and 2.18 grass. There were significant decreases in food intake as compared with the grouped reading period prior to the sanple after the second blood sample for all five groups. 181-1. -3. -5. -7 and -9 with aversge drops in food intake of 3.58. 6.87. 2.18. 1.79 and 1.92 grass. respectively. The mean urine output for each group across days is provided in Figures 7. 8 and 9. There were base rate differences between the groups for the eight days preceding the first blood sasple (Fb6.73. df 5/15. p<0.01). Groups ISI-i end -7 desonstrated a significantly altered urine eutuput after the first blood sample as compared with the base rate data. Group 181-1 dropped 2.66 ml while group ISL-7 increased its mean urine output by 2.79 a1. however the 131-? animals registered base line urine outputs that were very low (Figure 8) below a mean of 8 ml per 12 hour reading period. The first reading period urine output value after the initial blood sample was 9.83 for the ISI-7 group which is not different free the last base rate urine output mean for all five groups cosbined. 8.96 ml. Group ISI-3 showed a significant increase in urine output after the second blood sample. up 2.88 sl. while group 131-? decreased significantly by 3.38 al. The other three group revealed no significant change. Turning to urine sediua concentration and specific gravity. the findings are presented in like nanner as the previous volumetric intake-output data. The eight day base rate period revealed no differences between the five groups in terns of urine sodium concentration 27 Figure 5. Mean (1 5.8.) food consunption in 12 hour units for the intersaaple interval (131)-5 day and -7 day groups during the eight days prior to blood sampling and eleven days following the initial sample. Figure 6. Mean (1 S.E.) food consumption in 12 hour units for the intersample interval (ISI)-9 day group during the eight days prior to blood saapling and eleven days following the initial sample. 28 O H M . H _ H . . .9 I o A I A s L 5 7 i 7 9 g . Y . I I i A I S S . SAW 8 . s l 3 E 1 L P 1 I m I” , .‘.‘.‘..‘.‘.‘.‘.‘~‘.‘.-~‘ -‘...‘.“.' D ~“‘.“".II“.1 1 TOI. 1+8 8 V i a. D e O m L 6 . O i . L . B 1 ‘ A s \ \ L 2 1 { L P r E rp . b r . a 6 4 2 o 2 o 8 6 4 2 o 82(10 5 2:5 So: «3 mx<._.z_ coon. 32410 E 3:5 :3: N: mx0.10). All five groups showed tendencies to decreased urine sodius concentration following the first and second blood samples. Hith regard to the next reading periodhafter the first blood sample. groups 131-3. -5. -7 and -9 were significantly different from the last pro-sample day means. with decreases of 85.5. “0.6. 61.9 and 93.5 mEq/L. respectively. The sane groups registered significant drops again in urine sodium concentration after the second blood sasple. 61.5. 7b.2. 60.2 and 31.“ respectively. Urine specific gravities aregiven for the five groups in Figures 13. 1b and 15. In general the total solids changes are sore conservative than urine sodium alterations. however. the total solids changes are in the sane direction as those recorded for sodius. The eight day base rate period yielded non-significant differences between the groups (F-1.89. df b/15. p)0.10). Groups ISL-1. ~3. -7 and -9 revealed significant decreases in urine specific gravity following the first blood sample. Decreases of 0.00236. 0.00189. 0.00h80 and 0.00320 respectively. were registered. Following the second blood sasple urine specific gravity fell significantly in groups 151-3 and -5 by 0.002h9 and 0.00285 respectively. The blood sasples were analysed for sodiun. potassiua and protein concentrations and s heaatocrit value was deternined for each sample. The blood data are presented for each group beginning with the ISI-i day group through the ISI-9 day group. The open bars labelled “a“ represent the corresponding wean concentrations and S.E. for the first blood sample taken free the animals of a given group. The closed bars labelled "b” are scans and 8.19. for the secorrl blood saaple. To the 32 Figure 9. Mean (t S.E.) urine volume excretion in 12 hour units for the intersample interval (ISI)-9 day group during the eight days prior to blood sampling and eleven days following the initial sample. Figure 10. Mean (t S.E.) urine sodium concentration for the intersample interval (ISI)-1 day and -3 day groups during the eight days prior to blood sampling and eleven days following the initial sample. :8 52...: Leo: N: #3925 uz . c: H in 9 \I I. 17m S A 45w 8 13E L P ,1 ins D O 160 L B 14 12 I) p . ‘v 2 i ISI-l lSI-3 o-’----o O 3 2 .4\eusv 0 O 2 .0 2 I70 ‘...~--.-.".‘.‘- D “‘.“L 00 7 (DAYS) 02 szD BLOOD SAMPLES Figure 11. Mean (t S.E.) urine sodium concentration for the intersample interval (ISI)-5 day and -7 day groups during the eight days prior to blood sampling and eleven days following the initial sample. Figure 12. Mean (1 S.E.) urine sodium concentration for the intersample interval (ISI)-9 day group during the eight days prior to blood sampling and eleven days following the initial sample. 35 .oxeusc .0200 02 szD BLOOD SAMPLES lSl-9 e—e 2 2 2 AJ\awEV “ a m 0 m 0 .oz 00 oz MZED 9‘ ll 7 (DAYS) BLOOD SAMPLES 36 Only the last three numbers from the refractive index are presented on the ordinate of Figures 13. 1n and 15. Complete read cuts would take the form 1.3___. Figure 13. Mean (r S.E.) urine specific gravity for the intersample interval (ISI)-1 day and -3 day groups during the eight days prior to blood sampling and eleven days following the initial sample. Figure 1“. Mean (1 S.E.) urine specific gravity for the intersample interval (ISI)-5 day and -7 day groups during the eight days prior to blood sampling and eleven day following the initial sample. 37 O i I . I . . _ o .9 I 3 9! . . i I I 7 v- S s A I I D L 5 ( I em 8 i L P. i I‘M . A ' ‘-“- D ‘ ‘M...‘...‘~.“‘~.‘.L 1. 8 s D 0 J 6 O L B i 4 .. 2 b p e a? V) p a P p P »L 0 O O 0 O O O 8 6 4 2 O 8 4 4 4 4 4 3 3302. m>.._.o<¢uw5 mafia"... ._<._.o.r o 1 . . . . u G i 5 7 g - 1 I m s I I I L p b P p P a P .8 O m 0 O O O O 8 6 4 2 0 8 4 4 4 4 4 3 AwaZ_ w>_ku<¢mwm. mo...om 4_hu<¢uu~: majom ._<._.o.r BLOOD SAMPLES (DAYS) 41 Figure 16. The mean (! S.E.) serum sodium concentration for each 131 group on its first (a) and second (b) blood samples. The mean serum sodium concentration across all 181 groups for the first blood sample (a) is also provided. labelled MEAN. Figure 17. The mean (t S.E.) serun potassium concentration for each 181 group on its first (a) and second (b) blood samples. The mean serum potassium concentration across all 131 groups for the first blood sample (a) is also provided. labelled MEAN. 42 MEAN 6 5 4 4 . o q «JEONSV .ozoo oz Szcum INTER - SAMPLE INTERVAL (DAYS) LIIL 1 b 09 b7 0 .0 L05 t...‘...“'.‘.~-~-“‘.~.~.4 a AJ\.Om¢$ .0200 x 2...me MEAN fl [LFTLPbPrF-Pbt 0. o. o. o. o o. oo 7 6 5 4 3 2 I. INTERVAL (DAYS) INTER-SAMPLE 43 Figure 18. The mean (t S.E.) serum protein concentration for each 151 group on its first (a) and second (b) blood samples. The mean serum protein concentration across all 181 groups for the first blood sample (a) is also provided. labelled MEAN. Figure 19. The mean (t S.E.) hematocrit for each 181 group on its first (a) and second (b) blood samples. The mean hematocrit across all 181 groups for the first blood sample (a) is also provided. labelled MEAN. 0. 3 4. 3. 2. I. S 6 6 6 6 6 “J! CO. \43 255.01.“. 232mm MEAN INTER -SAMPLE INTERVAL (DAYS) T...‘.‘~.~.~-~"‘-.‘.“.‘E booms: h.¢uo._.<2mx :1 m E M .ll)» r r F pl? . . . i a 6 Q 2 O o INTER-SAMPLE INTERVAL (DAYS) 45 Group FBI-9 was significantly different from 181-3 and -5 in terms of mean hematocrit values for the first blood sample. There were signif- icant differences between group’ISI-1°s second blood sample mean and the other groups' second blood sampde mean. this also held for groups 181-3 and -5 compared with the other groups. and 131-7 and -9 versus the other groups. Hithin group comparisons between the means of the first and second blood samples showed groups 131-1. -3 and -5 to be significantly different. Discussion Experiment 1 was intended to answer the questions How long must the investigator wait between heart puncture blood samples in rats before the body fluids return to pro-sample levels as indicated by available indices? Although the results may appear sonewhat inconclusive on lesser questions there are ample indications from the data to sug- gest a preferred time delay between blood samples. In general the intake-output indices reveal drops in water and food intake following blood samples. Although such drops may or may not occur after the first sample. if they do not occur after the first sample they do occur after the second sample. Once a drop'is evidenced a recovery time of from one to four days is generally required before the pre- samplc level is reattained. This time period appears to vary with the degree of depression of food and water intake following the blood sample. The greater the nagnitude of the depression the longer the recovery time required. The consistent drops in urine sodium concentration after the blood samples are in large part due to the decrease in food intake and its interaction with other effects of the blood sample EEEEEE? 46 such as the two to five minutes under other anesthesia. Ihere food intake was most severely depressed. i.e.. group 131—). urinary sodium concentration was also maximally affected. A similar food deprivation effect upon urinary sodium concentration has been previously documented for normal rats (O'Kelly and Bright. 1971). The base rate level of urine sodium concentration was somewhat higher in the prior study (averaging about 240-250 mEq/L.) than in the present study during which these rats demonstrated mean values of approximately 180-200 mEq/L. Also in the earlier experiment the sodium concentration decrease under 24 hour food deprivation fell to around 40 ass/L. by the end of the deprivation period while the present decreases. even in the maximally affected group (ESI-B) was to a mean of about 90 mEq/L. during the 12 hours following the sample. The primary difference between the two studies in addition to blood samples in the present investigation is that Experiment 1 animals were free to increase food intake at any time following the blood sample. for the food was never removed as it was in the former study. Close inspection of the food intake data of the Experiment 1 animals further suggests that none of the groups completely stopped eating during the first 12 hours after the blood sample and they fully recovered to their former food ingestion levels by one to three days after the blood sample was taken. The data of most conern appear to be the blood sample measures proper. The serum sodium concentration values indicate recovery by nine days following the first sample. Serum protein offers a different picture with recovery by three days after the first sasple while hematocrit readings. which will be weighted heavily suggest seven days for the red bleed cell per cent volume to return to a value not different from the first blood sample values. 47 From this experiment it is recommended that a minimum of seven days be allowed to use between blood samples. To insure against confouniing effects introduced by sampling too soon the method to be followed in subsequent experiments of this series will be to allow at least ten days between blood samples or between surgery and blood samples . EXERIHENT 2 Posterior Medial Hypothalamic Lesions This experiment was an attempt to replicate previous findings concerning bilateral lesions of the area posterior hypothalami. Cort (1963a) has indicated urinary “salt wasting“ following such lesions in rats with death occurring within approximately ten days if a saline solution is not provided for purposes of body sodium replace- ment. This study closely inspected saline and water intake pre- and post-operatively and resulting alterations in blood osmolality. protein. sodium and potassium concentrations as well as urine total solids. sodium and potassium concentrations. Subjects Eighteen male albino rats of the Halts-an strain approximately 120 days of age were adapted to metabolism cages under constant light (the same cages as described in Experiment 1) for a pro-lesion period of 20 days. The animals were maintained on g libitum tap water. 0.87% saline solution ani powdered Hayne Breeder Blox. The tap water all saline drinking cyliniers were switched daily on the cages in a random sequence to avoid the establishment of position preference by the animals. Procedure The animals were stratified into three groups of 6 animals each. The first group received bilateral electrolytic lesions to the nucleus mediodorsalis thalami. The other two groups received bilateral damage to the area posterior hypothalami. In one group the damage was inflicted by the electrolytic lesion method; the other group received radio frequency lesions. During the 20 days of adaptation and subseq- uent 22 post-lesion days urine samples were taken from each animal once a day between 0800-0830 hours. At this time body weight. water. saline and food consumption and volume of urine excretion were also recorded. Urine samples were prepared for determimtion of sodium ard potassium concentration by flame photometry and total solids readings were also obtained. 0n F's-operative day 11 a blood sample was taken from each animal between 0830 am 1230 hours. The blood samples were treated in the same way as those described in Experiment 1. A second blood sample was taken from each animal on pest-operative day 11 and a third sample on post-operative day 22. Surgery was performed on all animals after the 0800-0830 readings of pro-operative day 20. The surgery lasted from approximately 0900 to 2000 hours. The lesions were applied in a randomised order of the eighteen animals ani with a raniom order applied to the lesioning of the hemispheres. i.e.. right-left or left-right. The electrode consisted of a 0.28 mm diameter stainless steel shaft entirely insulated except for a 0.5 mm uninsulated tip. The anode was grounded anally with the radio frequency an! electrolytic lesion techniques. 49 Following the conclusion of this experiment the animals were sacrificed by other overdose. perfused with 10% formalin arri the brains retained in 10% formalin for a minimum of two weeks prior to histology. Both the frosen and celloidin brain histological procedures were employed. ‘hlo brains from each of the three groups were celloidin embedded ani subsequently sectioned at 25,4. and stained with thionin (lissl method) for cell bodies a!!! hematoxylin (Heil arwi Heidenhein methods) for myelinated fibers. Approximately four days preceding frosen sectioning the other four brains from each group were placed in a sucrose-fornlin solution. They were sectioned at 40 ,2 and stained with cresyl violet acetate. a cell nissl body stain and by the Fink-Heimer method (Hjorth-Simonsen. 1970) for degenerating axons. Results The body metabolism data have been grouped across two day intervals. The last ten days of the twenty day pro-operative basal rate period is presented along with the entire twenty day post-operative period. The means and standard errors of the means are plotted across days for each of the three group. Once again the points are reliably different from one another at a minimum of the 0.05 probability level where standard error bars do not overlap. Tap water arr! 0.87% NaCl intakes are provided for each individual group in Figures 20. 21 ani 22. There were no differences for any of the groups between tap water versus saline intake during the pre- operative days 10-20. After the experimental lesions the thalamic. posterior hypothalamic radio frequency (PH-RF) an! posterior hypothalamic electrolytic (Hi-IRE) groups all showed significant differences between 50 water and saline intakes (F-25.97. 21.83. 11.15 respectively. df’1/8. p<0.01) with the saline consumption rising a mean of 24.4 ml to 52.0 ml by post-operative day 10 and the taptwater intake dropping a mean of 12.9 ml to 12.3 ml by post-operative day 10. On post-operative‘day 10 following the compilation of the daily intake-output records and urine sample collection. the saline cylinders were removed from the metabolism cages of all groups. Tap water consumption was then noted to rapidly rise a mean of 30.8 ml to 43.1 ml by post-operative day 20. Comparisons between groups concerning food intake (Figure 23) revealed no differences for the pro-operative period presented (Fb0.95. df 2/12. p>0.10). There was a substantial drop in food intake following the surgery for all groups.‘approximately 9 grams reduction comparing pro-operative days 19 and 20 grouped with post-operative days 1 and 2 grouped. Subsequently there was a food recovery period that resulted in the reestablishment of a food intake similar to pre- lesion levels by about post-operative day 8. Removal of the saline appeared to have minimal affects upon food intake. The groups were significantly different concerning urine output (Figure 24) registered during the pre-operative period presented (Ft-17.98. df 2/12, p<0.01). The major contribution to this deviation came from the PH-IRR group which evidenced urine output significantly below those of the other two groups. The post-operative urine outputs for the groups were non-significantly different (F-1.56. df 2/27. p>0.10). However. the mean urine output for all groups on post- operative day 10. the last day on both saline and water was significantly higher (1538.11) than the mean for all groups on the last day of tap water only (1520.44) post-operatively day 20 (t-8.51. df534. p<0.01). 51 Figure 20. Mean (1 S.E.) tap water and isotonic saline solution consumption exhibited by the control lesioned group for the last ten days of a twenty day pro-lesion period and twenty days post-operative. Figure 21. Mean (3 S.E.) tap water and isotonic saline solution consumption exhibited by the posterior medial hypothalamic radio frequency lesioned group for the last ten days of a twenty day pro-lesion period and twenty days post-operative. 52 60- ' \ THALAMIC LESION ' ‘\\ x 3 : E50. .‘ u: .' x .' <40 .' .— I z .‘ ‘ .' “so. -' z I 4 E < I m 20. CD o: u 2 lo» 3 WATER e——4 SALINE o ------ o n A A A l A A A A A L A A A |2|4|6l82024 s snonzulslezo PRE-OP POST-OP (DAY$ so ' PH(RF) A LESION _i 350 m x <40 .— z I“so E .J < "’20 fl a: w *- l0 4 3 WATER e———e SALINE O------O A 1 A A A A A a l2 l4 ‘6 IO 20 2 4 6 3 IO l2 I4 IS I. 20 PRE'OP POST-OP WAY$ 53 Figure 22. Mean (1 S.E.) tap water and isotonic saline solution consumption exhibited by the posterior sedial hypothalamic electrolytically lesioned group for the last ten days of a twenty day pro-lesion period and twenty days post-operative. Figure 23. Mean (2 S.E.) food intake in 2a hour units for the three lesioned groups for the last ten days of a twenty day pro-lesion period arr! twenty days post-operative. 54 5 I I PHHRR) q LESION .1 : V 3,50 . i f“ I (40 : g I _ I i u 230 I 3 i g I 20 g ‘ : E. u '- IO ' 4 I 3 : WATER e————e | sauna o ------ o of A A A A A I A J A A A A A A A J— I2 I4 Is no 20 z e e s IO l2 I4 l6 Is 20 PRE-OP POST-0P (DAYS) 24 : LESION OFF sum: 20 0’ 1 INTAKE (GM) l2? 0 O 8 O u. >- 4r THALAMIC .-——'. PH(RF) o- -------- o P PH(IRR) 0- ----- O 0‘ L 4 A L_A A A A A A A A A A A 12 I4 I6 IO 20 2 4 6 0 IO l2 l4 l6 I8 20 PRE-OP POST-OP (DAYS) 55 Comparisons between groups concerning urine sodius concentration (Figure 25) suggest significant differences during the ten day pre- operative period (Fh10.42. df 2/12. p(0.01). however. the post-operative twenty day period revealed non-significant differences between the groups (Ia-0.59. df 2/27, p>0.10). There was A significant difference between the mean sodius concentration for the last day on saline and tap water. post-operative day 10 and the last day on tap water only. post-operative day 20 (t for related measures. two tailed test-3.49. p(0.01) with a mean drop froa 184.35 to 133.12 aEq/L. Hith respect to total urinary sodium (Figure.26). originally cosputed as a urine concentration multiplied times urine values over unit time. both the ten day pre-operative and twenty day post-operative periods revealed non-significant differences between groups (F—2.40. 0.18. or 2/12. 2/27 respectively. p>0.1o). There was a general treni difference. however. over time with total sodiua decreasing after the lesion. followed by recovery and then a substantial drop in total sodius output when the saline was removed. This pattern is not too unlike that followed by sodius concentration (Figure 25) with the exception that total urinary sodius output recovered to a greater degree. This increased recovery on the part of total sodius appears to be primarily due to the increased urine output between post- operative days 8 and 12. Accompanying the withdrawal of the saline. post-operative day 10. came a decrease in urine output and urine sodius concentration that resulted in a dramatic decrease in total urinary sodium output registered during pest-operative days 12 to 20. It is of soae interest that urinary potassiun concentration (Figure 27) contrasts with this post-operative pattern evidenced by 56 Figure 24. Mean (i S.E.) urine volume output in 24 hour units for the three lesioned groups for the last ten days of a twenty day pro-lesion period and twenty days post-operative. Figure 25. Mean (3 S.E.) urine sodium concentration for the three lesioned groups for the last ten days of a twenty day pro-lesion period and twenty days post- operative. IN MEOJL. No CONC. URINE OUTPUT (ML) on o LESIOII 0 O Q 0 N O 0" SILINE 4‘ e 4‘. e~ . I a. I e. ’ ‘. 3.. I“ e ’ I, “ 0‘. s I, ‘ I . . . I, ‘ ‘3 O T O Ir’ ‘i” '0’ THALAMIC e————. PH(RF) O ---------- 0 PH (IRR) .- ----- Q 0 A A A A A A A A A A A A . L IZMIOIOZOZQGOUOIZMIBIOZO PRE'OP POST-OP (DAYS) I LESION OFF SALINE 280 ii I I I 240 §: I r- I i 200» ' , -_ x ‘ I ,y “ )- I .' ”.."‘-e- . \ ' I .‘a ‘\ I \‘ h . O\ 'a g \r. l ZOr ' ' L : THALAMIC F—4 I pwmr) o---------o 80- I ____ . PH(|RR) o- -e O L A A A A . A A A A A A A A A L l2l4l6|82024SBIOIZMISIBZO PRE‘OP POST-OP (DAYS) 58 urine output. sodium concentration and total sodiua. There were significant differences between the three groups during the ten day pre-operative period (F -13.41. df 2/12. p(0.01). There were non- significant differences between the groups during the post-operative twenty day period (F-O. 59. (if 2/27. p)0.10). The trend of urinary potassius concentration. however. was the opposite of that for the sodius concentration during this post-operative period. That is. while on both saline and tap water the potassium concentration fell sign. ificantly below the pre-operative level. Once the saline was removed the potassius concentration rapidly recovered to a level not different from the pre-lesion values. Hith reference to the total solids index (specific gravity. Figure 28) pro-operatively there were differences between the groups (F-14.28. df 2/12. po.io). The trend was very sisdlar to that of the urinary potassius concentration in that there were post-operative decreases in specific gravity after the lesion with a noticeable recovery upon the removal of the saline for all groups. The groups were non-significantly different in terns of'body weight (Figure 29) during the ten0.10). There was a substantial decrease in body weight for the groups following the surgery with a cosbined group scan loss of 29.22 grass fro: 391.72 to 362.50 grass. A constant weight recovery followed the lesion: but the weight gain appeared to be less than the pre-operative rate. At the coapletion of the twenty day post-operative 59 Figure 26. Mean (1 S.E.) total urinary sodium excreted per 24 hour units for the three lesioned groups for the last ten days of a twenty day pro-lesion period and twenty days post-operative. Figure 27. Mean (i S.E.) urine potassiun concentration for the three lesioned groups for the last ten days of a twenty day pro-lesion period and twenty days post-operative. TOTAL URINARY No (men/day) IN MEQJL. K CONC. ’ I I I THALamc e___. | PM Im o----------o : ‘ PH (IRR) e—----e I \ : , \ I . "° ’ ' I . ’ A | l .' I :I ‘ )‘ I o'l \ I .H a | ‘\ 0" ‘0 D o I ‘I I I J I I : x I. I a - 's : 2.0 LESIION orr sauna }_ ‘ " .; 0 AA A A A A I L A A A A A 4 A A A I2 I4 I6 I8 20 2 4 6 8 IO I2 I4 I6 I8 20 PRE-OP POST-OP (DAYS) j I '3 $ LESION OFF SALINE I\ I I ' \ I II : l x I; I ‘ 0' ‘ -. e ' I "0' ‘ . so : \\ ' \ I | 0 \ .. vo : . ‘ . I \ so I ‘ . ~ I “. THALAmce——e : thn o ------- 0 3° . pa (mm e—--—* 0 4L A A A ALA A A A A A A L 1 ‘ I2 I4 I6 I6 20 2 4 6 8 IO l2 I4 I6 I6 20 PRE-OP POST-0P (DAYS) 61 period the cosbined group sean body weight lacked 9.59 grams of being equal to the pre-operative day 20 seen weight. lean serus sodius values (Figures 30. 31 and 32) for the three groups were non-significantly different from one another at the pre- operative sasple with a seen between 132 and 134 aEq/L. On post- operative day 11 the thalasic and Pfl-IRR groups were not different but the mean sodium level of the PH-RF group was significantly lower than the other groups. On post-operative day 22 the three groups registered increased sodius levels to seen values between 138 and 139 aEq/L. The serum potassium changes (Figures 33. 34 end 35) corresponded relatively well with those of sodium across the three blood sasples for the groups. lean serum protein levels for the three groups on each of the three blood sasple days are given in Figures 33. 34’and 35. There were no differences between the three saspdes for the two posterior hypothalasic lesion groups which had means of about 6.3 crass/100 ml. The thalasic lesion group registered a serus protein scan of 6.4 pro-operatively and then fell significantly to 6.1 by post-operative day 11 and 6.2 for post-operative day 22. Iith regard to serua ossolality the three groups were alike evidencing significant changes fros pro-lesion ossolality of about 267-271 won/Ks. and post-operative day 11 osmolality increases to about 275 to 280 econ/Kg. However the post-operative day 22 some osmolality found the thalaaic and PH-IRR groups dropping back to mean values of between 271-273 while the Pfl-RF group's seen was 279 IDsm/Kg. Figure 28. Mean (* S.E.) urine specific gravity for the three lesioned groups for the last ten days of a twenty day pro-lesion period and twenty days post-operative. Figure 29. Mean (r S.E.) body weights exhibited by the three lesioned groups for the last ten days of a twenty day pro-lesion period and twenty days post-operative. TOTAL SOLIDS (REFRACTIVE INDEX) BODY WEIGHT (GIL) 63 I I ‘3 LES'ION on cause I I 46 : . l” ‘I I I 440 I I g i - ' \\ s‘ I’ I D ‘\ I’.o.\ )0 .. 420 : . . I II." ‘9, . . .‘ ‘ {no I . \‘ ’I’. 400 I " , : " THALAMIcr———e I PI-HRF) o -------- 0 33° : PH (IRRI e—-——«e o A A A A A | A A L A A A A A A A I2I4I6I8202468IOI2|4I6I820 PRE-OP POST‘OP (DAYS) " I.” THALAIIC e——e . PH (RF) 0 --------- o : PM (In) e------e 40 I : on sauna I I 390 : I I | O 38 I I ." I .... ' O I - ’ 370 | ___ ”‘0 ‘- ’ : " ' ‘ l 1’ I ’ . ° 360 I -. I ’ 1’ L I o A A A A A I A A 4 A A A L A A l I2I4I6I8202 4 6 8 IOIZ I4I6l820 PRE-OP POST- 0P (DAYS) Figure 30. Mean (1 S.E.) serus sodium arr! potassiuw concentration for the control lesioned group on one pre- lesion and two post-operative blood sample days. Figure 31. Mean (1! S.E.) serun sodium and potassium concentrations for the posterior redial radio frequency hypothalanic lesioned group on one pro-lesion and two post—operative blood sample days. 65 + .F —E -F- b----------------.--------------- 2.; + —F- I I n. t-e «2 9; it a. a a. + I 1‘2 _ ‘3 no u- e (‘1/b3w) (1/b3w) wmoos mass wmssuoc wnaas _F —E-—" 2 i 2 -F- -[: I P:---------------IF---------- + -F —L ‘ ‘ A A ‘ ‘ O ID 0 ID 0 In 0 g '2 :2 <2 .o' e“ e‘ (1/b3w) (1/b3w) WI’IIOOS “083$ NDISSVlOd “083$ 22 POST- O P (DAYS) PRE-OP 22 POST-OP (DAYS) PRE-OP Figure 32. Mean (1 S.E.) serun sodium and potassium concentrations for the posterior medial hypothalaaic electrolytically lesioned group on one pro-lesion and two post-operative blood saaple days. Figure 33. Mean (* S.E.) serum protein and osmolality for the control lesioned group on one pro-lesion and two post-operative blood sample days. 67 ESL—— THALAMIC E 'F b---------------- h--------------- o_ «u o n o hAL L P "- - co n n 8 h w '0 N N N N I'llOOl/s) (ow/meow) NIBLOUJ “083$ AlI'IV'IOVISO H0838 ea 4: ‘E-—-I Q e (1/b3w) (1/b3w) unloos muss umssuoe muss 22 POST-OP (DAYS) PRE'OP 22 PO ST'O P (DAYS) PRE-OP Figure 34. Mean (t S.E.) serum protein and ossolality for the posterior nedial hypothalamic radio frequency lesioned group on one pre-lesion and two post-operative blood sample days. Figure 35. Mean (1 S.E.) serum protein and osmolality for the posterior medial hypothalamic electrolytically lesioned group on one pro-lesion and two post-operative blood sample days. 69 PH (IRR) b---------------- 22 POST-OP (DAYS) 7.o|. ("1w ooue) NIBlOUd M1838 o In an I~ I~ co N e: N N (ow/meow) ALI'IV‘IOVISO N0 838 PH (RF) r---------------- b----—---------- ’. O. N. a on n ('1 w OOI I o) NIBLOUd H0838 In In 8 n 2 e N N N N (ow/meow) ALI'IV‘IOVISO “083$ PRE-OP POST-OP (DAY S) PRE-OP Antoni: IICII of 7O Anatomical Findings Photosicrographs of coronal sections prepared for the anisals of each of the three groups. i.e.. control. experimental (Radio Frequency). experimental (Electrolytic) utilised in the experisent are presented in the Appendix. Next to each photosicrograph is the corresponding de Great (1959) atlas section. The stain employed for each section presented in the photomicrographe is indicated. The area destroyed in each hesisphere of each animal presented in frosen section was estisated by the use of the cosponsating polar planimetery method and is included in Table 1. The extent of the lesions with reference to the de Groot atlas is also included. The control lesions were generally well placed bilaterally (refer to the Appendix). There was a tendency. however. for the lesions to be slightly skewed toward the left hemisphere. Lesion dassge in the control group was confined to the medial and lateral habenular nuclei. the stria medullaris of the thalasus. the para- ventricular thalssic nucleus and is sose cases the dorsomedial thalasic nucleus and hippocaspsl fissure. The scan volume dassge to the right hemisphere of the control anisals was 1.93 cubic as and to the left hesisphere 1.94 cubic ms. The experisental lesions were not as well placed bilaterally and in no case completely destroyed the posterior sedial hypothalasus. However some dassge was consistently inflicted upon the target area as well as nearby structures including the dorsal premasillary nucleus. fornix.and occassionally the dorsal longitudinal fasciculus. and arcuate nucleus of the hypothalasus. In almost all instances there was third ventricle damage. Ania? h .6 COLT! *4 \J‘ \J‘ 20 71 Table 1 Volume and Extent of Lesions Area Destroyed (mn3) Extent of Damage Animal Left Hemisphere Right Hemisphere (de Groot Atlas) controls 3 1.18 1.36 A u.u-5.u 6 0.29 (1.19) 0.72 (1.20) A 3.0-u.u 15 Celloldin Embedded A 3.6-u.u 16 1.96 (0.27) 1.84 (0.50) A u.0-u.6 19 0.86 (2.02) 0.35 (1.69) A n.0-u.6 21 Celloidin Embedded A u.2-u.8 experimentals (RF) 5 0.0u 0.70 A u.2-6.0 9 celloidin Embedded A h.4-h.8 10 0.32 (0.15) 0.80 (0.08) A u.0-5.0 11 0.21 (0.30) 0.10 A 3.8-4.8 12 Celloidin Embedded A n.6—5.0 1a . 0.31 (0.12) 0.32 (0.09) A a.u-u.6 experimentals (Elect.) 1 0.87 0.70 A a.2-5.u 7 1.20 (0.20) 1.01 (0.10) A u.0-5.0 8 Celloidin Embedded A 0.2-5.2 17 0.77 (0.37) 1.1a (0.1a) A 0.2-5.0 18 1.32 (0.17) 0.30 (0.04) A 0.2-5.0 20 Celloidin Embedded A u.u-5.0 The extent of gliosis for those animals in which it was encountered has been placed in parentheses. Total effects of the lesion would. therefore. be obtained by adding the area of the direct lesion and the gliosis area. 72 With reference to the experimental Radio Frequency group the mean volume damage to the right hemisphere was 0.51 cubic mm and to the left 0.46 cubic mm. For the experimental Electrolytic group the mean damage to the right hemisphere was 0.9? cubic mm and to the left 1.23 cubic mm. Discussion This experiment failed to replicate Cort‘s (i963a) findings concerning "salt wasting“ following posterior hypothalamic lesions. The post-lesion urinary total sodium output did not increase as predicted by Cort nor did the potassium concentration increase. However it is clear that such lesions result in a dramatic change in isotonic saline preference with a nearly two-fold increase while tap water intake decreased by almost one-half. The question ismediately arises as to the location of the sodiua and chloride in the bodies of these aniaals. Presumably storage of these ions must be occuring given the substantial increase in ingestion with no apparent change in urinary output after recovery from surgery. The ”sodiua reservoir“ hypothesis as offered by Stricker and Holf (1967: Rolf and Stricker. 1967) may warrant considerable attention. It is important to note that food intake remained constant daring this reaoval thus eliminating it as a source of sodium change. An additional consideration at the time of saline removal conerns the potassium concentration that increased. This pattern may be interpreted as a body stress reaction with the liberation of intracellular potassium from cells. however. the concentration quickly approaches an! levels off at values very similar’to pre-lesion concentrations. 73 The Electrolytic and Radio Frequency groups were comparable in their pest-lesion fluid metabolic patterns thus casting doubt on the possibility of attributing this increased isotonic saline intake to the irritative quality of the electrolytic lesion technique as demonstrated for the lateral hypothalamus by Barbara Rolls (1970). The increased isotonic saline intake by the dorsomedial thalamic lesioned group was of course unexpected for it was intended that these animals serve as a control group. It may be that electrolytic lesioning of this structure with possible damage to nearby and/or related areas. actually does precipitate alterations in body water regulation. This suggestion is indeed open to skepticism for there appears to be a complete lack of support for such findings in the literature. In several animals there was damage to the aedial habenular nucleus. Lengvari et al. (1969) have reported alterations in salt and water metabolism with lesions of this area. Specifically such lesions reduced aldosterone secretion while adrenalectomy resulted in the enlargement of cell nucleus sise in the medial habenular nucleus. In summary. this attempted replication of earlier findings concerning changes in body water balance fellowing electrolytic posterior hypothalamic lesions was at best only partially successful. Increased isotonic saline intake was noted with post-lesion recovery; however. accompanying increases in urine sodium and potassium excretion did not occur as would be expected on the basis of Cort's hypothesis. Also the removal of the isotonic saline for a period of twelve experimentally monitored days and several additional post-experiment days prior to sacrificing the animals revealed no predisposition on the part of these subjects 7“ toward an unhealthy physical coalition. nor did the animals increase their food intake which represented the only remaining source of sodium. A preliminary amlysis of the available data to this point would likely suggest an alteration in saline preference with posterior hypothalamic damage that persists in the form of increased tap water intake with saline removal but does not appear to have a noticeable absolute ”set point” for the sodium or potassium intake-output ratios. more: 3 Replication Lesions of the Posterior Medial Hypothalamus Experisent 2 established comistent and comparable post-lesion effects for damage to the area posterior medial hypothalami by both the electrolytic and radio frequency lesion methods. This supports the contention that the increased ingestion of physiological saline is not simply due to the irritative qualities of the electrolytic lesion method. However. the dorsomedial thalamic and medial habenular nucleus lesioned animals evidenced equally impressive physiological saline solution intakes thus cancelling their utility as a control group. The present study was designed as a replication attempt of Experiment 2 and focused conern upon the post-operative body sodium accusulation that was encountered in Experiment 2. Subtcts Thirty-six male albino rats of the Roltsman strain approximately 120 days of age were adapted to metabolism cages urder constant light for a pro-lesion period of 20 days. The animals were maintained on g libitum tap water and/or 0.87% saline and/or 2.001 saline am powdered Uayne Breeder Blox. The fluid cylinders were switchedthily on the cages in a rsxriom sequence. 75 Procedure The animalsUImestratified into three group of 12 animals each. Each of these groups were further split into 6 experimental arr! 6 control subjects. The first group was maintained on tap water only. The accord group on tap water and a 0.87% saline solution; and the third group 0.87% ani 2.00% saline solutions. Groups 1 and 2 were composed of normal animals while Group 3 subjects had been adrenalectomised a minimum of seven days prior to the initiation of pro-operative day l. The six experimental animals in each of the three groups received bilateral electrolytic lesions to the area posterior medial hypothalaai. The six control animals were treated in the same manner as the exper- imentals. however. sham lesions were administered. consisting of sterile dura punctures without the infliction of lesion damage. During the 20 days of adaptation to the metabolism cages am subsequent 20 post-lesion days a urine sample was retained from each animal am body weight determined once a day between 0800-0830 hours. At this tine as well as at 2000-2030. water. saline and food consumption and urine volume excretion were also recorded. Urine samples were prepared in the same. fashion as described in the previous experiments with regard to analysis. Also 45-hour readings on pro-operative day 15 and post- operative day 6 followed the pattern described in Experiment 2. Blood samples were taken on pre-operative day 10 and post-operative day 10 am! also conformed with the procedures described in the previous studies. Results Hetabolism data have been grouped across two day intervals. The last ten days of the twenty day pre-operative basal rate period is presented along with the entire twenty day post-operative period. 76 Figure 36. Mean (3 S.E.) body weights for posterior medial hypothalamic and sham lesioned groups maintained on tap water for the last ten days of a twenty day pre- lesion period and twenty days post-operative. Figure 37. Mesn(t S.E.) body weights for posterior medial hypothalamic and sham lesioned groups maintained on tap water and isotonic saline solution for the last ten days of a twenty day pre-lesion period and twenty days post-operative. 77 0 O b O .b O BODY WEIGHT (614.) a a m a PH GROUP WATER H—Q SHAH GROUP WATER O-‘o --q LESION S HAM GROUP WATER 400 12141616:% 2 4 6 e b e R G.e a) PRE- OP POST— OP (DAYS) 50 : LESION 46 i ' 2 _- ,, vH: -_ <946 T T * : ffxf f » w H) H” I I 844 ’é: % % ’ ,H E) 1 H HM 3420 §-§ : I g I : PH GROUP WATER 40 I .3793 SALINE '- ----- 0 E I .87 9: SALINE O-----‘° l2 I‘4 IAG PRE-O IO |2 l4 l6 IO 20 (DAYS) Id 202 4 6 8 P POST- OP 78 Figure 38. Mean (1 S.E.) body weights for the adrenal- ectomised posterior medial hypothalamic and sham lesioned groups maintained on isotonic and 2.00%»saline solutions for the bet ten days of a twenty day pre-lesion period and twenty days post-lesion. Figure 39. Mean (1 S.E.) tap water intake corrected for body weight differences by the posterior medial hypothalamic ani sham lesioned groups maintained on tap water for the last ten days of a twenty day pre-lesion period and twenty days post-operative. (GAL) BODY WEIGHT 79 500 } LESION i 48 | .2 1 -— I ’1 x ., 1% +1.) 46 I " . ', - :v -- 1 : ’I -- ‘ I I 44 . I 1 l 420 g I : PH .8716 SALINE 400 . 2.00m SALINEe ----- -e : swAw .8736 sauna 1 2.00%SALINE o------o OB ‘ ‘ '4 L A 4i i 1 12141616202 4 6 6101214161620 PRE-OP POST-0P (DAYS) PH GROUP O-——-O SHAH GROUPO—-——O r "I 0’ O ---------------. O 2 “16:23:. h OLAIAA‘LJAJ‘LAIL I2|4|6I82024GBIOIZMIGIBZO PRE-OP POST-OP (DAYS) WATER INTAKE/BODY WEIGHT a IOO 80 The meals and staxwlard errorsd‘ the means are plotted across days for each of the six groups. Four out of the six groups revealed sign- ificant drop in body weight following surgery (Figures 36. 37 and 38) therefore all subsequent intake-output measures were corrected for body weight differences. Liquid intakes are shown for each of the three experimental groups an! their correspouling sham lesion control groups in Figures 39. “D an! 41. In that these intakes have been corrected for differ- ences in body weight they are presented as percentages. The points on these figures represent the means of the percentages calculated for each animal for each reading period and grouped over two day periods for a given group. The pre-lesion water intake values (Figure 39) for the posterior medial hypothalamic lesioned group (PH group) versus the sham lesioned goup were statistically different (t-5.80. p<0.01). Post-operatively the water intakes between the two group were also different (tn-8.53. p<0.01). Compring the water intakes of the sham lesioned animals pre- and post-operatively there was no difference (t for related measures-0.57. P>0.10) however. the same comparison with the experimental lesioned group revealed significant increases in water intake during the post-lesion period (t for related measures-6.98. p<0.01). This increment in water intake is relatevely small representing a mean change of from 3.89% pre-op to 11.st post- operatively. The tap water and 0.87% saline solution intakes of the second posterior medial hypothalamic (PH) lesioned group and its sham lesion control group is offered in Figure #0. The tap water intake of the PR group was not different pre- and post-operatively. 81 Figure #0. Mean (1 S.E.) tap’water and isotonic saline solution consumption corrected for body weight differences by the posterior medial hypothalamic and shaa lesioned groups naintained on tap water and 0.87%.saline solution for the last ten days of a twenty day pro-lesion period and twenty days post-operative. Figure #1. Mean (2 S.E.) fluid intakes corrected for body weight differences of the adrenalectonized groups maintained on isotonic and 2.00% saline solutions for the last ten days of a twenty day pro-lesion period and twenty days post-operative. WATER 8 SALINE INTAKE/B. W. x IOO SALINE INTAKE/BODY WEIGHT x IOO 82 I PH GROUP WATER e———-e F a .8735 SALINE e- ------ e LESION . sum GROUP WATER o-——o I .ent sum: 0- ----- -0 I2 $- : I I .- I I u I 8 r- : I l I I I I 4 t g I I r : ""i--§--§ : c l 1 1 l J l A 1 i l + PRE-OP l2l4l8l82024 68l0|2|4|6|820 POST - OP (DAYS) PH .87% SALINE O------O 2.00% SALINE O—-—O swmernsnuws o------o 2.00%SALINE o-——o LES on HH/ “W I I I f ' I L A A ! g l I & l2l4l6l8202468i0l2l4|6|820 PRE-OP POST - OP (DAYS) 83 Rollover the 0.87% saline intake of this group was significantly greater post-operatively (t for related measures-17.32. p<0.01) representing a mean change of free 0.68% pro-lesion to 7.65% post-lesion. The shaa lesioned anissls evidenced no difference cospring pre- arr! post-lesion water and saline intakes (t for related ”nurse-0.57 arr! 0.53 res- pectively. p>0.10). The adrenalectonised PH lesioned group (Figure #1) demomtrated a significant increase in 0.87% saline intake during the post-operative period (t for related ensures-13.65. p<0.01). This rem-esents a scan change of from #327» pro-op to 10.21% post-operatively. Their 2.00% saline solution intakes were not different concerning the pre- and post-lesion periods (t for related ”seizes-0.98. p>0.10). Although the adrenalectosised shaa group iniicated no significant change in 2.00% saline intake post-operatively (t for related measures-0.71. p)0.10) their 0.87% saline intake did increase significantly (t for related measures-6.22. p<0.01). but the scan increase was less than for the sdrenalectomised PH group being h.87% pre-lesion and 6. 50% post-lesion. Hith regard to food intake corrected for body weight changes the pattern is such the ease with the aninals of this experisent as established by those of Experisent 2; nasely a significant decrease in food ccnsunptien following surgery. Significant decreases in post-operative food intake were registered by the PH group sain- tained on tap water only between day 20 pro-op and day 1 post-op (Figure uz. t for related mares-3.91. No.01). This was also true for the PH and shaa groups saintained on tap water an! 0.87% saline (Figure #3: t for related resource-6. 56 an! “.145 respectively. p0.05). This was a mean change of from 2.27% pre-op to 2.58% post-op. The tap water sham group demonstrated no difference between pre- aui post-lesion urine volume output (t for related measures-1&8. p>o.iO). This was likewise the case for the sham group maintained on tap water ani 0.87% saline solution (Figure #63 t for related measures-0.60. p>0.10) while the corresponding PI-I lesioned group revealed a significant increase in urine volume output post-operatively (t fer related messures-ih.71. p<0.01). This represents a mean change of from 2.h7% pre-lesion to 7.331 post-lesion. The adremlectomised groups (Figure II?) both evidenced increased urine output pest-operatively (t for related measures-10.19 and 6w37. PH and sham groups respectively. p<0.01). The mean change for the PH lesioned group was from 3.57% pre-op to 7.21% post-op. while the mean change for the sham lesioned group was from 3.1I2% pro-op to “.751 post-operative. Turning to the urine constituent data it must be noted that with regard to sodium concentration there was a significant drop 89 Figure #6. Mean (1 S.E.) urine volume outputs corrected for differences in body weight of the posterior medial hypothalamic and sham lesioned groups maintained on tap water and 0.87% saline solution for the last ten days Of a twenty day pre-lesion period and twenty days post-operative. Figure 47. Mean (1 S.E.) urine volume outputs corrected for differences in body weight Of the adrenalectomized posterior medial hypothalamic and sham lesioned groups maintained on 0.87% and 2.00% saline solutions for the last ten days Of a twenty day pro-lesion period and twenty days post-Operative. URINE OUTPUT/BODY WEIGHT x IOO URINE OUTPUT/BODY WEIGHT x IOO r "I (I! ON I. II ‘ \ \ I, ‘\ ’ ‘ \ . I i I \ I, 77%“.-- .3» .6” \§‘-§- -.§.v "8‘ WO é"§ PH GROUP WATER t 41: l. I 4.6. .8795 SALINE e------e SHAH GROUP WATER .0715 SALINE O-------O L A A A I I . . I I | I I I2I4IsI82024ssIOI2I4IeIszo PRE-OP POST-OP (DAYS) l .0; I ’ LESION I v {It 8» I I; ‘X ’1’ I “t ,+ I \ ’ J . I +uy' Y ' I 6|- ' I, ’-’§ -\+ I I . P ' 'I I’é’ I , I 4 '- *siéz\é : ;’§.-+ % “é 1' ‘ ' » I4 I? 2r- : PH .3735 SALINE I 2.00% SALIREe- ----- e . I saw 37% SALINE I I 2.OOII$AI.INEO- ----- O O . . . I L I2I4IGI8202 4 6 a IOIz I4IGIezo PRE-OP POST-0P (DAYS) 91 Figure #8. Mean (1 S.E.) urine sodium concentrations of the posterior medial hypothalamic and sham lesioned groups maintained on tap water for the last ten days of a twenty day pre-lesion period and twenty days post-operative. Figure 49. Mean (1 S.E.) urine sodium concentrations of the posterior nedial hypothalamic and sham lesioned groups maintained on tap»water and 0.87% saline solution for the last ten days of a twenty day pre-lesion period and twenty days post-operative. No couc. m mEq/L. $3; No CONC. IN mEq./L. 92 I u PH GROUP WATER G———e “94°" SRAw GROUP WATER o-———-o 400 300 200 I00 0 ‘ ‘ A IZMIGIBZOZ468l0l2l4|6l820 PRE-OP POST-OP (DAYS) PH GROUP WATER .e7s5 SALINE e- ------ O LES on SHAH GROUP WATER 400 .3795 SALINEé o- ----- O 300 If: i§I II IMI II MI “I {d ”W HI p .00; 0‘1 111 Pl A 111;. I2 MIG I8202 4 6 8 IOIZ I4I6I820 PRE-OP POST-OP (DAYS) -------------— -. F b 93 post-operatively denonetrated by the HI lesioned tap water only group (Figure #8; t for related seams-3.51. “0.01). The sean chenge wee froa 178.02 liq/L. pro-op to 138.25 sin/L. poet-opustive. The corresponding sha- lesion group wna not different in this respect. M was an initial pro-lesion difference in urine oodiun concentration between the erperinental and shn group with the she- group evidencing a significsntly elevated lean concentration (238.76 IEq/L. an conpnred with the m lesion group's 178.02 nEq/Lu 910.23. p<0.01). The urine sodiun concentrstion for the two groups phced on tap water an! 0.87% ssline solution (Figure It9) followed a sinilar gotten in thnt the sha- grcup showed no difference pre- and post-lesion (t for rented tenures-1A8. p>0.lo) while the PH lesion group remled a decreesed nean eodiun concentration Of fron 255.73 pro-op to 200.28 post-op (t for related mares-3.35, p<0.01). The urine scdiun concentrations for the adrenalectonised groups (Figure 50) are very nuch different fron the other four groups in thnt both the sha- and PI! lesioned groups' sodiun concentration pro-op wns such higher with learn of £533.22 am! 186.25 IBq/L. respectively. than post-operatively where the aeans were 359.78 and 29m? nEIq/L. respectively. For both the shun Ana exper- inentel lesioned groups there were significant decreases in urine sodiun concentration (t for rented mourn-6.75 and 8.3“ respectively. “0.01). The El and shan lesioned groups neintained on tsp water (Figure 51) revealed no chsnge in total urinary sodiun excreted pre- our! post- operatively (t for related neuures 1A2 snd 1.03 respectively, 1370.10). The PI! lesioned group provided tnp water erd isotonic saline solution Figure 50. Mean (1 S.E.) urinary sodium concentrations of the adrenalectomized posterior medial hypothalamic and sham lesioned groups maintained on 0.87%‘and 2.00% saline solutions for the last ten days of a twenty day pro-lesion period and twenty days post-operative. Figure 51. Mean (* S.E.) total urinary sodium excreted in 24 hour units By the posterior medial hypothalamic and sham lesioned groups maintained on tap water for the last ten days of a twenty day pro-lesion period and twenty days post-operative. No cow c. m mEqJL. TOTAL URINARY No (mEq/doy) 95 I I I I I 40 I : x‘H : \ I “ w ' +‘~{s~ ”§\ +.+ ' ‘i' ‘\ a" LESION \i’I’é I I I 20 n : I PH .8796 sauna : 2.00% SALINE .- ------- o ' : sum .B‘HISALINE . 2.00% SALINE o- ------ -o I l2 l4 l6 I820 2 4 s a no l2 :4 l6 IBZO PRE-OP POST-OP (DAYS) PH GROUP WATER O——O SHAH GROUP WATER O——-——O LE SION l2l4|6|8202 4 6 a IOIZ mustazo PRE-OP POST-OP (DAYS) 96 Figure 52. Mean (‘5: S.E.) total urinary sodium excreted per 2“ hour units by the posterior medial hypothalamic and sham lesioned groups maintained on tap water and 0.87% saline solution for the last ten days of a twenty day pre-lesion period and twenty days post-operative. Figure 53. Mean (* S.E.) total urinary sodium excreted per 24 hour units by the adrenalectomized posterior aedial and sham lesioned groups maintained on 0.87% an! 2.00% saline solutions for the last ten days of a twenty day pro-lesion period and twenty days post-operative. 97 I PH GROUP wATER - .8735 sum: 0- ..... e LES!“ sum GROUP WATER 2 : .OT'A SALINE o ----- O )Hi+{ §"§"§ {k ‘.§. § 0 ‘\ ’l I—o-I 2'" +499 T 356-: @T‘bwzzfi T? i TOTAL URINARY No (mEq/doy) i3 111 JLALali l2|4l6l8202 4 GOIOIZMIBIBZO PRE-OP POST- OP (DAYS) i' I I A I E LESION / \\ , \ 2 : } i] \\ - -- U | I ‘s I o‘ s u I I s . x , O ' i I \\ : I _%’ X \ | ’ z ’ ‘\%' I ’ s+ ’ \ - )- §’ I § C '2 y! : ‘2‘ I? - I ’ ¢ I :3 e - : -’ I ‘ I PH .8756 sum: '5 4» : 2.00% SALINE e ----- e " : 5mm .3795 SALINE ' 2.00% SALNE O ----- OJ 0 .1 L I L L IZMIIGIBZOZ 4 £3 8 ILOIAZ I4nslszo PRE-OP POST-OP (DAYS) 98 (Figure 52) amt-mud a significantly increased total urinary sodium output post-operatively while the corresponding sham group showed no change (t for related measures-21.99 an! 2.05; p(0.0i and p>0.05 respectively). The adrenalectosised group maintained on isotonic ani 2.00% saline solution (Figure 53) both evidenced increased total sodiua outputs post-operatively (t for related measures-7.405 and 6.91 m the PH ani shaa lesioned group respectively. p<0.01). The urine potassiua concentrations exhibited by the PH lesion group on tap water (Figure 54) was significantly lower post-lesion than pro-lesion (t for related ensures-3&2. p<0.01) depicting a mean drop of from 187.“? aEq/L. pre-op to 153.90 aEq/L. post-operatively (t for related measures-1.39. p>0.10). This same pattern persisted with the two groups maintained upon tap water an! 0.87‘ saline solution (Figure 55). The shaa lesion group displayed no change pre- and post-operatively (t for related measures-1.63. p)0.10) while the PM lesioned group decreased its potassius concentration significantly (t for related measures-8.30, p<0.01) from a scan of 168.15 aEa/L. pro-Op to “9.33 IEQ/L. post-operative. me adrenalect- oaised groups (Figure 56) in this imtance obeyed the pattern of the Other four groups in that the shaa group was not different in potassiua Concentration pre- and post-op (t for related measures-1.79. p>0.05). while the PH lesioned group significantly decreased its potassiua concentration (t for related measures-6.31. p<0.01) from a mean of 13“.? IEq/L. pro-Op to 59.05 aEq/L. post-lesion. There were pro-lesion basal rate differences in specific gravity bOtueen the two youps with tap water only available (Figure 57: t‘19.5I), p(0.01) with the sham lesion group evidencing a mean Figure 5b. Mean(t S.E.) urinary potassium concentrations of the posterior medial hypothalamic and sham lesioned groups maintained on tap water for the last ten days of a twenty day pre-lesion period and twenty days post-operative. Figure 55. Mean (1 S.E.) urinary potassium concentrations of the posterior medial hypothalamic and sham lesioned groups maintained on tap water and isotonic saline solution for the last ten days of a twenty day pro-lesion period and twenty days post-operative. K CONC. IN mEqJL. K CONC. IN mEq./L. 100 300 200;.- mol 9 I--------‘ 2 L--------- PH GROUP WATER .———C SHAM GROUP WATER O————O I2I4lelezoz4GsIOIzI4Isnszo PRE-OP POST-OP (DAYS) I PH GROUP WATER .sTx SALINE e- ------ e LES 0" sum GROUP WATER .3795 SALINE o------o ,a'. “x ._qi_. I‘ ’\ v-b-§~ \ I I2 I4 I6 I I -----—--------I - . #04 L 4 IO 20 2 L PRE -OP I I . “{K -u§~\§\§; § ‘§' ‘§-..§ \ \ \ \ . I“ ?'+~i‘~-,---§.~i’_” * O O 8 IO (2 I4 (‘6 IAG 2A0 P0 ST - OP (DAYS) 101 Figure 56. Mean (t S.E.) urinary potassium concentrations Of the adrenalectomized posterior medial hypothalamic and sham lesioned groups maintained on 0.87%Tand 2.00% saline solutions for the last ten days of a twenty day pre-lesion period and twenty days post-operative. Figure 57. Mean (1 S.E.) urinary specific gravity Of the posterior medial hypothalamic and sham lesioned groups maintained on tap water for the last ten days Of a twenty day pro-lesion period and twenty days post-operative. K CONC. IN mEqJL. TOTAL SOLIDS (REFRACTIVE INDEX) 102 200!- ”T oi A A 1 I2 I4 IS IS 20 PRE-OP )- ION PH .87'5 SALINE 2.00 7. SA LINE 0 ...... g SHAH .BTVGSALINE 2.00% SALINE o. ..... o “xi—H+—§~~.§-H-§ (“t-<1». A A A 2468IOI2I4IGI820 POST-OP Y4“? *4“? A A L . (DAYS) W I I so I I I I I I 46 | I I LESION I I 42 . I I I I I as I . PH GROUP WATER e——* : GHAIA GROUP WATER o——-O o L 1 a a A ' 1 1 a 4 1 1 A A A 1 I2I4IG|8202 4 6 s IOIZ I4IGIszo PRE -OP POST- OP (DAYS) 103 Figure 58. Mean (* S.E.) urinary specific gravity Of the posterior medial hypothalamic and sham lesioned groups maintained on tap water an! 0.87% saline solution for the last ten days of a twenty day pro-lesion period and twenty days post-operative. Figure 59. Mean (3 S.E.) urinary specific gravity of the adrenalectomized posterior medial hypothalamic and sham lesioned groups maintained on 0.87% and 2.00% saline solutions for the last ten days Of a twenty day pro-lesion period and twenty days post-operative. TOTAL SOLIDS (REFRACTIVE INDEX) TOTAL SOLIDS (REFRACTIVE INDEX) 104 SOOI PH GROUP WATER .eTx SALINE e- ...... . SHAM GROUP WATER .87 '5 SALINE O- ----- O r M (I) O 2 I I I I I I I I | . 460' I, ‘\ ‘I 1' '§” §X ’ ‘§-‘%C : “‘ P ' | t . a . I § H 420 g}, ' \ I ‘I I { § I‘x’ ' ‘ ” -- \ 1’ ‘f 330 I f \‘Q/é“ ,9 I \\§a l I o 1 1 L 1 1 I A l a a 4 4 1 A L L |2I4IGI8202468IOI2I4I6IOZO PRE-OP POST-OP (DAYS) I PH .ens SALINE 50 LESION 2.00% SALINE .' ------ C I SHAH .87%SALINE : 2.00%SALINE o- ----- o I x 4 *‘x I : I I '% ‘ I’ ‘ I ‘ I ‘I ’§'% “5 I I 42 g ‘. ' I I ' f i pm} i I I 38 I I I I .r) J J 1 l A ' a A I A A A a A A I2 PRE-OP I4 IG I8202 4 G 8 IO l2 MIG IazO POST-OP (DAYS) 105 refractive index of 1.3516 and the Pfl lesion group 1.3h68. The PH lesion group revealed significant decreases in specific gravity (t for related seasures-6.05. p<0.0i) with a lean refractive index of 1.3“69 pro-op to 1.3“h2 post-op. The corresponding shas group>did drop slightly but not significantly fros a lean refractive index of 1.3517 pro-op to 1.3503 post-operative (t for related seasures- 1.99, p)0.05). The groups placed on tap»water and 0.87%Isaline solution (Figure 58) both indicated drops in specific gravity post- operatively. The shaa lesion group decreased fros a refractive index of aean 1.3h56 pre-op to 1.3““5 post-op (t for related seasures-3.06. p<0.01) while the PH lesion group dropped free a scan refractive index of 1.3hél pro-op to 1.3390 Post-op (t for related seasures- 5.#l. p<0.01). The adrenalectosised shaa lesion group (Figure 59) revealed no difference between its pre- and post-lesion refractive index (t for related seasures-1.77, p>0.05) however. the PH lesion groupIdropped with a seen refractive index of 1.3““9 Pr0-op and 1.3“05 post-op (t for related seasures-5.77. p<0.01). Measuresents taken on blood constituents are offered in Table 2. Serus sodius and potassiun nean concentrations pre- (A) and post- lesion (B) are given for each of the six groups as are protein and hesatocrit values. The standard errors of the seans are also provided. One of the scat interesting aspects of the blood data concerns the pre-lesion depressed sodius. potassius and protein concentrations for the adrenalectosised groups and the recovery of these seasures after the experisental or shaa lesions. The hesatocrit however. 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 N0.0 0000 00 +1 +1 +I « HHmUOBwater intake. however. the eagnitude of lesion dassge 110 suffered by this PH lesion group was found to be less than that incurred by the other two PH lesion groups (Table 3). It is inportant to point out that with the PH lesion tap and 0.87% saline solution group there was likewise no change in tapnwater intake pre- to post- operatively. With reference to the adrenalectonised PH group again there was a significant increase in isotonic saline solution intake post-lesion but no change in 2.00% saline intake. Danage in the posterior nedial hypothalanic area. therefore does notlappear to affect tap water consunption in nornal anisals or 2.00% saline selution consunption in adrenalectonised rats but with both preparations the effect is specific to 0.87% saline solution intake. There is a corresponding increase in urine volune output acconpanying the increase in isotonic saline solution ingestion. Uith regard to urinary constituents there is a general decrease in concentration of the electrolytes neasured for the two PH lesion groups given access to 0.87% saline. Specific gravity of the urine also»decreased with these groups. The total urinary sodiun excreted per day increased significantly after dassge to the posterior nedial hypothalanus of these 1wo groups. This increase was in contrast to that found in.Experinent 2 in which conparable groups revealed no such post-lesion increase in.sodiun excretion. Analysing individual aninals with regard to total sodius consuned versus that excreted. the general conclusion of Experinent 2 is supported. i.e.. no “salt loss syndrone" as reported by Cort (i963a). Rather it appears that the excretion of sore sodiun accospsnies an exaggerated inhibition of isotonic saline post-operatively (refer to the individual records in the Appendix). The adrenalectonised groups recorded a very nuch 111 elevated pro-operative urinary sodiun concentration of.approxinately 000.050 nEq/L. conpared with nornal anisals' sodiun concentration of tron 200-300 IEq/L. This has been well docunented by Richter (1939) and Bare (1909) who suggest that there is a decrease in sodiun and chloride reabsorption fron the glonerular filtrate of the kidney resulting in the excretion of increased concentrations of these ions in the urine and a decrease in the concentration of these ions in the extracellular fluids. of the body including the blood. This is supported by the blood serun sodiun levels reported in this experisent. 0f additional interest is Richter's suggestion that the aninal's attenpt to relieve its body salt deficiency in the fore of an increased saline solution intake nay not be due to a learning process but rather a chenical change in the taste nechanisn in the oral cavity. Hhatever the physiological characteristics underlying this altered salt appetite it nay be further nodified by posterior nedial hypothalanic lesioning resulting in even greater quantities of isotonic saline solution intake. In that adrenalectonised as well as nornal anisals reveal this heightened saline intake following posterior nedial hypothalanic dassge the adrenal corticosteriods say be elininated as possible contributors to this effect. The tap and 0.87% saline solution PH group of this experinent subsequently underwent s 35Iday period with only taplwater available in the interin between Experinent 3 and Experinent u and during Experinents # and 5. The anisals survived well reducing their urinary electrolyte concentrations considerably. Once again as in Experinent 2 there were no signs of ill health as predicted by Cort (1963.) for posterior nedial hypothalanic lesioned anisals. 112 Recently Kawasura et a1. (1970) have reported cellular unit recording data that say be pertinent to the interpretation of the present findings. Rats were anesthetized with nesbutal and then paralysed with Flaxedil. Data on stosach distention was reported utilising the ballon nethod. One ventronedial hypothalanic neuron had a spontaneous discharge of 7 spikes/sec which rose to 25 spikes/sec during distention. This cell. however. did not respond to the application of a 6%.saline solution to the tongue. Other units. particularly posterior hypothalanic cells. were very such affected by such stisulation. A cell located in the nedial part of the posterior nucleus saintained a spontaneous discharge rate of 5 spikes/sec but with the application of 6% NaCl the rate accelerated to 10 spikes/sec. Electrical stisulation of the tongue also increased firing rate. In contrast a neuron at the sane anterior-posterior level but in the lateral hypothalasus evidenced a spontaneous firing rate of 12 spikes/sec which dropped to 6 spikes/sec with the placesent of 6% final on the tongue. Electrical stimulation of the tongue in this instance decreased the firing rate of the neuron. There was no response by either neuron when a 1% NaCl solution was placed on the tongue. The saline solutions were rinsed off with water between tongue applications. Kawanura's results suggest that hforsation fron tongue receptors say be channeled directly to the posterior nedial and lateral hypothalasi concerning salinity concentrations of solutions present in the south. Also of interest is the finding that a 1%»NaCl solution applied to the tongue resulted in no change with regard to unit discharge activity in these nerual regions. It say be that dassge inflicted upon the posterior nedial hypothalasus in sose way elevates existing thresholds governing the quantity of isotonic saline inbibed by rats. 113 EXPERIMENT “ Pitressin Influence upon the Experinental Lesion This experiment investigated the effect of subcutaneous pitressin injections upon the increased fluid intake revealed by posterior medial hypothalamic lesioned animals. There was concern over the possibility of extreme body hydration due to the interaction of pitressin tannate and the exaggerated saline intake. Therefore between post-operative day 20 of Experiment 3 and the first pitressin injection of this study a period of ten days with the non-adrenalectomised animals on tap water was isposed. The adrenalectomised groups were maintained on 0.87% saline solution during this period. Although an increased tap water intake was exhibited by some of the non-adrenalectomised animals the possibility of hyperhydration due to saline ingestion was reduced. Subiects The animals of Experiment 3 were employed in this experiment. Their maintenance in metabolism cages was continued and closely approximated the conditions of Experiment 3. The animals were of course older at the initiation of this experiment. approximately 170 days of age. Procedure The animals were kept in the same groups as assigned in Experiment 3. On post-lesion day 31. h hour readings were begun at 0100. These reading periods obeyed the format described in earlier experiments with the recording of metabolic intake-output measures and the retaining of a urine sample from each animal at each reading period. Urine analysis was also characteristic of the established procedure. Immediately following the 2100 hour reading period of post-operative day 31 one- 110 half of the animals received a subcutaneous 0.2 u/1oo grass dosage2 of pitressin tannate in oil (Ford-Davis a Company). The other half received an equivalent volume of pure peanut oil. Assignment of the animals to one of the two categories conformed with the following procedure. Each of the three groups had previously been randomly separated into six control animals which received sham lesions and six experimental animals which received posterior medial hypothalanic lesions. During this experiment. within each of these two subgroups the animals were further randomly divided into three experimental injection animals. to receive pitressin. and three control injection animals to receive peanut oil. During post-lesion day 32. b hour readings continued; these readings followed the injections. At the completion of post-lesion day 32 the animals were allowed a three day recovery period during which time metabolic intake-output measures were recorded twice daily at 0800-0830 and 2000-2030. A urine sample was retained and analysed for each animal at the morning reading period. On post-lesion day 36. 4 hour readings were once again initiated in the same fashion as five days earlier. Immediately following the 2100 hours reading period subcutaneous injections of pitressin were administered those animals that had earlier served as control injection subjects and an equal volume of peanut oil was provided these animals that had earlier served in the pitressin injection group. In this way each animal received a pitressin injection and a control injection separated by five days. Experimental and control data were thus collected for each animal. 2This pdtressin tannate dosage represents a comprosise between the dosages found to be effective for rats by other investigators (nubar et a1. 1969; Morrison et al.. 1967. Smith and HcGInn. 1960). 115 Results The four hour reading period data for the 20 hour period preceding injections and the 2“ hour period immediately following the injections are included in the figures of this section. Each point in these figures represents the mean of the six animals of the designated group. Standard errors of the means are also provided. The procedure called for a counter balanced design. i.e.. one-half of a group of six animals received the experimental treatment during one 5 day period and thealternate half of the group received the experimental treatment during the next 5 day period. Basal rate data during the pre- injection 2“ hour period was thus collected twice for a given group. These pro-injection data were combined for each group for each 0 hour reading period. During the post-injection 2“ hour period the results of the pitressin injection are compared with the results derived from the control injection of peanut oil for each group. Figure 60 represents the tap water intake recorded for the two groups of animals maintained on taptwater only through Experiment 3. During the pro-injection 20 hour period there were significant dif- ferences between the posterior medial hypothalamic lesioned group and the sham lesioned group with regard to tap'water intake (t-b.ub. p(0.01). The PH lesion group yielded a mean of 8.7 ml/h hour units and the sham group 5.“ ml. Comparing within groups for the pre- and post-peanut oil injection periods there were no differences (t for related measures- l.60 and 0.52. PH and sham groups respectively. p)0.10). There were likewise no differences between the pre- and post-pitressin injection periods within groups (t for related measures-0.6? and 0.33. PH and sham groups respectively. p>0.10). There was a difference between 116 Figure 60. Mean (1 S.E.) tap water intake in 0 hour units exhibited by the posterior nedial hypothalamic and sham lesioned groups 2“ hours prior to and 20 hours following independent applications of pitressin and peanut oil injections. Figure 61. Mean (e S.E.) food consumption in 0 hour units for the posterior nedial hypothalanic and sham lesioned groups maintained on tap water 20 hours prior to and 20 hours following independent applications of pitressin and peanut oil injections. TAP WATER INTAKE (ML/4hour units) 117 30F wATER PH e WATER SHAH O PITRESSIN ---- PEANUT OIL —— FOOD INTAKE (GM/4 hour unIIs) -----—-----------I 0 A a L A L A A P L L A OIOO 0500 0900 I300 I700 2|00 OIOO 0500 0900 |300 I700 2|00 PRE-INJ. POST- INJ. (hours) TAP WATER GROUP PH e: PITRESSIN ---- 9 TAP WATER GROUP SHAM o: PEANUT OIL -——— I I I I I l I 6" ' I I I I I I I 3_ I I I I I I I I o in . I . L A I L . J . . i OIOO 0500 0900 I300 I700 2I00 0I00 0500 0900 |300 I700 2I00 PRE-INJ. POST- INJ. (hours) 118 the two groups comparing their reactions to pitressin treatment (t-3.87. p(0.01). The PR lesion group revealed a mean of 7.12 ml/u hour units while the sham lesion group's mean was 4.99. Figure 61 considers food intake for the two groups maintained on tap water. During the pro-injection 2“ hours there were no differences between the PH and sham lesioned groups (t - 1.69. p>0.10). Conparing each group's food intake during pre- and post-peanut oil injection periods there were non-significant differences (t for related measures- 0.87 and 0.10. PH and sham lesion groups respectively. p>0.10). Considering each group's reaction to pdtressin injection the PH group revealed no change (t for related measures-1.38. p>0.10) and the sham lesioned group indicated no difference (I: for related measures- 1.38. p>0.10). Comparing the two groups during the post-pitressin period the PH group registered a significantly higher food intake than the sham lesioned group (t-2.28. p<0.05) while the groups were not different during this period following peanut oil injection (t-o.57.p>o.io). Figure 62 considers urine volume output of the two tapiwater groups. no differences existed between or within the groups during the post- injection period. Pro-injection the experimental and sham groups were significantly different (t—II.)o. p<0.01). The PIT lesioned group's mean urine volume output was 6.10 and the sham lesioned group's mean was 4.22 ml. Figure 63 indicates the sodium concentrations of the urine in mEq/L. during these two 2b hour periods for the groups maintained on taptwater. There were pro-injection differences between the two groups (t-7.33. P<0.01) with the PH group yielding a mean concentration of 119 Figure 62. Mean (1 S.E.) urine volune excretion in 4 hour units exhibited by the posterior nedial hypothalamic and sham lesioned groups maintained on tapuwater. for the 20 hours prior to and 2“ hours following independent appli- cations of pitressin and peanut oil injections. Figure 63. Mean (1 S.E.) urinary sodium concentration in 4 hour units for the posterior nedial hypothalanic and sham lesioned groups provided tap water. for the 24 hours prior to and 20 hours following independent applications of pitressin and peanut oil injections. URINE OUTPUT (ML/4hour units) IN mEq/L No CONC. 1 2’0 20f TAP GROUP PH 0 b TAP GROUP SHAM O PITRESSIN ---- PEANUT OIL '— OIOO 0500 0900 I300 I700 200 OIOO 0500 0900 I300 ITOO ZIOO PRE-IN»). POS T- INJ. (hours) 400 TAP GROUP PH 0 TAP GROUP SHAM 0 PITRESSIN ---- PEANUT OIL — OIOO 0500 0900 I300 I700 PRE-INJ. ZIOO OIOO 0500 0900 I300 I700 ZIOO POS T- INJ. (hours) 121 155.75 mEq/L. and the sham lesioned group 204.25 mEq/L. Each group revealed no change comparing pre- and post-peanut oil injection (t for related measures-0.38 and 0.83. PH and sham groups respectively. p>0.10). The PR group.also registered no change comparing pre- and post-pitressin injection (t for related measures-0.84. p>0.10) and the sham lesion group also demonstrated no significant change in sodium concentration during the post-pitressin injection period (t for related measures- 0.73. p>0.10). Comparing the two groups during the post-pitressin injection period there were differences indicated (t-2.73. p<0.05) with the PH group yielding a mean of 1h?.83 aEq/L. and the sham group 178.83. Figure 6“ considers the urine potassium concentrations registered by the two tap water groups during the periods under discussion. The groups were significantly different from one another on this measure during the pro-injection period (t-7.77. p<0.01). The PR lesion group indicated a mean of 128.00 mEq/L. while the sham group revealed a mean of 180.00 nEq/L. potassium. Each group registered no difference comparing pre- and post-peanut oil injection periods (t for related aeasures-0.87 and 0.89. PH and sham groups respectively. p>0.10). This was also true for each group comparing pre- and post-pitressin injection periods (t for related measures-0.15 and 0.55. PH and sham lesion groups respectively. p>0.10). Conparing the two groups during the post-pitressin injection period there was a significant difference (t-3.81. p<0.01) with the PH group yielding a mean of 131.50 and the sham group 168.33 nEq/L. Figure 65 concerns the urine total solids measures registered for each of the two groups kept on tapiwater. There was a significant difference between the two groups during the pro-injection period 122 Figure 6#. Mean (* S.E.) urinary potassiua concentration in 4 hour units for the posterior medial hypothalamic and sham lesioned groups maintained on tap water. for the 2“ hours prior to and 2“ hours following independent applications of pitressin and peanut oil injections. Figure 65. Mean (* S.E.) urinary specific gravity in 4 hour units for the posterior medial hypothalanic and sham lesioned groups saintained on tap water. for the 20 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. IN mEq./L. K CONC. INDEX) IREFRACT IVE TOTA L SOLIDS 123 200 ISO I00 50 TAP GROUP PH 0 TAP GROUP SHAM O 1 L A L PITRESSIN ---- PEANUT OIL — A I A A l J OIOO 05000900 I300 I700 ZIOO OIOO 0500 0900 I300 I700 ZIOO PRE- INJ. POST- INJ. (hours) 350; TAP WATER GROUP PH 0. TAP WATER SHAM o: PITRESSIN -—-— PEANUT OIL —- 4L A OIOO 05000900 I300 I700 ZIOO OIOO 0500 0900 I300 I700 2I00 PRE ~ INJ POST - INJ. (hours) 12b (t-8.75, p(0.01) with the PH group registering a mean of 1.31qu specific gravity and the sham group 1.3“85. Comparisons for each group concerning pre- and post-peanut oil injections resulted in a significant difference for the PH lesioned group but not for the sham group (t for related measures-2.03. and 1.96, p(0.01. p>0.05. PH and sham lesioned groups respectively). There was no difference for the sham group pre- to post-pitressin injection (t for related measures-1.71. pDO.10) nor did the PH group reveal a difference (t for related measures-1.77. p>0.10). Although not significant there were urine specific gravity increases for the PH group following pitressin injection that eventially decreased until by 1300 hours. i.e.. 16 hours after injection. the measures were equal wflh pre- injection values. The two groups were significantly different comparing their post-pitressin injection periods (t-2.?7. p<0.05) with the PH group yielding a scan specific gravity of 1.3965 and the she- sroup 1.3h89. Figure 66 begins an analysis of the two groups maintained on tap»water and isotonic saline solution during Experiment 3 but placed on tap water only during the present experiment. This figure concerns the water intake recorded for these groups once again during pre- and post-injection periods. Differences existed between the two groups con-poring the pre-injection period (tn-3.2a. p<0.01). The PH group had a mean water intake of 8.88 ml/h hour periods. the sham group 6.89. Comparing pre- and post-peanut oil injection periods for each group there were no differences (t for related measures- 1.03 and 1.20. PH and sham groups respectively. p>0.10). However 125 Figure 66. Mean (i S.E.) taprwater consumption in 4 hour units exhibited by the posterior nedial hypothalamic and sham lesioned groups provided tapiwater. no saline for the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. Figure 67. Mean (1 S.E.) food intake in 4 hour units for the posterior nedial hypothalamic and sham lesioned groups saintained on tap water. no saline for the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. WATER INTAKE (ML/4 hour units) FOOD INTAKE (GM/4 hour unIIs) um TAP NO SALII’E PH TAP N0 SALINE SHAH O O ---------------I PITRESSIN ---- PEAN. OIL — 5 _ o 1 1 A A A A A L L a n A 000 05030900 I3“) I700 ZIOO OIOO 0500 0900 Im I700 2'00 PRE-INJ. POST- INJ. (hours) TAP no SALINE PH e : PITRESSIN ---- TAP NO SALINE SHAM o : PEAN. OIL — I I I I I I I 6*- ' I I I I I I I 3.. : I I I I I I o 4 L L J A LI A . L A A . 0000 05000900 I300 I700 ZIOO OIOO 0500 0900 I300 I700 ZIOO PRE- INJ. POST- INJ. (hours) 127 coaparing the groups for pre- and post-pitressin injection periods each revealed significant decreases in water intake (t for related measures-2.87 and 2.77. PH and sham respectively. p<0.05) with the PH group dropping from a mean of 8.88 ml/4 hour units to 5.08. The sham group dropped from 6.89 to 3.97 ml/4 hour units. No differences existed between the two groups comparing their post- pitressin injection periods (t-1.68. p>0.10). Figure 67 concerns food intake for the two groups under consid- eration. Few differences existed within or between the groups for the upsrisons tested. There were significant drops in food intake pre- to post-pitressin injection (t for related measures-2.27. p<0.05); the PH group changed from 3.27 gm/4 hour units to 1.56 post-injection. The sham group's means dropped from 2.45 grams food/4 hour units to 1.53 post-injection however they were not significantly different (t for related measures-1.60. p>0.10). Figure 68 represents the urine volume output for these two groups. Again there were no differences between or within the two groups coaparing pre- arli post-peanut oil injection periods. Each group did reveal significant decreases in urine output comparing pre- and post- pitressin injection periods (t for related measures-5.32 and 5.84. PM and sham groups respectively. p<0.01). This represents a mean drop of from 5.81 to 2.77 Iii/II hour unite for the m group an! 5.22 to 3.24 ml/4 hour units for the shaa group. There were differences comparing the two groups during the post-pitressin injection period (t-2.97. p(0.01). This represents a mean difference of 2.77 Iii/II hour units for the PH group and 3.24 for the sham group. 128 Figure 68. Mean (r S.E.) urine volune excretion in 4 hour units for the posterior nedial hypothalanic and sham lesioned groups provided tap water. no saline for the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. Figure 69. Mean (1 S.E.) urinary sodiun concentration in 4 hour units for the medial posterior hypothalanic and shaa lesioned groups maintained on tap water. no saline for the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. IN mEq./L No CONC URINE OUTPUT (ML./4hour mi") 129 20 F TAP no SALINE PH TAP N0 SALINE sum 0 PITRESSIN ---- PEAN. OIL —— ----------------J a I A A A 1 A J OIOO 05000900 I300 I700 2I00 OIOO 0500 0900 I300 I700 2I00 PRE- INJ. POST- INJ. (hour8) 400 300 200 D .00; 0L TAP NO SALINE PH TAP N0 SALINE SHAM O OIOO 05000900 I300 PRE ~|NJ. PITRESSIN ---- PEAN. OIL — )---------------( 4 ITOO 2I00 OIOO 0500 0900 I300 I700 ZIOO POST- INJ. (hourS) 130 looking now at the urine sodium concentration for these two groups (Figure 69) there were dfferences between the groups during the pro-injection period (t-3.89. p<0.01). The PH group revealed a aean of 133.08 mEIq/L. while the sham group's mean was 156.92. Each group demonstrated no differences compring pre- and post-peanut oil injection periods (t for related measures-1.90 and 1.99. PH and sham groups respectively. p>0.05). Compring each group's pre- arxi post-pitressin injection period there were differences (t for related measures-2.90 and 3.47. PH and shaa groups respectively. p<0.01). The PH group increased its sodium concentration from a pro-injection mean of 125.16 to 182.1? mflq/L. The sham group's corresponiing means were from 153.33 to 208.67 aEq/L. By approximately 1300 hours or 16 hours after pitressin injection the PH group's urine sodium concentration had decreased to its pro-injection level. Comparing the two groups during their post-pitressin injection periods they were different (t-4.82. p<0.01) with the PH group indicating a mean of 182.17 mEq/L. and the sham group 208.67. I Figure 70 considers the urine potassium concentration of these two groups. There were differences between the two group for the pro-injection period (t-4.22. p<0.01) with the PH group yielding a mean of 115.03 aul the sham group 132.33 IEq/L. Compring each group with respect to pre- and post-peanut oil injection periods there was no difference for the PH group (t for related measures- 1.03. p>0.10). The sham group. huever. revealed a significant difference (t for related measures-5.17. p<0.01). There was a mean change of from 134.33 mEq/L. pro-injection to 149.1? postéinjection. 131 Figure 70. Mean (1 S.E.) urinary potassium concentration in 4 hour units for the medial posterior hypothalamic and sham lesioned groups maintained on tap water. no saline for the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. Figure 71. Mean (t S.E.) urinary specific gravity in 4 hour units for the groups maintained on tap water. no saline. medial posterior hypothalamic and sham lesioned for the 24 hour period prior to and the 24 hours following independent applications of pitressin and peanut oil injections. IN mEq./L. K CONC- INDEX) (REFRACTIVE TOTAL SOLIDS 2° TAP N0 SALINE PH e : TAP no SALINE sum 0 g I I I I50 . I I I I I I00 . I I I I I 50'- I I I . : PITRESSIN . PEANUT on. —— o A A A A A A ' A A A A 4 A OIOO 0500 0900 I300 I700 ZIOO OIOO 0500 0900 I300 I700 2I00 PRE-INJ. POST- INJ. (hours) SOOfiAP NO SALINE PH e : TAP N0 SALINE SHAM o g r I I I I 450-» : I I I . I I I I I 400.. | I I I I ’ I I : PITREssm ---- 350 I PEANUT on. -— O A s 4_ A A A ' A n n A 1 n OIOO 0500 0900 I300 I700 2IOO OIOO 0500 0900 l300 I700 2I00 PRE-INJT POST-INJ. (hours) 133 Comparing each group‘with regard to»pre- and post-pitressin injection periods both indicated differences (t for related seasures-6.22 and 8.65. PH and sham respectively. p<0.01). The PH group changed from a pro-injection mean of 115.03 to a post-injection mean of 170.17 mEq/L. while the corresponding change in the sham group was from a mean of 132.33 to 181.97 mEq/L. post-injection. The two groups were different from one another during the post- peanut oil injection period (t-6.25. p<0.01) with the PH group yielding a mean of 122.33 and the sham group 149.33 mEq/L. During the post- pitressin injection period they were also different (t-4.05. p<0.01) with the PH group revealing a mean of 170.17 and the sham group's mean was 181.97 mEq/L. During the post-pitressin 24 hour period neither group revealed a tendency to begin falling back to the urine potassium level registered during the precinjection periods. Figure 71 concerns the urine total solids recorded for each group during the indicated observation periods. There were pre- injection differences for the two groups (t-3.89. p<0.01). The PH group had a mean of 1.3425 and the sham group's urine specific gravity mean was 1.3437. Each group similarly registered differences comparing pre- and post-peanut oil injection periods (t for related measures-7.14 and 7.53. PH and sham groups respectively. p<0.01). The PH group's mean changed from 1.3425 pro-injection to 1.3439 post-injection while the sham group changed from 1.3“37 to 1.3455. Hithin each group there were significant changes comparing pre- and post-pitressin injection periods (t for related measures-5.44~and 7.10. PH and sham groups respectively. p<0.01). The PH group 134 Figure 72. Mean (* S.E.) isotonic saline consumption in 4 hour units for the medial posterior hypothalamic and sham lesioned adrenalectomised groups maintained on 0.87% saline solution. during the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. Figure 73. Mean (r S.E.) food consumption in 4 hour units for the medial posterior hypothalamic and sham lesioned adrenalectomized groups maintained on 0.87% saline solution during the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. 087% SALINE INTAKE (ML/4 hour units) FOOD INTAKE (GM/4 hour units) 135 PITRESSIN ---- PEAN. OIL — T 30? ADRENAL PH 0 AORENAL sum 0 v 20p ---------------+ \ I 3 \ I \\ el \ o A A L # J + J L 4* J L 0I00 05000900 l300 I700 ZIOO OIOO 0500 0900 ISOO I700 ZIOO PRE- INJ. POST- INJ. (hours) ADRENAL PH e : PITRESSIN ---— 9 ADRENAL sum 0 : PEAN. on. — I I I I I I E 5r a I I I I I : 3_ u I I I I I i o L L L J . L . 0'00 05000900 |300 I700 ZIOO OIOO 0500 0900 ISOO I700 2|00 PRE-INJ. POST- INJ. (hours) 136 changed free a aean of 1.3425 pro-injection to 1.3492 post-injection. Corresponding alterations for the shaa group were free 1.3437 to 1.3504. Coaparing the two groups during post-pitressin injection periods they were different (t-2.10 . “0.05). And they were different during the post-peanut oil injection periods (t-4.95. mom). with Figure 72 begins an analysis of the results derived free the two adrenalectoaised groups saintained on isotonic saline during this experisent. In this figure 0.87%.saline solution intake is considered. There were differences between the two groups during the pro-injection period (t-3.69. p<0.01). The PH group yielded a scan of 16.54 al/4 hour units while the shaa group's scan was 10.42. Coaparing each group'during pre- and post-peanut oil injection periods there were no significant changes (t for related seasures-0.0§ and 1.62, PH and shaa groups respectively. p>0.10). The shaa group revealed no change coaparing pre- and post-pitressin injection periods (t for related aeasures-1.86, p>0.05). The PH group revealed a non-sign- ificant decrease in isotonic saline intake (t for related aeasures- 1.37. p>0.10) of free a seen of 16.45 to 10.53 a1/4 hour units. There was no difference between the two groups coaparing the post-pitressin injection period (t-1.46. p>0.10). Figure 73 considers the food intakes of these groups. Coaparing the groups during the pro-injection periods there was no difference (t-0.08, p>0.10) with the PH group yielding a aean intake of 3.86 ga/4 hour units and the shaa group 3.82. Each group registered non- significant decreases in food intake coaparing pre- and post-peanut oil injection periods (t for related aeasures-1.15 and 1.38. PH and shaa groups respectively. p>0.10). For the PH group there was a scan 137 drop of free 3.86 ga/4 hour units pee-injection to 2.56 poet-injection. Corresponding values for the sham group were 3.82 to 2.08 ga/4 hour units. There were also non-significant drops for each group coaparing pre- and post-pitressin injection periods (t for related aeasures-1.23 and 1.54, PH and sham groups respectively. p>0.10). These changes were free a pro-injection aean of 3.86 to a post-injection aean of 2.33 ga/4 hour units for the PH group and flea 3.82 to 2.47 for the shaa group. The two groups were not different free one another'during the post-peanut oil injection period (t-1.34. p>0.10) or during the post-pitressin injection period (t-o.78. p>0.10). Figure 74 concerns the urine voluee output of the two adrenal- ectoeised groups. Coaparing the groups during the pro-injection periods there was a significant difference (t-6.10, p<0.01). The PH group's lean was 12.19 al/4 hour units while the loan of the shaa group was 7.72. Coaparing each group during pre- and post-peanut oil injection periods there was no difference for the PH group (t for related aeasures-0.05. p>0.10) with a pro-injection seen of 12.19 and post-injection of 12.n4 a1/4 hour units. The shaa grouprdid indicate a significant difference coapering these periods (t for related aeasures-2.65, p<0.05). The scan change was free 7.72 pro-injection to 5.61 a1/4 hour units post-injection. Coaparing within each group regarding pre- and post-pitressin injection periods there was a significant decrease in urine output registered free the PH group (t for related aeasures-2.73, p<0.05) representing a seen change of froa 12.19 to 6.85 al/4 hour units. Hhile the shaa group also indicated a significant change (t for reited aeasures-3.71. p<0.01) 138 Figure 74. Mean (i S.E.) urine volume excretion in 4 hour units for the medial posterior hypothalanic and sham lesioned adrenalectonized groups provided 0.87% saline solution during the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. Figure 75. Mean (e S.E.) urinary sodium concentration in 4 hour units for the medial posterior hypothalanic and shaa lesioned adrenalectonized groups provided 0.87% saline solution during the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. IN mEq./L. No CON C. 139 2° ADRENAL PH 0 :PITRESSIN ---- ADRENAL SHAH O IPEAN. OIL — V 1‘ i '7: ' :I ' P 5 )5 | 1 ‘ l . 3 I .J l [ z : J V I "' '0 : IL ,’ D I Q : ,' S | “ --_ I O : . \\ ’__: .‘ . \ \ . O . g 5 r- : . ‘\‘ l’ e-- e E ' ‘ I I D ' ‘. ' I I i.’ I I Oi l L A A A ' L l A L L L OIOO 05000900 I300 ITOO 2000 OIOO 0500 0900 I300 I700 ZIOO PRE-INJ. POST- INJ. (hours) T I I 400 g I I L : I I soot : I I I I I I I ZOOI- ' i _ :ADRENAL PH e .ADRENAL sum 0 'PITRESSIN ---- o . . . i n i I . . . . i i OIOO 05000900 I300 ITOO 200 OIOO 0500 0900 (300 I700 ZIOO PRE-INJ. POST- INJ. (hours) 140 with a scan change of free 7.72 al/4 hour units to 547 post-injection. The two groups differed significantly free one another during the post-peanut oil injection period (t-4.85. p0.10). Figure 75 concerns the urine sodiun concentrations recorded for the adrenalectoaised groups for the periods under consideration. During the pro-injection period the two groups differed significantly free one another (t-8.80. p<0.01) with the PH group yielding a scan of 298.75 aEq/L. and the shaa group 374.83. Coaparing each group during pre- and post-peanut oil injection periods there was no difference (t for related aeasures-0.92 and 0.31. PM and shaa res- pectively. p>0.10). There were differences within groups coaparing pre- and post-pitressin injection periods (t for related aeasures- 3.54 and 2.38. PH and shes groups respectively. p<0.01. p<0.05). This represents a aean change of free 298.75 IEQ/L. to 323.83 post- injection for the PH group and corresponding values for the shaa group‘were 374.83 and 402.67. There was a difference between the two groups coaparing post-pitressin injection periods (t-12.21. p<0.01). There was also a difference between the two groups for the post-peanut oil injection periods (t-7.76. p<0.01). Figure 76 considers the urine potassiua concentrations for the adrenalectoaised groups. During the pro-injection period there were significant differences between the two groups (t-11.44. p<0.01). The PH group's seen was 59.42 aEq/L. while the shaa group's aean was 107.33. Conparing each group during pre- and post-peanut oil injection there were no differences indicated for the PH group (t for related 141 Figure 76. Mean (1 S.E.) urinary potassium concentration in 4 hour units for the medial posterior hypothalanic and sham lesioned adrenalectonized groups provided 0.87% saline solution during the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. Figure 77. Mean (* S.E.) urinary specific gravity in 4 hour units for the medial posterior hypothalamic and sham lesioned adrenalectonized groups provided 0.87% saline solution during the 24 hours prior to and 24 hours following independent applications of pitressin and peanut oil injections. IN mEq. IL. K CONC. (REFRACTIVE INDEX) TOTAL SOLIDS 142 2 ADRENAL PH e : PITRESSIN ---- ADRENAL 5mm 0 : PEAN. on. — I I I IS . : I ° \ I ° \\ 0 I . _ . | \. --e e I ~ - ‘~ IOO ' .’ x x I ,' $ , g x I ‘x‘ I 50- . I I F . I I O i i r L . a | A A 1 L A A 000 0500 0900 I300 ITCX) le OIOO 0500 0900 I300 WOO 2'00 PRE-INJ. POST-(NJ. (hours) 5 ADRENAL PH 0 i I ADRENAL SHAM o : I I I I 450 g I I I I I I I I 400. W I -. I , I I I P I I : PITRESSIN ---- 350; I PEANOIL —- w L L i l i ' . A a 4 A OIOO 05000900 |300 (700 2|OO OIOO 0500 0900 |300 ”00 2)00 PRE-INJ POST- INJ. (hours) 143 seasures-0.01. p70.10). However. the shaa group revealed a significant difference (t for related aeasures-2.06. p(0.05) with a aean change of free 107.33IEq/L. pro-injection to 126.00 post-injection. There was a difference for the shaa group coaparing pre- and post-pitressin injection periods (t for related aeasures-4.14. p<0.01). The PH group indicated an increase in potassiua concentration (t for related aeasures-3.65. p<0.01) free a pro-injection seen of 59.42 aEq/L. to 83.67 post-injection. Coaparing the two groups during the post- peanut oil injection period there were significant differences (t-21.83. p(0.01) between the groups which were also apparent concerning the post-pitressin injection period (t-6.54. p<0.01). Figure 77 inspects the urinary total solids eeasures taken on the adrenalecteaised groups during the pre- and post-injection 24 hour periods. Coaparing the groups during the pro-injection period there were significant differences (t-12.26. p<0.01) with the PH group revealing a scan of 1.3398 specific gravity and the shaa group 1.3436. Hith reference to pre- and post-peanut oil injection periods the PH group indicated no difference while the shaa group‘did indicate a difference (t for related aeasures-0.10 and 2.91. PM and shaa groups respectively. p>0.10. p<0.01). The scan change for the shaa group was free 1.3436 pro-injection to 1.3455 post-injection. Coaparing pre- and post-pitressin injection periods for the PH and shaa groups there were significant differences registered (t for related aeasures-3.36 and 3.93. PH and shaa groups respectively. p<0.01). For the PH group this was a scan change of free 1.3398 specific gravity to 1.3435 post- injection. For the shaa group the change was free 1.3436 to 1.3459 144 post-injection. There were subsequent fall hacks for each group to their respective pro-injection levels by 2100 hours post-injection. Cosparing the two groups with respect to post-peanut oil injection periods there were significant differences (t-22.98. p 0.01). There was also a significant difference between groups coaparing post- pitressin injection periods (t-3.04. p 0.01). Between 0100 and 0900 hours the PH group's urinary specific gravity rose quickly free 1.3404 to 1.3482. Subsequently it fell back until by 2100 hours post-injection the seasure was 1.3403. Discussion This experisent was included as a scans of evaluating the effect of exogenous pitressin upon the increased fluid intake revealed by posterior sedial hypothalanic lesioned anisals. The shas and exper- isental lesioned groups originally saintained on tap»water thus offer little in the way of seaningful data in the present experisent for they did not increase their fluid intake to any substantial degree over that evidenced by their shaa lesioned control group. The next two groups designated tap water-no saline and the adrenalectosised anisals which were experisentally lesioned. revealed sean fluid intakes significantly above those of their corresponding control groups. Considering the tap-no saline groups the sajority of the statistical tests suggest that the experisental and shaa groups responded sisilarly to peanut oil and pitressin injections. The relative level of water intake is unchanged for each group following peanut oil injection. Hith pitressin injection both groups decreased their water intake. 145 Urine voluse output appeared to be depressed slightly be panut oil injection and only slightly sore by pitressin injection for the shaa and experisentally lesioned groups. Turning to urinary constituents. sodius concentration revealed a slight increase following peanut oil injection for the shaa group and slight increase or no change for the experisental group. Both groups indicated a significant increase in sodiun concentration following pitressin injection with a subsequent decline to pre- injection levels by case 16 to 20 hours after the injection. Urinary potassius concentrations were only sinisally elevated by peanut oil injection for the two groups but pitressin injection resulted in an increased potassius concentration occuring within 8 hours after injection and persisting throughout the resaining 24 hours post- injection period for both groups. A sisilar pattern held for the urinary specific gravity seasures. For the sajority of indices utilised in this experisent the non- adrenalectosised experisental versus shaa lesioned group cosparisons were very sisilar with regard to pitressin injection. Pros the data thus far considered there is no bases for the contention that exogenous pitressin adsinistration differentially affects the shas and exper- isentally lesioned groups. In general the data collected free the shaa and experisental lesioned adrenalectosised groups confers with the established pattern if allowance is sade for the altered values of the seasures taken due to the effects of adrenalectosy. 1&6 Urinary specific gravity offers the only sound difference between the shaa and experisental aninals' responses to pitressin injection. The shaa group»evidenced very sisilar reactions to both peanut oil and pitressin injection. The PH group revealed a substantial increase in urinary specific gravity following pitressin injection which was not apparent with peanut oil injection. The conclusion to be offered froa Experiaent u is that exogenous pitressin adainistration results in only superficial response differences between the eXperiaental and shaa lesion groups. It nay therefore be suggested that bilateral posterior nedial hypothalanic lesions do not inpair the antidiuretic horaone systea's influence upon kidney water reabsorption. However. this experisent did not address the question of whether the heightened isotonic saline intake noted to follow such lesions nay be due to a direct iapairnent of ADH synthesis and/or release. The eaploynent of an ADH bioassay nethod (Gil-ore and Vane, 1970) would. presunably be valuable in such an analysis. EXPERIMENT 5 The Effect of Hater Deprivation To this point it has been denonstrated that posterior nedial hypothalanic lesioned anisals increase their isotonic saline solution intake to a significantly greater degree than shaa lesioned controls. Such lesions applied to previously adrenalectoaised anisals likewise result in increased isotonic saline solution intake. The exper- inentally lesioned groups responded to exogenous pitressin adainistration with an increased urine concentration. The shaa lesioned groups 1n? evidenced a sisilar urine concentrating ability. A water’deprivation reginen is a stressful condition in that body water and electrolyte saving aechanisas are required to function rather well in aaintaining ECF voluae and tonicity. Coaparisons between the orperiaental and shaa lesioned groups in response to a 23% hour water’deprivation schedule was thus esployed as an additional scans of identifying body water regulatory changes due to the experisental lesions. Subjects The anisals of Experiments 3 and u were used in the present experisent. At the initiation of this experisent the anisals were approxinately 18“ days of age. Procedure Following the conclusion of Experiaent u a five day recovery period was provided. Experiaent 5 directed attention upon the isposition of a 23% hour water deprivation schedule and concoaitant changes in aetabolic seasures. 0n post-lesion day 43, 4 hour readings began following the design earlier described. Iwaediately after the 2100 hours reading period water was reaoved froa all three aajor groups of anisals and h hour reading periods continued through post-lesion day an. Uith the conclusion of day an. 12 hour readings were initiated and water provided for a 30 ainute period once a day iaaediately following the 0900 reading period. Results The forest established in the results section of Experiaent 4 will be followed in the presentation of these findings. The data for the a hour reading periods during the 2“ hours preceding water 148 deprivation and the 2“ hour period issediately following the initiation of water deprivation are included in the figures of this section. Each point represents the loan of the six anisals of the designated group. Standard errors of the seans are also provided. Figure 78 indicates the food intake registered by the two groups saintained on tap water prior to and after the application of water deprivation. During the 2“ hour pre-deprivation period the posterior nedial hypothalanic lesioned group and the shaa lesioned group were not significantly different with regard to food intake (t-o.u1, p>0.10). Each group revealed significant decreases in food intake coaparing the pre- and post-deprivation periods (t for related neasures-3.h3 and 2.22. PH and shaa groups respectively. p<0.01. p(0.05). However, the two groups were not different during the first day of water dep- rivation with reference to food intake (t-0.57. p>0.10). The food intakes of the groups earlier supplied both tap and 0.87% saline solution (Figure 79)evidenced no difference during the pre-deprivation period (t-1.53. p>0.10). Coapring each group's food intake during the pro-deprivation and deprivation periods the posterior nedial hypothalanic group’deaonstrated no difference (t for related seasures- o.26. p>0.10). Again with the two adrenalectonised groups saintained on 0.87% saline solution (Figure 80) there were no differences between their food intakes during the pro-deprivation period (t-0.8h. p>0.10) or during the isotonic saline deprivation period (t-1.27, p)0.10). However each groupldeaonstrated a significantly decreased food intake coaparing pro-deprivation and first day fluid deprivation periods 11.9 Figure 78. Mean (e S.E.) food consumption in 4 hour units of the nedial posterior hypothalanic and shaa lesioned groups saintained on tap water during the 24 hours prior to. and 2h hours of fluid deprivation. Figure 79. Mean (* S.E.) food consumption in h hour units of the nedial posterior hypothalanic and sham lesioned groups provided tap water. no saline during the Zn hours prior to. and 2“ hours of fluid deprivation. FOOD INTAKE (GM/4 hour units) F000 INTAKE (GM/4 hour units) 150 :PH GROUP WATER e——. 'SHAM GROUP wanna—.0 L A a A J OIOO 0500 0900 ISOO ITOO moo 000 0500 0900 ISOO ITOO ZIOO PRE - DEP WATER DEP (hours) 1 4 4A_ 1 1 PH GROUP WATER NO-8796 SALINE H SHAH GOUP WATER N0 .87 % SALINE H L l 1 L A L a a OIOO 05000900 l300 ITOO 200 0000 0500 0900 BOO WOO ZIOO PRE-DEF WATER DEP (hours) 151 Figure 80. Mean (1 S.E.) food consumption in h hour units of the nedial posterior hypothalamic and shaa adrenalectonized groups maintained on 0.87% saline solution during the 2h hours prior to and 2“ hours of fluid deprivation. Figure 81. Mean (t S.E.) urine volune output in a hour units for the nedial posterior hypothalanic and shaa lesioned groups provided tap water during the 2“ hours prior to and 2h hours of fluid deprivation. F000 INTAKE (GM/4 hour units) URINE OUTPUT (ML/4 hour units) 152 :PH .8796 SALINE r .NO 2.0% sumac“--. :SRAM .s'm SALINE 6i- : NO 2.0% SALINE O ----- o I 1’ “ I I- I’ \‘ : .. \ ' I i \\ ' 4 I ' \\\ 1 ' " | I \ ‘ ' I I \ ‘ I \ ' \ \ fi ' ‘I ’ \ ‘ , I F ‘ I \ l ' ’ I I \ \‘ I, : ’1 ‘\ 2 p \\ ’o \I I : ’I \\ \\ I ” I \‘\‘ “é" \‘ ’ I% ' y \ I, ‘f I, \ ’I . \ I \\ I k? ' \¥/’ I O A l l L L 4 ' l l 1 4 4 l l OIOO 0500 ONO 6% I700 2I00 0m 0500 0900 I“ ITOO Zlm PRE-DEF FLUID DEP (hours) iOF EPR GROUP WATER e———e :SHAM GROUP WATERO———-O : s . : I I I s. : I I I I 4 , : I I I I 2 - g I I I e 4 4 L . L I i A . . . OIOO 0500 0900 l300 I700 ZIOO OK!) 0500 0900 I300 I700 ZIOO PRE-DEP WATER DEP (hours) 153 (t for related seasures-3.77 and 2.76. PH and shaa lesioned groups respectively. p<0.01. p(0.05). Turning to urine voluae output. for the two groups saintained on tap water (Figure 81) there were no differences coaparing then during the we-deprivation period (t-o.66. p)0.10) and the zone was true for the first day of water deprivation (t-o.03. p>0.10). Each group»did reveal significantly decreased urine output coaparing pre- deprivation and first day deprivation values (t for related seasures- 6.37 and 6.68. PH and shaa lesioned groups respectively. p<0.01). The urine veluae output of the two groups originally saintained on tap water and isotonic saline solution (Figure 82) indicated significant differences during the pro-deprivation period (t-2.72. p<0.05). This probably reflected the heightened intake of tap‘water by the PH group. The aean output for the PH lesioned group was 6.87 al/u hour units while the shaa group's scan was “.58 al/h hour units. These two groups were however not different with respect to urine output voluae during the first day of water deprivation (t-o.31. p>0.10). Conpnring pro-deprivation and first day deprivation periods the PH lesioned group registered a significantly decreased urine voluae output during water deprivation free a aean of 6.8? III“ hour units to 2.62 al/u hour units (t for related seasures- 4.42. p<0.01). The shaa lesioned group also indicated a change free a aean of 4.58 al/b hour units pro-deprivation to 2.72 during dep- rivation (t for related seasures-6.67. p<0.01). The urine fer-ation of the adrenalectoaised groups (Figure 83) also revealed differences between the two groups during the pre- 15“ deprivation period (t-13.82. pd0.01). There were significant drops in urine voluae output for the PH lesioned group froa 9.83 al/4 hour periods pro-deprivation to 3.02 al/li hour unite during the first day of deprivation (t fer related seasures-12.06. p<0.01). This was true of the shaa lesioned grouptas well. free a aean of 6.35 el urine/ h hour period pro-deprivation to 2.56 al/h hour units during the first day of'deprivation (t for related seasures-8.56. p<0.01). The urine constituents in general revealed a pattern of increased concentration during the iaposition of fluid deprivation. At odds with this stateaent were the adrenalectoaised groups particularly with reference to sodiun concentration. Figure 84 displays the sodiun concentrations in aEq/L. of the urine excreted by the groups placed on tap water during the pre- deprivation and first day of water deprivation. There was a significant difference indicated between these two groups during the pre-deprivation period with the shaa lesion group averaging 19#.83 aEq/L. sodiun and the PH lesion group yielding a seas of 182.33 IEQ/L. sodiun (t-b9.72. p<0.01). The two groups were. likewise different during the first day of deprivation. The shaa group's urinary sodiun concentration registered a lean of 289.63 aEq/L. while the m group averaged 227.83 aEq/L. (t-9.8o. p<0.01). Each group evidenced significant increases in sodiun concentration coaparing pro-deprivation and first day deprivation periods. with the shaa group loving free a seas of 1914.83 aEq/L. to 289.63 aEq/L. (t for related seasures-5.36. p<0.01) and the PH group froa 142.33 IEq/L. to 227.83 aEq/L. (t for related seasures-5.77. p<0.01). 155 Figure 82. Mean (1 S.E.) urine volume output in u hour units for the medial posterior hypothalanic and sham lesioned groups provided tap water. no saline. during the 2b hours prior to and 2h hours of fluid deprivation. Figure 83. Mean (1 S.E.) urine volume output in h hour units for the medial posterior hypothalanic and sham lesioned adrenalectonised groups maintained on 0.87% saline solution during the 2“ hours prior to and 2a hours of fluid deprivation. URINE OUTPUT (ML/4 hour units) URINE OUTPUT (ML/4 hour units) 156 I0 » : PR GROUP WATER : Moons SALINE '——O I SRAM GROUP WATER a : no.8? w. SALINE °——0 I I I I G. : I I I I 4. I I I I : 2 - , I I I o l i L A i i | A a a A a A OIOO 05000900 I300 I700 2IOO OIOO 0500 0900 i300 I700 200 PRE-DEF WATER DEP (hours) iPR .sTst SALINE :NO 2.0% SALINEe----e :SHAM ens SALINE . NO 2.0% SALINE o ----- o I I I I I I I I I I \ I \ I \ \ I \ ‘ ' \ x I I I I I I I I I a a L A A 1 A s L a A -4 OIOO 0500 0900 I300 I700 2I00 OIOO 0500 0900 I300 I700 ZIOO PRE-DEF FLUID DEP (hours) 157 Figure 84. Mean (* S.E.) urinary sodiun concentration in 4 hour units for the medial posterior hypothalanic and sham lesioned groups provided tap water during the 24 hours prior to and 24 hours of fluid deprivation. Figure 85. Mean (t S.E.) urinary sodium concentration in 4 hour units of the medial posterior hypothalamic and sham lesioned groups provided tapiwater. no saline. during the 24 hours prior to and 24 hours of fluid deprivation. IN mEq/L No CON C. IN mEq./L. No CON C. 158 400': :PH GROUP WATER e-——e :SHAM GROUP WATERo———o I p I I I I 300T ' I I ’ I I 200). I I I E ' I I I I00 I { l A L L A l ' J A A 4 A L OIOO osooosoo I300 ITOO ZIOO OIOO 0500 0900 I300 I700 ZIOO PRE- DEP WATER DEP (hours) 400- PH GROUP WATER NO-87% SALINE ’—0 SHAH GROUP WATER > N0 .87 It SALINE H 300 +- 200 r- ---------------I IOOE O 4 A A A42 4 A A a .1. OIOO 0500 0900 I3“) I7“) ZIOO 0m 05m 0900 I300 I700 ZIOO PRE- DEP WATER DEP (hours) 159 This sane pattern was established by the groups earlier saintained on tap water and 0.87%.saline solution (Figure 85). There were sig- ‘nificant differences between the groups during the pre-deprivation period (t-5.99. p<0.01) with the shaa group'displaying a aean urine sodiun concentration of 177.33 aEq/L. and the PH group 126.67 nEq/L. Differences also existed within each group coaparing pre-deprivation and deprivation periods (t for related seasures-4.22 and 3.95. shaa and PH groups respectively. p<0.01). The shaa group changed free a pre-deprivation level of 177.33 aEq/L. to 279.67 nEq/L. sodiun while the PH group altered its eean urinary sodiun concentration froa 126.67 lEQ/L. pro-deprivation to 224.75 lEQ/L. during the first day of deprivation. The two groups were also different froa one another during the deprivation periods (t-4.61. p<0.01). The adrenalectoaiaed groups (Figure 86) did not subscribe to the forest thus far established in that both groups while deaonstrating between differences during the pro-deprivation period (t-2.90. p<0.01). revealed decreased urinary sodiun concentrations during the deprivation period. The shaa groupidropped from a nean of 387.33 aEq/L. sodiun lire-deprivation to 344.17 during deprivation (t for related seasures- 3.30. p<0.01). And the PH grouptdropped free a scan sodiun concentration pro-deprivation of 331.33 lEq/L. to 286.17 aEq/L. during deprivation (t for related seasures-4.29. p<0.01). Again the groups were different froe one another during the first day of fluid deprivation (t-8.35. P<0.01). The urinary potassius concentrations displayed by the two groups provided tap water is given in Figure 87. There were no differences between the shaa and PH lesioned groups during the pre-deprivation 160 Figure 86. Mean (1 S.E.) urinary sodiun concentration in 4 hour units for the medial posterior hypothalamic and sham lesioned adrenalectomised groups provided 0.87% saline solution during the 24 hours prior tO and 24 hours of fluid deprivation. Figure 87. Mean (* S.E.) urinary potassium concentration in 4 hour units for the medial posterior hypothalamic and sham lesioned groups maintained on tap water during the 24 hours prior to and 24 hours of fluid deprivation. IN mEq./L. No CON C. IN mEq./L. K CON C. 161 40 I I r I I I I—T’o- b—‘z’o- N i ’l I \ \ \ -------------‘ PH .87.]. SALINE NO 2.0% SALINEO-----O: goo SHAH .8796 SALINE : CL "<1 2°: SALINE °-:---.°. I . i A 1 1.4 OIOO 05000900 1300 I700 zioo OIOO 050009001300 1700 2100 PRE-DEP FLUID DEP (hours) 300 _ I I I I r I I I : 20 . I I I I I I I I I0 ' I I I I .PR GROUP WATER e—e : SRAM GROUP WATER o——<> 0k A A a A A A ' a L A A L OIOO 0500M I300 I700 ZIOO OIOO osoo ONO I300 I700 ZIOO PRE - DEP WATER DEP (hours) 162 period (t-0.93. p>0.10). Each group evidenced significant changes from pro-deprivation to the deprivation period. The shaa group altered its potassius concentration fros a mean of 174.33 IEq/L. pro-deprivation to 231.17 aEq/L. during the deprivation period (t for related seasures-5.97. p<0.01). The PH group also increased its concentration from a mean of 102.00 aEq/L. potassius pre- deprivation to 234.83 aEq/L. during the deprivation period (t for related seasures-4.91. p<0.01). There were no differences between the two groups during the first day of water deprivation (t-0.42. p>0.10). The adrenalectosised anisals (Figure 89) acre disely followed the pattern established by the other groups for altered urinary potassius concentration during fluid deprivation. The shaa and PH lesion groups were different from one another during the pro-deprivation period (t-9.41. p(0.01). The shaa group altered its potassius concentration from a seen of 110.00 sEq/L. pro-deprivation to 200.50 during fluid deprivation (t for related seasures-5.98. p<0.01). The PH lesioned group also showed a difference with a potassius concentration mean of 69.83 aEq/L. pro-deprivation to 154.33 during the first day of deprivation (t for related seasures-3.81. p<0.01). The two groups were different fron one another during the first day of fluid deprivation (t-5.19. p<0.01) with the shaa group evidencing a higher potassius concentration in its urine. Changes in specific gravity were in the sane direction for all three aajor groups. The two grOUps supplied tap water (Figure 90) revealed differences during the pro-deprivation period (t—4.59. p<0.01). 163 Figure 88. Mean (* S.E.) urinary potassium concentration in 4 hour units for the medial posterior hypothalamic and sham lesioned groups provided tap water. no saline during the 24 hours prior to and 24 hours of fluid deprivation. Figure 89. Mean (* S.E.) urinary potassium concentration in 4 hour units of the medial posterior hypothalamic and sham lesioned adrenalectomised groups provided 0.87% saline solution during the 24 hours prior to and 24 hours of fluid deprivation. IN mEq./L. K CON C. IN mEq./L. K CONC. 164 300!- J PH GROUP WATER NO .8796 SALINE ..__. SHAM GROUP WATER NO .87 % SALINE °—‘—'° ---------------i A A A OIOO 05000900 I300 I700 200 OIOO 0500 0900 I300 I700 ZIOO PRE-DEP WATER DEF (hours) 30% 2001- b iOOIL. I--'I'”I\\ XI" fa .L I \ I I PH .8736 SALINE NO 2.0% SALINEO----O SHAM .8796 SALINE NO 2.0% SALINE O ----- O 1--.}- if I---------------- o\ \ \ \ \I \ A A A A OIOO 0500 0900 ISOO I700 ZIOO OIOO 0500 0900 1300 I700 ZIOO PRE-DEF FLUID DEP (hours) 165 Each group indicated significant increases in specific gravity comparing pre-deprivation and deprivation periods (t for related measures-5.87. and 3.20. sham and PH groups respectively. p<0.01). The sham group changed from a mean specific gravity of 1.3486 pre- deprivation to 1.3549 during deprivation and the PH lesioned group from 1.3464 to 1.3509 during the first day of deprivation. The two groups originally maintained on tapiwater and isotonic saline solution (Figure 91) were different during the pro-deprivation period with regard to urinary specific gravity (t-7.29. p<0.01). Each group evidenced a significant increase in specific gravity comparing the pro-deprivation and deprivation periods (t for related measures-4.35 and 4.99. sham and PH groups respectively. p&0.01). The changes were in the magnitude of from a mean of 1.3472 to 1.3531 during deprivation for the sham group and from 1.3411 pre-deprivation to 1.3511 during deprivation for the PH lesioned group. The two groups revealed differences for the first day of deprivation with regard to urinary specific gravity (t-3.65. p(0.01). Figure 92 represents the urinary specific gravity fer the two adrenalectomised groups maintained on isotonic saline solution during this experiment. These groups were significantly different during the pre-deprivation period (t-11.78. p(0.01). Each group increased its urinary specific gravity during the first day of fluid deprivation (t for related measures-6.47 and 4.54. sham and PH lesioned groups respectively. p<0.01). The sham lesioned group changed from a mean of 1.3440 to 1.3508 during the deprivation 166 Figure 90. Mean (* S.E.) urinary specific gravity in 4 hour units of the medial posterior hypothalamic and sham lesioned groups provided tap water during the 24 hours prior to and 24 hours of fluid deprivation. Figure 91. Mean (t S.E.) urinary specific gravity in 4 hour units of the medial posterior hypothalamic and sham lesioned groups provided tap water. no saline during the 24 hours prior to and 24 hours of fluid deprivation. TOTAL SOLIDS (REFRACTIVE INDEX) (REFRACT IVE INDEX) TOTAL SOLIDS 167 «I 500 I- v PH GROUP WATER I sum GROUP WATER o—-—<> I A L L A A A A A 4A A A OIOO 0500 0900 I300 I700 ZIOO 000 0500 ONO ISOO I700 ZIOO PRE - DEP WATER DEP (hours) 8 O O O 1— .L. -1 OIOO 0500 0900 l300 I700 ZIOO OIOO 0500 0800 I300 I700 ZIOO PRE-DEP WATER DEP (hours) PI-I GROUP WATER 110.877. SALINE H SHAM GROUP WATER NO .87 % SALINE H ---------------* A A A A A 168 Figure 92. Mean (* S.E.) urinary specific gravity in 4 hour units of the medial posterior hypothalamic and sham lesioned adrenalectomised groups maintained on 0.87%Isaline solution during the 24 hours prior to and 24 hours of fluid deprivation. 169 I PH .87 96 SALINE NO 2095 SALINE on--. sum .8796 SALINE NO 2095 SALINE O-----o 500 .4 TOTAL SOLIDS (REFRACTIVE INDEX) “\§-_’§_ __§_..- OIOO 0500 0900 I300 I700 ZIOO OIOO 0500 0900 I300 I700 2IOO PRE-DEP FLUID DEP (hours) A 170 period while the PH lesioned group‘altered its urinary specific gravity from 1.3402 pro-deprivation to 1.3480 during the first day of fluid deprivation. There were differences between the two groups during the first day of deprivation with the sham group displaying a higher urinary specific gravity (t-6.11. p(0.01). Discussion During the first day of water deprivation. food intake sign- ificantly decreased for all groups. This is in contrast with water intake during the first day of food deprivation (O'Kelly and Bright. 1971). The urine volume also declined during water deprivation for all groups. All three major groups revealed no difference between urine volume output during the first day of water deprivation comparing the experimental and control subgroups. This is of some interest in that the experimentally lesioned animals of the water- no 0.87%.saline group and of the adrenalectomised group revealed significantly higher urine volumes prior to deprivation compared with their control groups. This. of course. reflected the heightened fluid intake of these animals. Regarding urinary sodium concentrations the experimentally lesioned animals were consistently below the values indicated by their corresponding control groups both prior to and during the first day of water deprivation. The non-adrenalectomised groups maintained on tap water revealed increases in sodium concentration during water deprivation. The experimentally lesioned animals. however. did not concentrate their urine to the same degree as the controls. This may represent an altered kidney ability due to the hypothalamic lesions. This interpretation is not supported by the 171 potassium ion results. however. for the experimental water-no 0.87% saline group was not different from its control group«during the deprivation period in spite of its depressed potassium concentration level during the preodeprivation period. The adrenalectomised animals maintained on 0.87%.saline solution actually decreased their urinary sodium concentration during depr- ivation. This may reflect a decreased food intake. Uayne Breeder Blox have about a 1% sodium chloride content. The sodium intake provided by the isotonic saline solution was. of course. not available during fluid deprivation. Their potassium concentration. however. did increase substantially with the experimentally lesioned animals again revealing a low concentration during the pro-deprivation period and an inhibited ability to increase its urinary potassium concentration as compared with its control group. The urinary specific gravity data closely approximated the potassium results. For all groups the experimentally lesioned animals demonstrated low pro-deprivation urinary specific gravities and during deprivation maintained levels somewhat below their corresponding control groups. Thus even though the three major groups revealed few differences in urine volume output during deprivation. coaparing each experimental group with its control group. the former in general failed to concentrate its urine as efficiently as the control groups. This may represent an impaired kidney concentrating ability. These results may deny support for Cort's ”Substance X” in that damage to the presumed production site of this neurohumor should result in a natriuresis or urinary sodium loss that would probably be unchanged or enhanced during water deprivation rather than being inhibited as demonstrated in this experiment. 172 General Discussion Given the intricate nature of the ADH-Aldosterone systems in maintaining body water balance the voluntary ingestion of isotonic or near isotonic saline soltuion in excess of actual need is puasling (Bare. 19l+93 Nelson. 19"]: O'Kelly. 19516; Stellar and HoCleary. 1952; Young and Chaplin. 1949). The present series of experiments has identified a contribution to thissaline ingestion at the site of the posterior medial hypothalamus. Bilateral lesions at this locus by either electrolytic or radio frequency procedures yields two- to three-fold increases in isotonic saline solution imbibition. Adren- alectomy prior to lesioning does not alter this pattern thus eliminating the influence of corticosteriods. The adrenalectomised animals were ingesting more isotonic saline than normal animals due to the decreased ability of their kidneys to reabsorb sodium. In spite of this increased intake. lesions of the posterior nedial hypothalamus caused a still greater intake of saline soltuion. This isotonic saline polydipsia evidenced by the experimentally lesioned animals was not due to an inability of the kidney to conserve fluids for the administration of exogenous pitressin tannate resulted in a decreased fluid intake and increased urinary specific gravity and electrolyte concentrations not appreciably different from control animals. A close exaaination of day l of a 23% hour water deprivation schedule reflected differences between the experimentally and control lesioned subjects. Hith the initiation of water deprivation all animals evidenced decreased food intake and urine volume output. There were urinary constituency differences between the control and 173 experimental groups. In general the experimental subjects revealed a reduced ability to concentrate their urinary electrolytes. as compared with their corresponding control groups. Urinary specific gravity showed a similar trend. The possibility of changes upon aldosterone secretion is relevant to the following discussion and therefore several brief summary paragraphs concerning the adrenal-pituitary system have been included. Steroids which function as mineralocorticoids increase reabsorption of sodium not only at the tubule level but also from sweat. saliva and gastric juice. The primary mineralocorticoid influencing renal tubular exdllge of K" and m for Na+ ions is aldosterone (Ganeng. 1967). It is certainly true that sodium excretion is affected by other factors in addition to aldosterone such as glomerular filtration rate. osmotic diuresis and fluctuations in tubular sodium reabsorption independent of aldosterone. However. with chronic mineralocorticoid excess. i.e.. hyperaldosteronism (Conn's Syndrome) there is a marked ECF volume expansion. potassium,depletion and hypernatremia. Hith continued potassium depletion there is kidney damage resulting in a loss of concentrating ability (hypohlamic nephropathy) and a polyuric-polydipic coalition results (Nocenti. 1968. p. 979). Adrenalectomy or adrenal insufficiency (Addison's Disease) causes severe loss of both sodium and chloride by the kidneys due to a lack of aldosterone thus a serum hyponatremia and increased potassium level emues. There is also a decreased plasma volume with increased blood viscosity. with the initial sodium loss goes an obligatory water diuresis and consequently a dehydration state occurs. Following this 17“ initial diuresis water loss begins to lag behind salt loss and cellular overhydration occurs with the subject becoming oliguric (Canong. 1967. pp. 978-979). The effect of adrenalectomy upon the isotonic saline polydipsia shown to accompany bilateral posterior medial hypothalamic lesions is an important consideration for several reasons. It has been shown that the polyuria-polydipsia accompanying food deprivation (O'Kelly and wright. 1971) may be prevented in rabbits by prior'adrenalectomy. If these adrenalectomised animals are provided exogenous hydrocortisone acetate during food deprivation the polyuric-polydipsic syndrome.appears. Thus an adrenocortical hormone seems to be responsible for a polydipsic condition with urinary sodium loss during food deprivation in rabbits (Nbcenti and Cisek. 1970). As mentioned earlier in this paper it is clear that brain lesions may have an affect upon the adrenal gland. nedial habenular lesions result in a reduction in the nucleus sise of the sons glomerulosa cells suggesting a decreased aldosterone secretion (Lengvari. et al.. 1970). The above considerations serve as a prelude to an alternative inter- pretation of Cort's (1963a) findings that bilateral electrolytic lesions of the posterior nucleus of the hypothalamus result in a ”salt wasting“ syndrome due to the absence of a natriuretic substance. released from the diencephalon (Cort et al.. 1966: 1968). In that Cort's 1963a article is his sole and often referenced attempt to monitor this ”salt wasting“ syndrome for any extended length of time. seven post-operative days. it deserves particular attention. 175 Cort (1963‘) indicated a serum hyponatremia following the lesions. no‘data were given for serum potassium. There was also a urinary sodium loss increasing three to five-fold by the end of post-operative day 1. The animals increased their intake of isotonic saline and returned their serum sodium level from a low of 135 mEq/L. on day one to 1&5 by post-operative day five. Cort's conclusion was that with a post- lesion sodium depletion the animals drank isotonic saline in response to an EC? volume depletion rather than 3% saline to correct a pure sodium loss. No histological data were provided. There is nothing in the article to suggest that these lesions did not interfere with aldosterone secretion in a similar way as Lengvari's HHN lesions. Gort's animals displayed the symptoms evidenced by hypoaldosterone animals. i.e.. hyponatremia. urinary sodium loss. increased isotonic saline intake which prevented Cort's animals from revealing severe sodium depletion terminating in death. In other papers. however. Cort and his colleagues appear to have ruled out changes in endogenous aldosterone secretion as a factor in the “salt wasting” syndrome for a carotid occlusion stimulus in cats (Cort and Lichardus. 1963a; Lichardus and Cort. 1963). Following adrenalectomy the animals were maintained on daily injections of 5 mg DOCA and 15 mg Hydrocortisone (Fae). Nocenti am Cisek's F'sc dosage for rabbits was 2.5 mg/day. H1th this replacesent therapy Cort has failed to control the influence of hydrocortisone upon the increased tubular rejection fraction and urinary natriuresis accompanying carotid occlusion. 0f greater significance is his failure to test the interaction of posterior hypothalamic lesions with prior adrenalectomy stretched over several days. 176 The present series of experiments failed to replicate Cort's "salt wasting” syndrome following posterior hypothalanic lesions. This may have been a function of the small lesions used by the present investigator. However. an increase in isotonic saline ingestion was demonstrated in the lesioned animals. and prior adrenalectomy did not prevent this increased physiological saline intake. Thus Cort is supported on his assumption that adrenalectomy does not affect the heightened isotonic saline intake noted to occur following posterior hypothalamic lesions. but his proposal that the intake comes in response to a decreaSe in EC? volume accompanying acute sodium depletion is not supported. In fact the present results seriously question Cort's lesion findings with rats for it is clear that small bilateral posterior hypothalamic lesions nay yeild isotonic saline polydipsia with no true“salt wasting” syndrome. If Cort's lesions were larger than these it could be that neural efferents passing through the mesencephalon were severed possibly in a similar manner with those presumably interrupted by Lengvari et al. (1970) lesions of the HUN. Thus in addition to the isotonic saline polydipsia resulting from PH lesions Cort in some way may actually have interfered with aldosterone secretion. An alternative possible explanation concerns adrenocorticotropic hormone (ACTH). It is known that the hypothalamus has some influence over ACTH release by the anterior pituitary (Goldfien and Ganong. 1962). ACTH stimulates aldosterone secretion. however. hypophysectomy. and thus the loss of ACTH. does not result in the profound body fluid electrolyte disturbances present with adrenalectomy (Pitts. 1968. p.218). Even so ACTH influence should not be overlooked. 177 ADH also may play a role in that with the liberation of ADH from the median eminence there is passage of the hormone into the portal system of the adenohypophysis which stimulates ACTH. ACTH then activates the sons glomerulosa of the adrenal gland and aldost- erone output increases (Sawyer. Hunsick and Van Dyke. 1960). Arginine vasopressin influences the adrenal gland to increase its secretion of both aldosterone and hydrocortisone (Hilton. 1960). Sufficient damage to any one of the above systems could possibly result in a reduced level of aldosterone secretion. It is of some importance that posterior hypothalamic stimulation yeilds increased gastric secretion presumably by influencing the pituitary-adrenal systems in the form of increased corticoid release and thus a greater tubular rejection fraction for sodiun. This would also explain Cort's ”salt wasting“ syndrome. The present observation of an increased fluid intake following the lesioning of a nedially located brain area is certainly not original with this investigator. In fact there are indications from the literature that relatively homogenous neural regions may be functionally divided into medial versus lateral subdivisions that are complementary with respect to body water maintanence. Bilateral lesions of the ventro- nedial hypothalamus (VMH) yield an increased 1.0% saline solution intake (Kawamura et al.. 1970). Lesions of the arcuate nucleus of the hypothalamus have been shown to result in an increased 2.0% saline intake (Covian all! Antunes-Rodrigues. 1963). And bilateral lesions of what this author would like to refer to as the posterior lateral hypothalasus (LHP). i.e.. the region imaediately lateral 178 to the area under investigation in this dissertation. resulted in an adipsic condition with post-operative excretion of a dilute urine (o'xolly and Hatton. 1969). There are indications that the anygdaloid complex may also be similarly divided on such a basis. Bilateral lesions of the corti- coeedial nucleus result in an increased 1.5% saline intake while such lesions of the lateral nucleus. with some damage to the medial and basolateral nuclei. yielded decreased 1. 5% saline intake (Gentil et al.. 1968). The application of lesions to the septal area has not been conclusive. It appears that very large lesions affecting both the lateral and medial septal areas result in an increased intake of 0.82% (Donovich et al.. 1969) and 1.5% (Vilar et al.. 1967) saline solutions. Although Wolf (1967) claims to have lesioned only the lateral septal area and found no change in 2.0% saline solution intake. several of his lesions also damaged the medial septum and none of his lesions included sore than about two-thirds of the lateral septal (due to the elongated curving shape of this structure complete destruction is indeed a difficult task). An unexpected result of the control lesions utilised in Experiment 2 of this dissertation was increased isotonic saline intake. It appears that these bilateral lesions in addition to damaging the intended target. the dorsomedial thalamic nucleus. also affected the medial habenular nucleus (HRH). The subcommissural organ did not appear to be damaged. Although the exaggerated 0.87%»NaCl intake following such lesions does not appear to have been previously reported. Lengvari et a1. (1970) have demonstrated that such lesions result in a reduction 179 in nucleus sise of the sons glomerulosa cells with no effect upon the sons fasciculata suggesting a reduced aldosterone secretion. Adrenalectomy initiated enlargement of the nuclei of the HHH cells. The results of bilateral destruction of the lateral habenular nucleus (um) appears to be unavailable in the literature. There is neuroanatomical support for the above functional division of these structures into medial and lateral groupings. Fiber tracts‘appear to arise from the ventromedial. anteriomedial and posteriomedial hypothalamic nuclei and contribute to the peri- ventricular system (Morgana. 1969) at least a portion of which flows into the atria terminalis which courses around the anterior commissure terminating primarily in the central nucleus and medial portion of the basal nucleus of the amygdala. There is also a branching from the stria terminalis in the form of the atria medularis which establishes connections with the medial habenular nucleus (Haymaker et al.. 1969). On the other hand. fibers originating from the posterior and anterior lateral hypothalamus pass through the medial forebrain bundle forming the ventral anygdalofugal pathway which appears to terminate in and about the lateral amygdaloid nucleus (Haymaker et al.. 1969). Reci- procating fibers have been identified between the posterior lateral hypothalamus and the lateral septal nucleus (Cuillery. 1957) and between the lateral septal nucleus and the lateral habenular nucleus (Gurdjian. 1925). Although the above indications concerning the function of medial and lateral structures are certainly notconelusive there is ample evidence to suggest that the medial portions of the above listed 180 neural structures may be excercising an inhibitive influence upon fluid intake. lhen such regions are dassged or destroyed a polydipsic condition ensues which say represent the release of said inhibition. Conversely the lateral portions of these structures may exert a predominately facilitory influence upon fluid intake which. when removed via ablation. yields a aarked hypodipsic or adipsic condition. As previously indicated there resain several major pieces of information lacking from the above schemata. Hopefully knowledge concerning these untested portions of this hypothesis will be made available in the near future. LIST OF REFERENCES LIST 0? REFERENCES Antunee-Rodrigues. J. and Covian. H. R. Specific changes in water intake an! adipaia for water and sodiun chloride after hypothalamic lesions. Acta Phfiiologioa Latineaaericana. 1965. 15. 251-259. Bare. J. K. The specific hungar for sodiun chloride in normal and adrenalectoaised white rats. Journal of Comparative and Phfliolggical Bergstroa. H. H. The prticipation of bone in total body sodium metabolisa in the rat. Journal of Clinical Investigation. 1955. 3“! 997'10Me Bonjour. J. Ph. and Peters. G. Non-occurrence of a natriuretic factor in circulating blood of rats after expansion of the extracellular or the intravascular space. Pflugers Archives. 1970. 318. 21-3“. Brooks. 0.. Ushiyaaa. J. and Lange. G. Reactions of neurons in or near the supraoptic nuclei. American Jourml of Phgiologz. 1962. 202. l+87-‘490. Brookshire. K. H. Reinforce-ant value of water and hypotonic saline in discrete trial situations. Journal of Coaparative'and Physiological Pszcholog. 1967a. 63. 1h5-1h8. Brookshire. K. H. Inversion of discrete water-saline preference as a function of past drinking experience. Journal g_f_ Cosmrative and RysioloLical Psychom. 196%. 63. 2h-29. Chiang. H. arrl Wilson. H. A. Soae tests of the diluted-water hypothesis of saline consusption in rats. Journal of Coelmrative and Physiological Psychology. 1963. 56. 660-665. Conway. E. J. an! Ceoghehan. H. Molecular concentration of kidney cortex slices. Jourml 93 Physiolog. 1955. 130. “BB-M2. Cort. J. H. Central nervous control of the values of extracellular fluid. Pilgiologa Bohemoslovenic_a_. 1955. 1+. 14-31. Cort. J. H. Spontaneous salt intake in the rat following lesions in the posterior hypothalamus. Physiologa Boheaoslovenica. 1963a. 12 0 502‘505e Cort. J. H. Relation of the central nervous systea to water and electrolyte setabolism: Physiologic and clinical aspects. In Bland. J. H. Clinical Hetabolisa of Body Hater and Electrolytes. Philadelphia: I. B. Saunders Coapany. 19631:. Gupter 19. 181 182 Cort. J. H. Electrolytes. Fluid Mania and the Nervous Systes. New York: Acadeaic Press. 1965. p. 155. Cort. J. H. and Keeler. R. J. The effects of discrete hypothalamic lesions on the renal excretion of electrolytes in the rat. Journal gg‘EEIEEEEEEZe 195“. 1250 SOP. Cort. J. H. and Lichardus. B. The natriuretic activity of jugular vein blood during carotid occlusion. Physiologia Bohesoslovenica. 1963a. 12. “97-501. Cort. J. H. and Lichardus. B. The nature of the real response to the carotid sinus pressor reflex. In Horaones and the Kidney. Ed. P. C. Hilliams. New York: Acsdenic Press. 1963b. pp. 25-29. Cort. J. H. and Lichardus. B. The effect of dibensyline and hypertensin on saluretic pressor and “volume” reflexes. P11210129: Bohemoslovenica. 1963c. 12. 3015-909. Cort. J. H. and Liciurdus. B. The effect of the carotid sinus pressor reflex on reml function and electrolyte excretion. 0n the nature of the afferent signal. mysiologia Boheaoslovenica. 1963d. 12. 291-299. Cort. J. H. ani Lichardus. B. Natriuretic hormone. m. 1968. 50 “Cl-#09. Cort. J. H.. Hageaann. I. and Lichardus. B. The effect of aethyl alcohol and vasopressin on the pressor and renal response to carotid occlusion in the cat. filysiologia Bohemoslovenioa. 1965. 11+. 130-133. Cort. J. H.. Dcusa. T.. Pliska. F.. Lichardus. B.. Safarova. J.. Vranesic. H. and Rudinger. J. Saluretic activity of blood during carotid occlusion in the cat. American Journal of PhEiologz. 19680 2150 921'927e Cort. J. H.. Rudinger. J.. Lichardus. B. ani Hageaann. I. Effects of oxytocin antagonists on the saluresis accoapanying carotid occlusion. American Journal of Physiolog. 1966. 210. 162-168. Covian. H. R. and Antunes-Rodrigues. J. Specific alterations in sodiun chloride intake after hypothalamic lesions in the rat. Agerican Journal 9_f_ Physicbg. 1963. 205. 922-926. Crabbi. J. and DeHeer. P. Action of aldosterone on the toad bladder and skin of the toad. hture. 19646. 202. 298-299. Cross. B. A. and Green. J. D. Activity of single neurones in the hypothalasus. Effect of ossotic and other stiauli. Journal 93: 23,3101251. 19590 148, 55“-569. 183 Dahl. E. and Ursin. H. Obesity prcduced by iron and tissue destruction in the ventronedial hypothalasus. Physiology and Behavior. 1969. ‘5. 315-317. de Groot. J. The rat forebrain in stereotaxic coordinates. Verhandelingen der Koninkli ke Hederlanise Akademie van Heternsrnppen. Afd. Hatuurkunde. 1959! 520 1 e Deutsch. J. A. A new type of behavior theory. British Journal of Deutsch. J. A. and Jones. A. D. The water-salt receptor and preference in the rat. Nature. 1959. 183. 1:172. Deutsch. J. A. and Jones. A. D. Diluted water: An explanation of the rat's preference for saline. Journal of Coaparative an! Physiological Psychology. 1960. 53. 122-127. Donovick. P. J.. Burright. R. C. and Lustbader. S. Isotonic and hypertonic saline ingestion following septal lesions. Conunications in Behavioral Biology. 1969. 1+. 17-22. Edleman. I. S.. Bogoroch. R. ani Porter. G. A. On the mechanism of action of aldosterone on sodiun transport: The role of protein synthesis. Proceedings of the National Acedeny. 1963. 50. 1169-1177. Falk. J. L. and Titlebaua. L. F. Saline solution preference in the rat: Further demonstrations. Journal 91 Cdaprative and Elsielogical Psychology. 1963. 56. 337.3%. Fieognari. 0.. Fanestil. D. D. and Eielman. I. 3. Induction of RNA and protein synthesis in the action of aldosterone in the rat. Aaeriosn Journal g_f_' Physiology. 1967. 213. 9515-962. Fisher. 6. L. Saline preference in rats determined by contingent licking. Journal 9: the Experimental Analysis of Behavior. 1965. 8. 295-303. Forte. L. R. and Landon. E. J. RNA fromation associated with nineralo- corticoid activity of phenylbutasone and aldosterone. Federation Proceedings. 1968. 27. 1602. Fregly. H. J .. Yates. R. E. and Landis. E. H. Eriua sodiun concentration of hypertensive rats: Relation to NaCl intake. blood pressure aid age. Proceedi of the Society for Experimental Biolggy and Hedicine. 1955. 90. Era. Caable. J. C. Cheaical Anatosy. Physiolog am litholog of Extra- cellular Fluids. Cambridgeo Harvard Press. 195“. 18“ Canong. H. F. Review of Medical Ph 101 . Los Altos. California: Lange Hedical PublicatIEns. 1967. pp. - 14. Gentil. C. 6.. Antunes-Rcdrigues. J.. Negro-Vilar. A. and Covian. H. R. Role of amygdaloid coaplex in sodiun chloride and water intake in the rat. Physiology and Behavior. 1968. 3. 981-985. Gil-ore. J. J. and Vane. J. R. A sensitive and specific assay for vasopressin in the circulating blood. British Journal of Pharaacologz. 19700 389 633‘652e Goldfish. A. and Canong. H. F. Adrenal aedullary and adrenal cortical response to stiaulation of diencephalon. American Journal of Physiology. 1962. 202. 205-211. Gottschalk. C. H. Osmotic concentration and dilution in the aaamalian nephron. Circulation. 1960. 21. 861-868. Cuillery. R. H. Afferent fibers to the dares-medial thalamic nucleus in the cat. Journal of Anatosy. 1957. 93. b03-u19. Gurdjian. E. S. Olfactory connections in the albino rat. with special reference to the atria aedullaris and the anterior coamissure. Journal of Comparative Neurolggy. 1925. 38. 127-163. Hatton. C. I. Drive shifts during extinctions Effects on extinction and spontaneous recovery of bar-pressing behavior. Journal of Comparative end Physiolgeicnl Pamholm. 1965. 59. 385-391. Hatton. G. I. and Thornton. L. H. Hypertonic injections. blood changes. and initiation of drinking. Journal of Comparative and Physiolggical PI hol . 1968. 66. 503-506. Haymaker. 8.. Anderson. E. and Nauta. H. J. H. The Hypothalanus. Springfield. Illinois: Charles C. Thomas 00.. 1969. pp. 37. 137. Heller. J. and Stule. J. The physiology of the antidiuretic horsonel I. A sisple titration nethod. Physiologia Boheaoslovenica. 1959. 8 o 558-56“. Hilton. J. C. Andrenocorticotropic action of antidiuretic hormone. Circulation. 1960. 21. 1038-1047. HJorth-Simonsen. A. Fink-Heimer silver iapregnation of degenerating axons and terninals in aounted cryostat sections of fresh and fixed Jalowiec. J. E. and Stricker. E. H. Restoration of body fluid balance following acute sodiua.deficiency in rats. Journal of Comparative and Physiological Psychology. 1970. 70. 94-102. 185 Jewell. P. A. The occurrence of vesiculated neurones in the hypothalamus of the dog. Journal of Physiology. 1953. 121. 167-181. Jewell. P. A. and Verney. E. B. An experisental attenpt to deteraine the site of the neurohypophyseal osmoreceptors in the dog. Philosophical Transactions 25.222 Roygl Society of London. 1957. 240. 197-32h. Kawaaura. Y.. Kasahara. Y. and Funakoshi. H. A possible brain nechanisn for rejection behavior to strong salt solution. Physiology ani Behavior. 1970. 5. 67-7h. Koepoed-Johnsen. V. and Ussing. H. H. The contributions of diffusion and flow to the passage of D20 through living aesbranes. Effect of neurohypophysial horaones on isolated anuran skin. Acta Physiolggig Scandinavia. 1953. 28. 60-68. Leaf. A. and Hasby. A. R. An antidiuretic nechanisn not regulated by extracellular fluid tonicity. Journal of Clinical Investigstion. 1952. 31 . 60-71. Lengvari. 1.. Koves. K. and Halass. B. The medial habenular nucleus and the control of salt and water balance. Acta Biologica Acadeaiae Scientiarua Hungaricae. 1970. 21. 75-83. Leveque. T. F. and Sharrer. E. Pituicytes and the origin of antidiuretic hormone. Endocrinology. 1953. 52. 436-4hh. Lichardus. B. and Cort. J. H. The effect of adrenalectomy on the renal response to the carotid sinus pressor reflex. Physiologia Boheao- slovanica. 1963. 12. 397-399. Lichardus. B. and Jonec. V. Vplyv jednostranych lesii hypotalaau Ha vylucovanie nicktorych electrolytiv. Sunsarised in Cort. J. H. and Lichardus. B. The role of the hypothalaaus in the renal response to the carotid sinus pressor reflex. Physiologia Boheaoslovanica. 1963. 12. 389-395. Lichardus. B. and Pearce. J. H. Evidence for a humeral natriuretic factor released by blood voluae expansion. Nature. 1966. 209. “07-h09. Lichardus. B.. Hitro. A. and Cort. J. H. Size of cell nuclei in hypothalasus of rat as a function of salt loading. Aaerican Journal 9; Elysiology. 1965. 208. 1075-1077. Lichardus. B.. Jones. V.. Hitro. A. and Cort. J. H. The effect of a posterior hypothalanic lesion on the reaction to a salt retaining stiaulus in the rat. Physiologia Bohemoslovenica. 1965. 1“. 126-129. Lubar. J. F.. Schaefer. C. F. and Hells. D. G. The role of the septal area in the regulation of water intake and associated motivational behavior. Annals of the New York Acadeay g£_Sciences. 1969. 157. 875-93. 186 Mook. D. C. Oral and postingestional deteninants of the intake of various soltuions in rats with esophageal fistulas. Journal of Comprative and Physiologcsl Psychology. 1963. 56. 655-659. Moran. H. H. alrl Ziaaermann. B. Mechanisms of antidiuretic horaone (ADH) control of isportance to the surgical ptient. Surggy. 1967. &I 639'Me Morgans. P. J. The function of the limbic and rhinic forebrain—liabic aidbrain systess and reticular formation in the regulation of food and water intake. Annals of _t_he_ New York Acadeaj 9_i_‘ Sciences. 1969. 157. 806-858. Morrison. S. D.. Mackay. C.. Hurlbrink. E.. Hier. J. K.. Nick. M. S. and Millar. F. K. The water exchange and polyuria of rats deprived of food. Quarterly Journal of Experiaental Physiology. 1967. 52. 51’67e Myer. J. S. and Van Heael. P. E. Saline as a reinforcer of bar pressing by thirsty rats. Journal of Comparative and Physiological hlcayana. T. Hypothalamic electrical activities produced by factors causing discharge of pituitary hormones. Jannese Journal g_f_ Physiology. 1965. '5. 311-316. Nelson. D. Do rats select more sodiun than they need? Federation Proceedig. 19‘”. 6. 169. Nocenti. H. R. Adrenal Cortex. In Medical Ph 1010 . Ed. by V. B. Mounteaase. St. Louis: Mosby Comp. 166W 01H. . Chapter #7. pp. 979. 987. Nocenti. M. R. and Cisek. L. J. Effects of hydrocortisone acetate and estradiol in normal ani adrenalectoaised salt deficient rabbits. Novin. D. The relation between electrical conductivity of brain tissue and thirst in the rat. Journal of Comparative and Physiological Psychology. 1962. 55. 1135-15“. Nevin. B.. Fox. A. and Berger. M. The relation between saline solution ingested and tissue conductivity. Physiology and Behavior. 1966. 1. 167-170. O'Kelly. L. I. The effects of preloads of water and sodiun chloride on voluntary water intake of thirsty rats. Journal of Coaparative and Physiological Psycholog. 195k. 47. 7-13. O'Kelly. L. I.. Falk. J. L. and Flint. D. Hater regulation in the rat: I. Gastrointestinal exchange rates of water ani sodiun chloride in thirsty anisals. Journal of Captive and Physiological Psycholm. 1958. 51 .m '— 187 O'Kelly. L. I. and Hatton. G. I. Effects on ingestion ani excretion of water of lesions in a single hypothalamic area. Physiong and Behavior. 1969. “. 769-776. O'Kelly. L. I. and Wright. J. N. The effect of food deprivation upon body water balance in rats. Paper presented at Midwestern Psychological Association Meeting. May 6. 1971. Detroit. Michigan. Peters. J. P. Body Hater. Springfield Illinois: Charles C. Thomas. 1935. Pitts. R. F. Ph 1010 of the Kidne and Body Fluids. Year Book Medical Publishers no. szeaaa Edit en?‘"19 . Efiiiizre 7 and 12. Porter. C. A.. Bogeresch. R. and Edelsan. I. S. On the nechanisn of action of aldosterone on sodium transport: The role of RNA synthesis. Proceeding g_f_ £12 National Academy of Sciences. 196“. 52. 1326-1333. Raisman. C. Neural connexions of the hypothalasus. British Medical Bulletin. 1966. 22. 197-201. Richter. C. P. Salt taste thresholds of normal and adrenalectoaised rats. Endocrinolcg. 1939. 216. 367-371. Robinson. J. R. Reflections 9_n_ Renal Function. Oxford: Blackwell Scientific Publications. 1951+. Rolls. B. J. Drinking by rats after irritative lesions in the hypothalasus. Physiolog and Behavior. 1970. 12. 1385-139“. Sawyer. G. H. and Cernandt. B. E. Effects of intracarotid and intra- ventricular injections of hypertonic solutions on electrical activity of the rabbit brain. American Journal of PhEiolog. 1956. 185. 209-216. Sawyer. H. H.. Munsick. R. A. and Van Dyke. H. B. Antidiuretic hormones. Sharp. C. H. C. and Leaf. A. Studies on the sods of action of aldosterone. Recent Progress in Horaone Research. 1966. 22. 101-#71. Sims. E. A. H. and Soloson. S. The role of antidiuretic hormone and of aldosterone in control of water and electrolyte balance. In Blani. J. H. Clincal Metabolis- 9_f_ Bod Hater and Electrolfies. Philadelphia: H. B. Saunders all! Coapany. 133. Chapter b. Smith. D. F. and Stricker. E. M. The influence of need on the rat's preference for dilute NaCl solutions. Physiolggy all! Behavior. 1969. I}. “074510. 188 Saith. R. N. ani McCann. S. M. Increased and decreased water intake in the rat with hypothalanic lesions. In Thirst. Ed. M. J. Rayner. New York: Macmillan Coapany. 196“. pp. 381-3W. Starling. E. H. Physiological factors involved in the causation of dropsy. Lancet. 1896. 1. 1h07-1h10. Stellar. E. and McCleary. R. A. Food preferencesas a function of the method of seasureaent. Anerican Psychologist. 1952. 7. 256. Stellar. E.. Hyman. R. and Sanet. S. Gastric factors controlling water- and salt-solution drinking. Journal of Conprative and Physiological Psychology. 1959. 1&7. 220-225. Strauss. M. B.. Davis. R. K.. Rosenbaum. J. D. and Rossmeisl. E. C. ”Hater Diuresis“ produced during recunbency by the intravenous infusion of isotonic saline solutions. Journal of Clinical Investigation. 1951. 30. 862-868. Stricker. E. M. Extracellular fluid voluae and thirst. Aaerican Jourml 21: Physiology. 1966. 211. 232-238. Stricker. E. M. and Holf. 6. Blood voluae and tonicity in relation to sodiun appetite. Journal of Cogsrstive and Physiolgical Psycholog. 19660 62' 275’279e Stricker. E. M. and Half. C. Hypovolemic thirst in coaparison with thirst induced by hyperosmolarity. Physiology and Behavior. 1967. 2. 33" 37 e Stricker. E. M. and Holf. G. Behavioral control of intravascular fluid volume: Thirst and sodium appetite. Annals of the New York Acadeay 9_i_‘ Sciences. 1969. 157. 553-568. Verney. E. B. The antidiuretic horaone and the factors which deter-inc its release. Pmceedings of the Royal Society. 1997. 135. 25-106. Vilar. A. N.. Centil. C. G. and Covian. M. R. Alterations in sodiun chloride and water intake after septal lesions in the rat. Physiolog and Behavior. 1967. 2. 167-170. Von Euler. C. A preliainary note on slow hypothalamic ”osmopotentials". Acta flysiologia Scandinavia. 1953. 29. 133-136. Neiner. I. H. and Stellar. E. Salt preference of the rat determined by a single stiaulus nethod. Journal g_f_ Co_aparative and Physiological psychology. 1951. M. 39mm. Heiner. N. am Deutsch. J. A. Effects of salt deprivation and strain differences on tests of a diluted water hypothesis. Journal of Comparative and Physiological Psychology . 196?. 6+. 4m 189 Hirs. H. The location of antidiuretic action in the massalian kidney. Proceedings of the 8th Sysposiua 2£ the Colstcn Research Society. 1956. 8. 157:- Rolf. C. Hypothalaaic regulation of sodiun intakes Relation to preoptic and tegmental function. American Journal 2: Physiology. 1967. 213. 1433-1438. Rolf. C. Thalaaic and tegmental aechanisms for sodiun intake: Anatoaical and functional relations to lateral hypothalamus. Physiology and Behavior. 1968. 3. 997-1002. Rolf. G. and Quartermain. D. Sodiua chloride intake of'adrenalectoaised rats with lateral hypothalanic lesions. American Journal 2: Physiology. 1967. 212. 113-118. Holf. G. and Steinbaua. E. A. Sodiua appetite elicited by subcutaneous fornalinl Mechaniss of action. Journal 2: Conparative and Physiolqgical Psychology. 1965. 59. 335-339. Wolf. 6. and Stricker. E. A. Sodiua appetite elicited by hypovolenia in.adrenalectoaized rats: Reevaluation of the “reservoir” hypothesis. Journal sf Cospsrative and Physiological Psychology. 1967. 63. 252-257e Young. P. T. and Chaplin. J. P. Studies of food preference. appetite and dietary habit. X. Preferences of adrenalectoaised rats for salt soltuions of different concentrations. Coaparative Psychological Monographs. 1949. 19. No. 102. 45-74. Young. P. T. and Falk. J. L. The acceptability of tap water and distilled water to nonthirsty rats. Journal sf Coaparative and Physiological PBych01951. 1956. 49. 33 -33 . Zotterman. Y. Species differences in the water taste. Acta Physiologia Scandinavica. 1956. 37. 60-70. Zotterman. Y. and Diasant. H. Has water a specific taste? Nature. 1959. 183. 191. APPENDIX APPENDIX The following photosicrographs were taken with a Zeiss microscope and 35 an camera attachment. A 5x eye piece was inserted iasediately infront of the camera shutter and the nicroscope's variable magnification lens (1-5x) was adjusted with a given brain section for optinal exposure area. The microscope light source consisted of a Hotan 12V. 60w bulb powered by a Zeiss Hegel-Transformer (type 39 25 333 3-15V) set at 3.5 volts. Yellow and red filters were placed between the light source and the nicroscope stage. Exposure tines varied from 1/15 to 1/60 second depending upon the stain characteristics of the brain sections. The condenser was not used. High contrast copy file was esployed. developed for seven ninutes in D-76 and prints made on Kodak F-5 print ptper. 190 ___fl_._.-‘-.—e EXPERIMENT 2 ANIMALS Rat #1: The de Groot insert is at A 4.4. The photoaicrograph is of a frozen section stained by the Fink-Heiner method. Magnification x 30. Rat #3: The de Groot section is at A 4.6. The photonicrograph is of a frozen section stained by the Fink-Heimer nethod. Magnification x 30. Rat #5: The de Groot insert is at A 4.8. The photonicrograph is of a frozen section stained by the Fink-Heiner nethod. Magnification x 32. Rat #6: The de Groot insert is at A 3.4. The photoaicrograph is of a frozen section stained by cresyl violet acetate. Magnification x 30. 191 192 Fig. I7 . x../|. Rat #7: The de Croot insert is at A 4.2. The photoaicrograph is of a frozen section stained by the Fink-Heiasr nethod. Magnification x 30. Rat #8: The de Groot insert is at A 4.6. The photosicrograph is of a celloidin enbedded section thionin stained. Magnification x 28. Hat #9: The de Croot insert is at A 4.6 The photonicrograph is of a celloidin enbedded section thionin stained. Magnification x 28. Rat #10: The de Groot insert is at A 4.2. The photosicrograph is of a frozen brain section stained by cresyl violet acetate. Magnification x 30. 193 I a ‘1" 7 -\\.° —’ ‘a II a I ‘\ l li,’ VI ’I I a |‘_’ 19a Rat #11: The de Groot insert is at A 4.0. The photomicrograph is of a frozen section stained by cresyl violet acetate. Magnification x 30. Rat #12: The de Groot insert is at 4.8. The photomicrograph is of a celloidin embedded section thionin stained. Magnification x 28. Rat #14: The de Groot insert is at A 4.6. The photoaicrograph is of a frozen section cresyl violet acetate stained. Magnification x 30. Rat #15. The de Groot insert is at A 3.8. The photomicrograph is of a celloidin embedded section thionin stainded. Magnification x 28. 195 / I r 'r t in #1 - ‘ ‘. ' u} l ' _ . _ -{z".'“n’ . I :' (\'\ VI / ml '3 k- 196 Rat #16: The de Groot is of a frozen section Magnification x 30. Rat #17: The de Groot is of a frozen section Rat #18: The de Groot is of a frozen section Magnification x 30. Rat #19: The de Groot is of a frozen section Magnification x 30. insert is at A 4.2. The photomicrogrsph stained in cresyl violet acetate. insert is at A 4.6. The photomicrograph stained in cresyl violet acetate. insert is at A 4.6. The photomicrograph stained in cresyl violet acetate. insert is at A 4.2. The photomicrograph stained in cresyl violet acetate. 197 ---" , - . q I {‘ ’- ids/.- I I e.a . ‘ : asi"\ - . \ ‘ | 2s- .-5 t a 198 Rat #20: The de Croot insert is at A 4.6. The photoaicrograph is of a celloidin embedded section thionin stained. Magnification x 28. EXPERIMENT 3 ANIMALS Rat #A: The de Croot insert is at A 4.6. The photonicrograph is of a frozen section stained in cresyl violet acetate. Magnification x 30. Rat #2: The de Groot insert is at A 5.0. The photonicrograph is of a frozen section stained incresyl violet acetate. Magnification x 30. Rat #3: The de Croat insert is at A 5.0. The photomicrograph is of a frozen section stained in cresyl violet acetate. Magnification x 30. 199 '--—- I O M -.:. . I I I -_lg-~.' '\‘~'.£’I ’ \. I I ‘ I I r ’r l b m ’ n - .' I _ 91,], VI , -: ' x a ’\ a - .K e ‘12:; .. . . ' 5y - 200 Rat #4: The de Groot insert is at A 4.4. The photoaicrograph is of a frozen section stained by cresyl violet acetate. Magnification x 30. Rat #5: The de Groot insert is at A 4.2. The photomicrograph is of a frozen section stained by cresyl violet acetate. Magnification x 30. Rat #14b: The de Groot insert is at A 4.4. The photmicrograph is of a frozen section stained in cresyl violet acetate. Magnification x 30. Rat #15b: The de Croot insert is at A 4.8. The photomicrograph is of a frozen section stained in cresyl violet acetate. Magnification x 30. 201 _ a“. _—-—— 202 Rat #16b: The de Croot insert is at A 4.4. The photoaicrograph is of a frozen section stained in cresyl violet acetate. Magnification x 30. Rat #25: The de Groot insert is at A 4.2. The photcnicrograph is of a frozen section stained by cresyl violet acetate. Magnification x 30. Rat #29: The de Groot insert is at A 3.8. The photomicrograph is of a frozen section stained in cresyl violet acetate. Magnification x 30. Rat #30: The de Groot insert is at A 3.8. The photomicrograph is of a frozen section stained in cresyl violet acetate. Magnification x 30. 203 ‘__ --.~ 204 Total sodium intake as represented by food and 0.87%lsaline solution consumption minus total urinary sodium output presented in + or - mEq. of Na+. Rats #19b and 13b were experimental animals utilized in Experiment 3 and maintained on s2 libitum Hayne Breeder Blox (Approximately 1% NaCl content). tap water and isotonic saline solution. Rats #20 and 24 were control subjects provided the same diet as described for the experimental animals. 205 mi a 7 L f PRE-0P J. 7 p ‘ 7 /o air-of M /7/l ll )0