A QUANYtTATE‘x/E WUOY OF ‘FHE SUPRAQPTEC NEURORS Q? FHE ALENG EAT AND E‘WG QESEE? aonems, GERREL AND KANGARGO RA? Thesis gm“ if?“ Define 3% M. A. MEWS/{Ii SMTE UNIVERSE? Caro}; Lynn lander 1969 LIBRARY Michigan State I ‘8: Univcxsi Icy ‘ IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIOIIIIIIII 3 1293 105284 ABSTRACT A QUANTITATIVE STUDY OF THE SUPRAOPTIC NEURONS OF THE ALBINO RAT AND TWO DESERT RODENTS, GERBIL AND KANGAROO RAT BY Carol Lynn Zander Desert—dwelling rodents are, of necessity, efficient water economizers. Much of the recent interest in these animals has been focused on the renal and hormonal factors associated with the water conservation process. The im-' portant role of the supraOptic nucleus in elaborating the hormones essential for appropriate renal function, is a well-documented fact in the laboratory rat. The present study addresses itself to a quantitative analysis of these neurons in two desert rodents, the gerbil and kangaroo rat. After apprOpriate histological preparation, the brains of these animals and those of two normal albino rats and two water-deprived albino rats were examined for signs of neurosecretory activity. The cells of the supraOptic nucleus (both the anterior nucleus and the much-neglected posterior portion, or "tuberal supraOptic nucleus") which reacted positively to neurosecretory staining, were then examined to assess the extent of activity in these nuclei. Carol Lynn Zander The quantitative measures applied included estimates of the number of cells per nucleus, cell size, and number of nucleoli per cell. The kangaroo rat, one of nature's most competent water economizers, with its ability to gain weight on a diet free from exogenous water, seems to demonstrate a functionally more active supraOptic nucleus than that found in either the gerbil or laboratory rat. This desert rodent has relatively more supraOptic neurons per gram body weight and more double nucleoli per cell than either of the other two animals. There is also some indication that cell size (relative to body weight) is greater in this animal than in the gerbil or labo- ratory rat. Correspondingly, the gerbil has more supraOptic cells, relative to body weight, than the laboratory rat; the data suggests, in addition, that gerbil supraOptic cells are larger than those in the rat. The fact that the supraoptic cells of a normal laboratory rat deprived of water for five days demonstrate changes that approach the conditions found in desert rodents, indicates that increases in cell size and number of nucleoli may be adaptive mechanisms which desert rodents have capitalized upon. A QUANTITATIVE STUDY OF THE SUPRAOPTIC NEURONS ' OF THE ALBINO RAT AND TWO DESERT RODENTS, GERBIL AND KANGAROO RAT BY Carol Lynn Zander A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Psychology 1969 ACKNOWLEDGEMENTS The author wishes to thank Dr. Glenn I. Hatton for his abiding support and good humor throughout the preparation of this thesis, and Dr. John I. Johnson Jr. and Dr. Martin Balaban for their helpful guidance and criticism. This research was supported by NIH fellowship l-Fl-MH-37, 738-01,02. ii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . iv LIST OF FIGURES. . . . . . . . . . . .-. . . . . . . v ABBREVIATIONS USED IN FIGURES 1-5. . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 METHOD . . . . . . . . . . . . . . . . . . . . . . . 10' RESULTS. . . . . . . . . . . . . . . . . . . . . . . 12 DISCUSSION . . . . . . . . . . . . . . . . . . . . . 32,. SUMMARY. . . . . .-. . . . . . . . . . . . . . . . . 43 REFERENCES . . . . .,. . . . . . . . . . . . . . . . 45 APPENDIX A (MATERIALS) . . . . . . . . . . . . . . . 49 APPENDIX B (PROCEDURE) . . . . . . . . . . . . . . . 51 APPENDIX C (RAW DATA). . . . . . . . . . . . . . . . 61 iii Table 1. LIST OF TABLES Page Mean cell areas of supraOptic neurons. . . . . 20 Cell area ratios computed as the ratio between tuberal and anterior portions Of the N30 0 O O O O O O O O O 0 O O O O O O O 22 A summary of Student's t values from a comparison of means condUcted on cell area results . . . . . . . . . . . . . . . . . 23 iv Figure 1. 6a. 6b. 7a. 7b. LIST OF FIGURES Photomicrograph of a sagittal Thionin- stained section through the hypothalamus of a normal albino rat at the level of the supraOptic nuclei. . . . . . . . . . Photomicrograph of a sagittal Thionin- stained section through the hypothalamus of a five-day water-deprived albino rat at the level of the supraOptic nuclei. . Photomicrograph of a sagittal Thionin- stained section through the hypothalamus of a gerbil at the level of the supra- Optic nuclei . . . . . . . . . . . . . . Photomicrograph of a sagittal Thionin- stained section through the hypothalamus of a kangaroo rat at the level of the supraoptic nuclei. . -.- . . . . . . . . Photomicrograph of a sagittal Aldehyde- Fuchsin stained section through the hypothalamus of a normal albino rat at the level of the supraOptic nuclei. . Number of N80 cells by nuclear division in four animals. . . . . . . . . . . . . Number of N80 cells, expressed as number of cells per gram body weight, by nuclear division, in four animals. . . . . . . . Absolute values of mean cell area and standard error of supraoptic neurons in four animals. . . . . . . . . . . . . Cell area of supraoptic neurons in four animals, expressed as number of units per gram body weight . . . . . . . . . . Page 13 14 15 l6 17 18 18 24 24 Figure 8. 10. 11. LIST OF FIGURES (Cont'd.) Mean cell area and standard error of anterior N80 and hippocampal neurons. Results of cell area ratios between N80 and hippocampal neurons . Number of nucleoli per cell, for both anterior (A) and tuberal (T) divisions of N30, in four animals . . Photomicrographs of Thionin-stained tuberal supraoptic neurons. vi Page 26 28 29 31 NSO-A. NSO-T. OFT. OT ABBREVIATIONS USED IN vii FIGURES 1-5 . . .mammillary area . . .supraOptic nucleus, anterior division . . .SupraOptic nucleus, tuberal division . . .olfactory tubercle . . .Optic tract . . .area of the pons INTRODUCTION A desert environment would be swiftly fatal to the albino rat. The physiological systems that this animal has evolved for survival in his native temperate environ- ment would be inadequate to handle the stress imposed by an arid climate. Lack of water, or more precisely, lack of a system adequate to COpe with a harsh restriction of water, would certainly be a major factor contributing to its demise.‘ Desert rodents, on the other hand, display a remarkable capacity to conserve water, thus ensuring their existence. Animals such as Australian desert mice (MacMillen and Lee, 1967), the Peruvian desert mouse, Phyllotis gerbillus (Koford, 1968), the kangaroo rat, DIpodomys merriami (Schmidt-Nielsen and Schmidt-Nielsen,- 1950), the Egyptian gerbil, Gerbillus gerbillus (Burns,. 1956; Schmidt-Nielsen, 1962) and the Mongolian gerbil, Meriones unguiculatus (Winkelmann and Getz, 1962), can sub- sist on a diet of air-dried seeds and other dry plant sub- stances without additional drinking water. What is it, then, about the desert rodent's physi- ology, that allows him to actually thrive in an environment unsuitable for many other species? For the desert animm; existing on a diet of dry grain, water is available from the preformed water absorbed in the grain and from metabolic water. These animals concurrently minimize body water loss by the production of a very con- centrated urine, dry feces, absence of sweating, and restriction of skin and respiratory losses (Lockwood, 1963). Renal Factors The production of a very concentrated urine, much higher than that of the laboratory rat (MacMillen and Lee, 1967) is probably one of the desert rodent's most valuable means of economizing water loss. The kidney of the desert rodent comes equipped with certain adaptive features which seem to account for its heightened ability to restrict water loss. Gollschalk and Mylle (1959) have found that there is a strong correlation between the lengths of the segments of the renal tubule and the ability to concentrate urine. Three species of desert rodents, with capacities for high urine concentration, g. gerbillus, J. jaculus (Khalil and Tawfic, 1963) and Dipodomys merriami (Vimtrup and Schmidt- Nielsen, 1952), have distal and collecting ducts which dif- fer in length and structure from those of the albino rat; they have a distinctive morphology and are much longer. Hormonal Factors Tubular readsorption efficiency is, of course, also dependent on the level of circulating antidiuretic hormone (ADH), a hormone whose storage site is in the posterior lobe of the pituitary. Neurosecretory stains which.stain the material of the posterior pituitary also selectively stain certain regions of the hypothalamus. The currently accepted hypothesis is that neurosecretory cells of the hypothalamus elaborate ADH (or its precursor), which then diffuses down the axons of the hypothalamic cells to the terminals in the neurohypOphysis (Scharrer and Scharrer,. 1963). While three distinct areas in the vertebrate brain have been identified which react to neurosecretory stains, viz., the paraventricular, supraoptic and mammillo-infundi— bular nuclei (Smith, 1951), the main ADH manufacturing plant seems to be the supraOptic nucleus (NSO), since con— tinued antidiuretic function is dependent on the integrity of the NSC, the hypothalamico—hypOphyseal tract, and pos— terior pituitary (Fisher, Ingram and Ranson, 1938). In desert rodents, with their "superior" kidneys, one would also expect to find evidence of a highly developed ADH system. Some evidence to this effect has already been adduced. Enemar and Hanstrbm, 1956 (as cited in Thorn, 1958), have noted that the neuro-hypOphyseal lobe of desert rodents and hibernating rodents is relatively larger than in species living in more temperate regions. Ames and van Dyke (1958) have demonstrated that the pituitary of Q. merriami contains more ADH (0.9 milliunits/ug.) than does the pituitary of the albino rat (0.3 milliunits/pg.); they have also found that the concentration of ADH excreted in the urine of this desert mammal is considerably higher than that found in the urine from the laboratory rat or dog. Howe and Jewell (1959) found that the posterior lobe of the pituitary of the desert rat, Meriones meriones, occupies about 17 percent of the total volume of the hypOphysis, while in the laboratory rat it only accounts for about 10.5 percent of the total gland. They also noted that in lO-day water-deprived animals of this Species, there was only a scant amount of neurosecretory material in the hypo- thalamus, indicating a depletion in response to the depri- vation stress. Khalil and Tawfic (1963), who have examined the hypothalamo-hypOphyseal neurosecretory systems of two desert rodents, J. jaculus and §.gerbillus, assert that there is a higher amount of active ADH synthesis in the cells of the NSC of these animals under normal conditions than there is in the albino rat. Their evaluation was based on certain criteria-~staining characteristics, the large Size of the cells, size and position of the nuclei, the large Size of the nucleoli, and the position of the Nissl substance. Castel and Abraham (1969) have investigated the hypothalamic neuro-hypOphyseal system in two species of spiny mice. Their results show marked changes in the system with in- creasing days of water deprivation on a seed diet. Other than the evidence cited above, little has been contributed to our knowledge of N80 influence on water con- servation function in desert rodents. The supraOptic nu- cleus deserves specific attention. Neuroanatomical Considerations First of all, within the supraOptic nucleus, an anatomical distinction should be made. It consists of two Spatially separated portions: the anterior nucleus, which lies slightly caudal to the most rostral point of the optic chiasma, and the tuberal nucleus, a region which commences immediately behind the Optic chiasma with cells clustering on the medial side of the optic tract. The tuberal portion of the supraOptic nucleus is seldom expressly considered; when mention is made of the N80, unless otherwise specified, the authors are presumably referring to the main portion-- the anterior supraOptic nucleus. In most mammals, the tuberal NSO would be considered part of the anterior NSO if not for the discontinuity be- tween the two areas, the small number of cells in certain species (Auer, 1951), and its diffuse arrangement of cells in some species (Auer, 1951; Bodian and Maren, 1951). How- ever, the cells themselves appear to be morphologically similar to those of the anterior nucleus; both portions of the nucleus feature large polngnal or bipolar cells (Westwood, 1962; Malone, 1916; Legait, 1955). The small amount of data that presently exists indi- cates that the tuberal N80 is not only morphologically but also functionally similar to the anterior NSO. Peterson" (1966) noted that this area in the albino rat stains much: in the same manner as the cells of the anterior portion, indicating neurosecretory activity. (Only Cotte and Picard (1968) report that the tuberal portion of the nucleus fails to show signs of neurosecretion.) Cells in both areas show signs of cellular distortion and a general chromatolytic reSponse when the animals have been deprived of water. Cells of both areas degenerate in much the same fashion when hypOphysectomy is performed (Bodian and Maren, 1951), or when the hypothalamus is isolated (Bleier, Bard and Woods, 1966). For the above reasons, any study that involves itself with a description of the supraoptic hor- monal system, must concern itself with both portions of the nucleus. Statement_g£ Purpose The purpose of the present study was to provide a systematic comparison between the neurosecretory supraoptic cells of desert rodents, both anterior and tuberal portions, with those of the laboratory rat. The comparisons were based on certain quantitative measures, i.e., cell size, number of cells in a nucleus, and number of nucleoli per cell. Caspersson's extensive work with various cell types (1950), has led him to the conclusion that conspicuous changes in protein metabolism within a cell's nucleus are correlated with nerve function. Edstrom, Eichner and SchOr (1961) have demonstrated that the ribonucleic acid content (and therefore nuclear protein metabolism) in N80 cells increases during water deprivation. It was therefore anticipated that the NSC cells in the animals selected for study would show signs of nucleolar activity in corres- pondence to the ability of these animals to resist dehy- dration. It was also expected that the number and size of the NSC cells would reveal a correlation with the differ— ing abilities of these animals to withstand water deprivation. The cells were first tested for reaction with neuro- secretory stains to establish that the regions under study in the desert rodents were the functional homologues, that is, neurosecretory centers, of those regions in the labora- tory rat. Reliance on these stains for demonstrating neuro- secretion was based on its correlation with information derived from several sources. Bachrach (1964) has demon- strated a secretory cycle in the NSC cells of the laboratory rat. He placed his rats on a dehydration treatment, since this is a well-known method for inducing significant ADH mobilization. Single animal groups were sacrificed at eight days in the dehydration period and after periods of 12 days of subsequent rehydration. From the changes of ribonucleic acid content and Nissl pattern of the cells and their change in size, a systematic pattern emerged which indicated a definite secretory cycle; changes in amount of Gomorivposi- tive material reflected the same cycle. Dawson's work (1966) indicates a correlation between microsc0pic findings and bio-assay determinations of neurosecretory activity. He investigated the staining pattern of the neuro-hypOphysis and hypothalamus of fetal and postnatal rats, and found that the onset of ADH production in fetal rats, as detected by staining, occurs on the eighteenth gestational day, which. coincides with the findings by bio—assay technique. In addition, the two techniques demonstrate a parallel por- trayal of progressive amounts of neurosecretion in maturing rats, increased production reflected by density of staining, or, in the case of bio-assay, by the number of units of extractable hormone. In the present Study, both neurosecretory staining characteristics, and three quantitative measures of NSO cells, were used to provide a composite picture of cellular activity in two desert rodents and the laboratory rat. The gerbil and kangaroo rat were the two desert animals chosen. The hypothesis was that desert animals compensate for re- stricted water-availability by being able to conserve the water that is available to them, through the use of the anti-diuretic hormone-producing system. While both the gerbil and kangaroo rat are able water economizers, the kangaroo rat is superior to the gerbil in its ability to concentrate urine and gain weight on a diet free from exo- genous water (Winkelmann, 1962; Schmidt-Nielsen, 1951). It was therefore anticipated that the kangaroo rat would reflect this difference in the measures of neurosecretory cellular activity. METHOD Subjects Subjects were two, adult, male kangaroo rats. (Dipodomys merriami), two, adult, male gerbils (Meriones unguiculatus), and four male Holtzman albino rats, 100-110 days old on arrival to the laboratory. Procedure The eight animals were housed in individual wire cages and maintained on a diet similar to their normal regi- men; for the desert animals, mixed seeds (approximately 12% preformed water by weight) were available ad libidum; the albino rats were allowed access to Wayne Mouse-Breeder BlOX and water. When their weights had stabilized, the gerbils, kanga- roo rats and two of the albino rats were anesthetized with ether and perfused through the left ventricle with physio- logical saline and formalin; the two treatment animals were kept in their cages five additional days, without access to water, before being sacrificed. The brains were removed and embedded in celloidin. Serial sections (15 u thick) through the hypothalamus were taken; one brain of each Species was sectioned in a horizontal plane, the other 10 11 in a sagittal plane. Alternate sections of each brain were stained with the following: (1) Thionin was used as the~ standard cell stain, (2) Bargmann's modification of Gomori's Chrome-Alum-Hematoxylin for neurosecretion, and (3) Gomori's Aldehyde-Fuchsin for neurosecretion. Two neurosecretory stains were used for comparative purposes; however, the Chrome-Alum-Hematoxylin stain prepared for this study did not act selectively, and therefore could not be used for purposes of demonstrating neurosecretion. (For details on histological preparation of the tissue refer to Appendix B.) The stained sections were mounted on glass slides and covered with "1" Size cover-slips (0.13-0.16 mm thick). Estimates of cell populations of the N80 were ob- tained. A sample of supraoptic cells was examined to determine cell size and number of nucleoli. (Refer to Appendix B for a description of the sampling procedure and methods of obtaining measurements.) RESULTS NeurosecretoryMStaining. Figures 1-4 show the relationship between the anterior and tuberal portions Of the NS0, and the surrounding.hypo- thalamic tissue in the normal albino rat (NR), the five- day deprived albino rat (DR), the gerbil (G), and the kangaroo rat (KR). Figure 5 is an example of the same nuclei stained with Aldehyde-Fuchsin.‘ The darkly stained supraOptic nuclei are readily distinguishable from the paler surrounding tissue which remained unreactive. Under high power the cells are usually not recognizable as dis- crete units but as irregularly-shaped packets or clumps of stained material. When individual cells can be identified, the darkest staining material appears massed at the periphery. The supraOptic nuclei in all animals reacted with the neuro- secretory stain. Number 2£.92£l§ Results of cell counts are presented in Figures 6a and 6b. Figure 6a indicates the absolute number of cells for each animal; Figure 6b shows number of cells per gram body weight. Because of the fact that some sections of N50 tissue in the normal rat were lost in histological preparation, 12 .‘ o r ‘97.; . .‘ ' _u .y ’.t$}‘ o I OT NSO-T NS "A <;_O_FT.___..— Figure 1. Photomicrograph of a sagittal Thionin-stained section through the hypothalamus of a normal albino rat at the level of the supraOptic nuclei. Magnification: x 30. l4 .. ...............t ...x“... . ..... .....w....._arw.. .... y . d a e e d h n . t I - ... .... W... yuan... t .1 0 #9 .... S f .... a a .1 V n f e O O l .1 h S e T u w 1 m a l t t a a ... ......... .. t h «mfi. ,zw .i+tt _. 56.253»? g 0 a n..orv... a P r h 0 a n e .1 f h b O t 1 a h h 0.. 9d a u 6 gm w. 0 h r r t p m n m may. 0.1 r t t e O C t h e a P S W 2 e r u g .1 F x 30. Magnification: supraOptic nuclei. 15 5’ U' ~ 5 ‘ . vs :- ’ K ~' .‘ . I J‘l’ a «a! u-“.‘ I . ‘. f I I?~_ . . "5‘ . .’-. ..‘a‘ ‘1 2; v u! \ 9:... «.4. . '373-0‘ . ‘3’ y‘. . Q ‘3‘”.7.‘ u ‘k 947' ' ...; I. s - « f ... d taine Magnification: ln-S ion lei. ttal Th section through the hypothalamus of a gerbil at the level of the supraOptic nuc Photomicrograph of a sagi x 30. 3 Figure 16 1... ...... .. . fl F1... ..4 ... L. 4i.3£im . . 4.... - . M . ...... ....... .s .. Q. O‘n _ . .. 9.“ . x5... . . . VHS..de ....(I .- .. .n t... Iv: .... . . .,. xiiiiusw;2.~ 1?, 69..-... . .. ,_ ...... .4 ..4 «- ... ... . 1. . . 0‘ ... .... ...o.$.. tug-1. 9...” \s .. . .. v ~c l A... i ... W. .... I o I 7:.H‘\Wvfi.m0m .., .. at.“ MM”! 3‘: In 0 .. t 1..) 5 O .4. v a . . .... ... D I 1 3.9.. ... L .... . a In , . ”A c .. . ‘ r .5 .. ‘1‘ . I I . . l .’- . a \s. .\ . \. s. s . . n . ...... )I: ... .. . . . ...» ISO 0. al'u v a\..J.IW . I... ‘3.\\R4-VA.LT.‘ u... Isa? W. . . ‘0 8 1 . S c. O .. s A, S. «.L n .. a I! I 3.16,); ....\..¢..h. .. 30...... a..... .12.... .. . ... ....-. . ....u Math“. h». ma): «.3 ... . . n . l. . ,I- n. . 0.. .. a h .. .I..JL..« ....u..P\..€ .... sir)”. letw.-.,.$ . u... . Ir \ I A I . ... .Vo. ...... . stained ionin- ittal Th Photomicrograph of a sag 4 Figure section through the hypothalamus of a kangaroo lei. 1C nuc rat at the level of the supraopt Magnification: x 30. Figure 5. Photomicrograph of a sagittal Aldehyde-Fuchsin stained section through the hypothalamus of a normal albino rat at the level of the supraOptic nuclei. Magnification: x 30. NUMBER OF CELLS NUMBER OF CELLS/GRAN BODY WEIGHT 15000 A 18 ANTERIOR NSO |:| TUBERAL NSO lllll 13000 a 11000 - "——‘ 9000 - 7000 - 5000 J 3000 - L_h__ DR NR G Figure 6a. Number of NSO cells by nuclear division in four animals. 120. 110. 100- 90. 80~ 70. 601 so; 40. 30. 20. 10I NR Figure 6b. Number of N50 cells,expressed as number of cells per gram body weight, by nuclear division, in four animals. 19 we relied on the data of Hatton and Johnson (1968). The fact that their estimate of the number of cells in the albino rat is smaller than ours cannot be attributed to the deprived state of our rats, but is probably due to section thickness differences; since their sections were 10 u thicker, it is assumed that a percentage of their cells escaped observation. Bodian and Maren's estimates (1951) came closer to ours, but the weights of their rats could not be obtained. In any case, the absolute number of cells in the rat, normal or deprived, exceeds that of either the kangaroo rat or gerbil; KR has a greater number than G. In relation to body weight, KR has the largest number of cells, G next, followed by DR and NR. (The deprived rat does not actually have more cells; the apparent differences between DR and NR is only a result of a large weight loss in the deprived rat, causing, by way of numerical transformation of the data, the deprived rat to have more cells per unit weight.) Cell Size Mean cell areas in horizontal and sagittal planes for each animal are presented in Table 1. Differences in cell area between planes appeared to be due to differential shrink- age of the brains rather than to the orientation of the cells, since (1) ratio comparisons of tuberal to anterior NSO remained Table 1. 20 Mean cell areas of supraOptic neurons. Animal Anterior NSO Tuberal NSO Deprived Rat 198.64 u2 207.11 Sagittal (S=l.55) (S=l.83) Deprived Rat 191.86 u2 230.8702 Horizontal (S=l.31) (S=l.92) Normal Rat 131.491.2 131.6902 Sagittal (S=l.21) (S=l.21) Normal Rat 151.2802 156.1202 Horizontal (S=l.02) (S=l.4l) Gerbil 73.721.2 73.76“2 Sagittal (S=.59) (S=.59) Gerbil 136.53u2 140.29112 Horizontal (S=l.43) (S=l.25) Kangaroo Rat 154.3802 160.25p2 Sagittal (S=1.40) (S=l.24) Kangaroo Rat 132.81112 148.80112 Horizontal (S=l.37) (S=l.02) 21 essentially constant except for the deprived rat (Table 2), and (2) there was no consistent biasing toward larger cells in one particular plane. The basis for selecting planes,' then, for purposes of inter-animal comparisons, was the: plane with the least amount of shrinkage. For the normal . rat this was the horizontal plane; for the gerbil, the hori- zontal; for the kangaroo rat, the sagittal. Selecting the plane for the deprived rat posed a problem, for while the sagittal DR anterior appeared to suffer less shrinkage than the horizontal, the situation in the tuberal portion was reversed. The sagittal plane was finally chosen over the horizontal on the basis of two observations: (1) The ranges of the horizontal anterior and tuberal cells were contained within the ranges of the sagittal anterior and tuberal cells (horizontal anterior: 142.56 - 256.20 uz, sagittal anterior: 136.36 - 287.19; horizontal tuberal: 152.89 - 305.79, sagittal tuberal: 140.50 - 367.77) and (2) a cOmparison of means between anterior and tuberal supra- Optic cells revealed significant differences in only the deprived rat horizontal, and possibly deprived rat sagittal or kangaroo rat sagittal. (See Table 3, * items.) Mean cell areas and standard errors of the four ani- mals in the planes selected are displayed in Figures 7a and 7b. Figure 7a depicts the absolute values of cell area, while Figure 7b Shows cell areas expressed as number of units per gram body weight. 22 Table 2. Cell area ratios computed as the ratio between tuberal and anterior portions of the NSC. ANIMAL HORIZONTAL SAGITTAL DEPRIVED RAT 1.20 1.04 NORMAL RAT 1.03 1.00 GERBIL 1.03 _l.00 KANGAROO RAT 1.12 1.04 23 Table 3. A summary of Student's E values from a comparison of means conducted on cell area results.* Kangaroo- lNormal IDeprived Deprived Rat Gerbil Rat Rat Rat figgi tal Hori'onta Hnri7nn+a Sagittal. A T A T A T A T A T 8 8 a u u m u o+)-g 54 A 88 (0 1.07 mm m ‘ITH 73 d 44 u 3.06 fi 1: 3 £3 E u 3.89 .68* :8 Q 4-) ‘3 .61 2.88 H c E O OH 8 5' .75 16.29 .95*» 2:1: :1: o PI m o E 5.70 9.77 8.32 .3 O 34-) .E E" * mm o 10.55 13.48 10.70 5.74 on: a: '8 73 a 7.25 13.60 8.71 1.14 :> u -H u 34J°3~ * on: a a 7.25 I10.43 7.57 .07 1.21 am (0 . ', .— x: Values_of E greater than 2.62 are significant at the .01” level of confidence (df=98). 1.98 .05 1.29 .20 1.04 .30 *: Designates anterior to tuberal comparisons within species and plane. CELL AREA IN 02 AREA (02)/GRAM BODY WEIGHT 200 190 180 170 160 150 140 24 .____ ANTERIOR NSO TUBERAL NSO DR KR NR G’ Figure 7a. Absolute values of mean cell area and ,standard error of supraOptic neurons in four animals. ANTERIOR NSO I I TUBERAL NSO I'll KR G DR NR Figure 7b. Cell area of supraOptic neurons in four animals, expressed as number of units per gram body weight. 25 A comparison of cell size means between animals (Tables 1 and 3) revealed that KR=NR, G an individual experiment. Enestrom (1967) reports that cell volume of laboratory rat NSO cells changes with suc-. cessive days of water deprivation; in his rats, cell size had increased by the fourth day, had further increased by the seventh day, but receded by the twelfth day. Peterson (1966) reports nucleolar enlargements with a five-day deprivation period. Bachrach (1964) also reports cell com- ponent enlargements (cytOplasm, nucleus, and nucleolus) with deprivation. Our results of cell size enlargement coincide with the above findings, and moreover, specify that these cell enlargements take place equally in both the anterior and tuberal portions of the nucleus. Desert rodents: Relatively little data is available for inter-Species comparisons.‘ Khalil and Tawfic (1963) describe "large" cells in the two desert rodents J. jaculus and g. gerbillus, but no quantitative comparisons are avail- able. On the basis of absolute size, our results show that KR cells are the same size as NR cells, both being larger than those of G and smaller than DR cells. When cell size 37 is expressed in relation to body weight, KR has the largest cells, followed by G, DR, and NR, in that order. This . structural relationship parallels the data concerning.the ability of these animals to tolerate restriction of water,‘ with the KR as most competent, followed by G, and labora- tory rat finishing as a poor third. The cell size value. for DR moves in the direction of the value for the desert. rodents; in other words, G and KR under natural environ- ‘mental conditions function in the way that a laboratory rat would when placed under similar, but for him,.unnatural and stressful conditions. The fact that a laboratory.rat will soon expire when maintained without exogenous water, while the kangaroo rat thrives and the gerbil manages to get by, suggests that increasing cell size (and presumably metabolic and neurosecretory function) is both a species and an individual survival mechanism. It should also be noted that family differences may play a part in cell size differences among the animals. This possibility will have to be investigated in other ro- dent species. The fact that the cells of the laboratory rat increase in size from the normal to the deprivation condition, lends support to the habitat-adaptation hypothesis. When anterior NSO cells were covaried with hippocampal cells, the results failed to corroborate the cell size relationships among animals previously indicated--ratio 38 comparisons of cell size means within animals across planes did not remain constant. This would seem cause for question- ing the validity of the cell Size distinctions previously made, until other factors are taken into account. Figure 9 represents the results of a transformation of N50 cell size. data based on an arbitrarily chosen nuclear group, the hippocampal cells. The actual meaning of these results is not totally apprehensible. First of all, it is not apparent which, if any, of the inter-plane, intra-animal cell size values are statistically different; the sample size (number of animals) is not large enough to allow such distinctions. One must also question the suitability of using the hippo- campal cells as the covariate. Although we assumed no speci- fic neurosecretory function on the part of hippocampal cells and, indeed, these cells did not react with Aldehyde-Fuchsin stain in the manner that supraOptic neurons do, we must con- sider the possibility of differential effect on these cells from other sources. Furthermore, the possibility that there may be cell size differences among different portions of the hippocampus itself, must certainly be considered. Since the validity of this measure itself must be called into question, any conclusions as to "real" cell size distinctions, based on this measurement and subsequent transformation, would be equivocal. Cell size comparisons based on selection of the plane with least apparent shrink- age and normalized for the size of the animal by ratios of 39 cell area to body weight (see Figure 7b), do not, then, appear to have been invalidated by the conflicting hippo- campal results. Furthermore, the fact that the cell size distinctions originally made are in harmony with other structural-functional considerations (relative number of N80 cells, ability of the animals to withstand water deprivation), should enhance the credibility of such dis- tinctions. However, since the hippocampal results did -.- deviate from expectation, and do not substantiate the earlier measure, the definitive statement on cell size distinctions must be suspended until larger groups of animals have been tested. Numbergf Nucleoli The nucleolus of a cell plays a central role in cytoplasmic protein synthesis. Caspersson's work (1950) with various types of nerve cells, and the results cited by other researchers, have lead him to the conclusion that conspicuous changes in protein metabolism are correlated with nerve function. During intense activity, a cell's protein-forming; system must be able to replace expended proteins at a rapid rate. Cells such as the supraOptic neurons, which, in addition to normal cellular functions, must produce protein-rich secretions, can be expected to place a great demand on their protein-synthesizing centers. 40 Edstrom, Eichner and SchOr (1961), determined the concen- tration of ribonucleic acid (RNA) in isolated hypothalamic neurons. (RNA is an important element in the biosynthesis of protein.) When rats were deprived of water for seven“ days, a treatment known to stimulate increased production of ADH, RNA content of supraoptic neurons increased to ‘over double the original amount. Bachrach (1964) has also demonstrated increased RNA production in the supraoptic neurons of deprived rats; he reported a corresponding.in- crease in the size of the nucleoli of these cells. In the present study no quantitative measure was made of the size of the nucleoli; however, a rather striking difference between animals, in the number of nucleoli per cell, became apparent. The normal laboratory rat supraOptic neurons are predominently uni-nucleolar. Only 5 percent (anterior N80) and 7 percent (tuberal NSO) of the cells had double nucleoli. After five days of water deprivation, the figures increased to 18 percent in the anterior and 14 per- cent in the tuberal. Fifty-eight and 57 percent of KR neurons had double nucleoli; there was also a small percent of cells possessing triple nucleoli. It would appear that the large number of cells with double nucleoli in KR, and increased numbers in DR, were results of cellular hyper- function, i.e., responses to physiological demands for a high level of hormonal production. An examination of the 41 cells with double nucleoli revealed that each nucleolus was approximately one half the size of the nucleoli of single nucleolar cells in the same animal. Why these particular cells develOped two nucleoli instead of merely increasing the size of the existing one is cause only for speculation: IS it a case of a degenerative phenomenon? The KR would seem to indicate that this is not so; these animals can live indefinitely on a diet of dried seeds with continual ADH production. Our animals, which had been maintained for approximately twenty days on a seed diet, were uniformly high in cells with double nucleoli. Perhaps two small nucleoli are more efficient than one large one for intensive protein synthesis. (Recall that under deprivation conditions a small percent- age of rat supraOptic neurons develOp double nucleoli.) This is all, of course, only Speculation. It seems a likely possibility until the gerbil is considered. The number of cells with double nucleoli in the ger- bils we examined, is just as small as in the normal labora- tory rat. Here is an animal who is able to get along almost as well as the kangaroo rat in a desert environment; however, the majority of the gerbils' cells were uni-nucleolar. Several things must be taken into consideration be- fore basing any assumptions on these data. Bachrach (1964) has demonstrated that supraOptic neurons go through a 42 number of phasic responses during deprivation and rehy-‘ dration. The size of the cytOplasm, nucleus, and nucle- olus changes during these treatments. Enestrom (1967) has shown that as days of deprivation increase, the cell expands to a certain size, and then decreases somewhat. Perhaps individual cells are able to increase the nucleo- lar size up to a certain point without impairing function; after that point has been reached, division of the nucleo- lus takes place. (Recall that double-nucleolar cells have nucleoli which are about one half the size of those in Single-nucleolar cells.) Both nucleoli may start to develop in size, competing for materials in the nucleus, such as the nucleolus-associated chromatin; this competition may eventually cause one of the nucleoli to degenerate and allow the remaining one to assume full function and to de- velop to the size of the original nucleolus, until it too undergoes division and the cycle is repeated. (Cases of cells with triple nucleoli may be due to a failure of one nucleolus to degenerate.) If this is indeed the case, the disparate appearance of the G and KR cells can be accounted for: The KR and G may have been sacrificed at what were actually different periods in a phasic cycle of the cells. Even though both species were maintained on the same diet, and were not sacrificed until their weights had stabilized, the conditions for the two animals were 43 not necessarily equivalent in terms of physiological de- mands imposed on the cells for ADH production. Castel and‘ Abraham (1969) have shown that the spiny mouse, Acomys. . russatus, displays a marked increase in N80 cells with multiple nucleoli as days of deprivation are increased- In order to clarify the meaning of the nucleolar. results in the gerbil, it would be desirable to investi- gate NSO functioning at different intervals along a depri- vation continuum. Such data may reveal that the picture furnished by the specimens used in this study is an in- complete one, the complete picture revealing a phasic response to deprivation. Summary The kangaroo rat, one of nature's most competent water economizers, with its ability to gain weight on a. diet free from exogenous water, seems to demonstrate a functionally more active supraOptic nucleus than that found in either the gerbil or laboratory rat. This desert rodent has relatively more supraoptic neurons per gram_body weight and more double nucleoli per cell than either of the other two animals. There is also some indication that cell size (relative to body weight) is greater in this animal than in the gerbil or laboratory rat. Correspondingly, the gerbil has more NSO cells, relative to body weight, than the laboratory rat; the data suggests, in addition, that 44 gerbil NSO cells are larger than those in the rat. The fact that the supraoptic cells of a normal laboratory rat deprived of water for five days demonstrate changes that approach the conditions found in desert rodents, indicates that increases in cell size and number of nucleoli may be adaptive mechanisms which desert rodents have capitalized upon. REFERENCES Ames, R. G. and van Dyke, H. B. Antidiuretic hormone in the urine and pituitary of the kangaroo rat. Proc. Soc. Exper. Biol._Med., 1950, lg, 417-420. Auer, J. Postnatal cell differentiation in the hypothalamus of the hamster. J. Comp. Neurol., 1951, 22, 17-41. Bachrach, D. "The relation of structure and function in the anterior hypothalamic nuclei." In Major problems .12 neuroendocrinology (E. Bajusz and G. Jasmin, eds.). Switzerland: S. Karger, 1964. Bleier, R., Bard, P. and Woods, J. W. Cytoarchitectonic appearance of the isolated hypothalamus of the cat. J. Comp. Neurol., 1966, 128, 255-312. Bodian, D. and Maren, T. H. The effect of neuro- and adeno— hypophysectomy on retrograde degeneration in hypo- thalmic nuclei of the rat. J. Comp. Neurol., 1951, 14, 485-512. " Burns, T. W. Endocrine factors in the water metabolism of the desert mammal, G. gerbillus. Endocr., 1956, 58, 243-254. Caspersson, T. D. "The organization of the system for cytoplasmic protein formation in the normal metzaoan cell." In Cell growth and function. New York: W. W. Norton & Co. Inc., 1950. Chastel, M. and Abraham, M. Effects of a dry diet on the hypothalamic neurohypophyseal neurosecretory system in spiny mice as compared to the albino rat and mouse. Gen. Comp. Endocr., 1969, JJ, 231-241. Conn, H. J., Darrow, M. A. and Emmel, V. M. Staining Pro- cedures. Baltimore: Williams and Wilkins Co., 1962. Cotte, G. and Picard, D. Etude ultrastructural des neurons ‘ du noyau supraOptique du rat. C. R. Assoc. des Anat. 1968, 141, 738-747. 45 46 Dawson, A. B. Early secretory activity in the hypothalamic ’ nuclei and neurohypophysis in the rat; determined by selective staining. J. Morph., 1966, 118, 519- 529. Edstrom, J. E., Eichner, D., and SchOr, N. "Quantitative ribonucleic acid measurements in functional studies of nucleus supraOpticuS." In Regional neurochemistry (S. S. Kety and J. Elkes, eds.) New York: Pergamon Press, 1961. Enemar, A., and Hanstrom, R. Egl. Fysiograf. Salisk Handl. Lund (NF.), 1956, El, 1. Enestrom, S. Nucleus supraopticus. A morphological and experimental study in the rat. Acta Path. Microbiol. Scand., Suppl., 1967, 186, 1-99. Fisher, C., Ingram, W. R., and Ranson, S. W. Diabetes instiduS and the neuro-hormonal controI of water balance: a contribution to the structure and function of the hypothalamico-hypophyseal system. Ann Arbor, Michigan: Edwards Brothers, 1938. Gottschalk, C. W., and Mylle, M. Micro-puncture study of the mammalian urinary evidence for the countercurrent concentrating mechanism hypothesis. Am. J. Physiol., 1959, I99, 927-936. Hatton, G. and Johnson J. Unpublished research.‘ Michigan State University, 1968. (Order of authorship to be determined.) Howe, A. and Jewell, P. A. Effects of water deprivation upon the neurosecretory material of the desert rat (Meriones meriones) compared with the laboratory rat. J. Endocr., 1959, JJ, 118-124. Khalil, F., and Tawfic, J. Some observations on the kidney of the desert J. jaculus and G. gerbillus and their possible bearing on t e water— economy of these ani- mals. J. Exp. Zool., 1963, I25, 259- 268. Khalil, F., and Tawfic, J. The hypothalamo-hypOphySial neurosecretory system of the desert rodents J. jaculus (Oliv. ) and G. gerbillus (Linn. ). J. Exp. Zool., 1963, .liir 189- 195. - 47 Koford, C. B. Peruvian desert mice: water independence, competition, and breeding cycle near the equator. Science, 1968, 160, 552-553. Legait, H. Correlations endocriniennes de l'hypothalamus et de la neurohypOphyse chez la poule Rhode-Island. C. R. Sgg. Biol. (Paris), 1955, 149, 1016-1018. Lockwood, A. P. M. Animal fluids and their regglation. London: Heinemann, 196% (p. 64). MacMillen, R. E., and Lee, A. K. Australian desert mice: independence of exogenous water. Science, 1967, 158, 383-385. Malone, E. F. The nuclei tuberis laterales and the so- called ganglion Opticus basale. Johns ngk. Hosp. Rep., 1916, J1, 441-511. Pearse, A. G. E. Histochemistry: theoretical and applied. London: _J. and A. Churchi1l, Ltd., 1961. Peterson, R. P. Magnocellular neurosecretory centers, in the rat hypothalamus. J. Comp. Neurol., 1966, 128, 181-190. Scharrer, E. and Scharrer, B. Neuroendocrinology. New York: Columbia University Press, 1963 (p. 116). Schmidt-Nielsen, K. Animal h siolo . Englewood Cliffs, New Jersey: Prentice-HalI, 1362 (p. 168). Schmidt-Nielsen, B. and Schmidt-Nielsen, K. A complete account of the water metabolism in kangaroo rats and an experimental verification. J. cell. comp. Physiol., 1951, 38, 165-181. Schmidt-Nielsen, B. and Schmidt-Nielsen, K. Do kangaroo rats thrive when drinking sea water? Am. J. Physiol., 1950, 160, 291-294. Smith, S. W. The correspondence between hypothalamic neuro- secretory material in vertebrates. Am, J. Anat., 1951, JJ, 195-232. Thorn, N. A. Mammalian antidiuretic hormone. Physiol. Revs., 1958, J8, 169-195. 48 Vimtrup, B. and Schmidt-Nielsen, B. Histology of the kid- ney of kangaroo rats. Anat. Rec., 1952, 114, 515- 530. Westwood, W. J. A. Anatomy of the hypothalamus of the ferret. J. Comp. Neurol., 1962, 118, 323-341. Winkelmann, J. R. and Getz, L. L. Water balance in the mongolian gerbil. J. Mammology, 1962,ng, 150— 154. APPENDIX A Materials APPENDIX A Materials Animals 1. 2. 3. Kangaroo rats obtained from The Pet Farm, Miami, Florida. Gerbils ordered from Chickline, Vineland, New Jersey. Albino rats, Holtzman strain, from Madison, Wisconsin. Miscrosopic Eguipment 1. Zeiss microsc0pe Photochanger Extension tube Whipple-Hauser disc Photographic Equipment l. 2. Cameras: Zeiss Icon micrOSQOpe camera and a 5" x 7" plate camera and Optical behch arrangement. Film: (1) Kodak High Contrast Copy, (2) Kodak Metal- lographic Plates. Printing paper: (1) Kodak Kodabromide - F-S, (2) Kodak Photographic Paper - AZO F-S. Contact Printer Photo Enlarger 50 APPENDIX B Procedure APPENDIX B Procedure Celloidin Embedding l. Perfuse brain as soon as anima1 is anesthetized, or as soon as possible after death. Perfuse first with saline (.87% Na C1), followed by a formalin mixture (10% formalin, .87% saline). Remove brain from skull, immerse in 10% formalin in .9% saline for five days. Place brain in running tap water overnight. Dehydrate through graded alcohols: 80% alcohol ' 1 day fresh 80% 1 95% alcohol 2 fresh 95% 3 used absolute alc. 1 fresh absolute 1 ether-alcohol (50/50) 1/2 thin celloidin 7 medium celloidin 7 thick celloidin 14 52 Wide mouth specimen jars are satisfactory for the de— hydration process and for the first three celloidins. To make thin celloidin use 5 gms Nitrocellulose to 100 cc ether alcohol; medium celloidin is 15 gms Nitrocellulose to 100 cc ether alcohol; thick is 25 _gms Nitrocellulose to 100 cc ether alcohol. The ether alcohol is made with 2 parts ether to 1 part absolute alcohol. On the last day in thick celloidin place brain in a paper box filled slightly with thick celloidin. Cover with thick celloidin and position brain with a probe. When the celloidin becomes thick enough not to adhere to the finger when pressed, place the block in an air- tight container and this within another airtight con- tainer. After bubbles in the celloidin have disappeared, place the block in a desiccator along with several small vials of chloroform. Place the lid on the desiccator and seal tightly. After the celloidin has become firm cover the block with 70% alcohol and let stand until the block can be handled easily. Store the block in fresh 70% alcohol until ready to mount and section. ‘ 53 Stainin Neurosecreto Material (for use with CeIIoidin embedded tissue) The following procedure works satisfactorily with sections of tissue 15 u thick; if the sections are much thinner or thicker, experimental modifications will be necessary to determine the procedure best suited for the material. A. Removing the celloidin 1. Select sections and place them in a petri dish filled with 95% alcohol. Agitate the paper under solution and float the sections off. Place the sections serially in a second petri dish filled with clove oil. The celloidin will melt away from the edges of the tissue almost immediately, but the celloidin in the tissue takes longer to come out. Fifteen u sections take about 2-2 1/2 hours using fresh clove oil. Transfer the sections to zylene for 15 minutes. Repeat this with three more changes of zylene. Rehydrating the tissue 1. Move the sections at 20 minute intervals through a series of graded alcohols, i.e., from 100% to 90, to 70, to 50, to 30. Place the sections in a dish filled with distilled water for 20 minutes and then transfer them to a fresh change of water for another 20 minutes. 54 Mounting the sections Arrange sections on gelatinized slides. Keep the water on the slide down to a minimum in order to keep the ‘gelatin from becoming too dilute to hold the sections on when they dry; after placing each individual section on the slide, blot brush on paper toweling at intervals while orienting the sections--when it is in the desired position blot the section with brush until it adheres. without moving when the slide is tilted. By the time most of the sections are on the slide, some will have begun to dry out and if allowed to get too dry, the edges will begin to curl up. Do not allow this to happen or sections will stain unevenly. When the sections have all dried at least once, place the slide in a tray and submerge in distilled water. Take slides through the staining baths. Gomori's Aldehyde-Fuchsin (Adapted after Conn, H. J., Darrow, M. A., and Emmel, V. M. Staining Procedures. Biological Stain Commission, 1962, Williams and Wilkins Co., Baltimore.) 1. Oxidize in potassium permanganate one minute. (.3 g KMn0 100 ml distilled water, .3 ml concen- 4' trated H2504. 2. Rinse in distilled water. 3. Bleach in 2% sodium bisulphite two minutes. 55 4. Wash in running tap water five minutes. 5. Stain in aldehyde fuchsin mixture overnight. (Add 1 9 basic fuchsin to 200 m1 boiling water. Boil 1 minute; cool and filter. Add 2 m1 concentrated HCl and 2 m1 paraldehyde. Leave stoppered at room. temperature. When mixture has lost reddish fuchsin color and is deep purple (3-4 days), filter and discard filtrate. Dry1precipitate on filter paper in oven. Remove and store. Dissolve 0.25 g in 50 ml of 70% alcohol. Stain may be used for several months, but filter before each new use.) 6. Leave in acid alcohol 15-20 minutes. (0.5% HCL in 70% alcohol.) 7. Leave in 95% alcohol from 6-12 hours until light enough so that stained neurosecretory areas will stand out from the background when placed under the microsc0pe. 8. Take through 3 changes of xylene (10 minutes in each). 9. Cover slip. Alternative Stain: Bargmann's Modification of Chrome-Alum- Haematoxylin Method of Gomori (Adapted after Pearse, A. G. E. Histochemistpy: theoretical and a lied. 1961, J. and A. Churchill, Lt§.,wLondon) 1. Leave sections 12-24 hours in 1 part Bouin's with O 1 part of 3-5% chrome-alum at 37 (Bouins: 50 ml 56 10. ll. 12. picric acid, 10 m1 commercial formaline, 5 ml glacial acetic acid, 35 ml distilled water.) Wash in tap water until colorless. 1 minute in potassium permanganate — sulphuric acid solution: 41.3% KMnO in 0.3% H280 4 4‘ Rinse in distilled water. Decolorize in 1% oxalic acid, 1 1/2 min. Wash in tap water, 1 minute. Stain 12 minutes in haematoxylin solution: haematoxylin 0.5 g distilled water 50.0 ml when dissolved add potassium dichromate (5%) 2 ml 2.5% sulphuric acid 2 ml ripen 48 hours--can be used as long as a metallic film is present. Store in refrigerator. Filter before use. Differentiate for 1 1/2 minutes in 0.5% HCL in 70% alcohol. Wash in tap water, 2-3 minutes. Dehydrate rapidly, 2 minutes in each solution. (30, 50, 70, 80, 90, 100% alcohol). Take through 3 changes xylene (10 minutes in each). Cover slip. 57 Thionin Stain (Nissl Method) 1. Float sections off paper into distilled water. 2. Place sections in a steaming bath of 1% aqueous thionin solution (buffered to pH 4.0) and leave in 54° oven 15 minutes. 3. Transfer the sections through two changes of distilled water. 4. Place sections in 80% alcohol and agitate for 1-2 minutes then to fresh analine alcohol (50cc aniline, 450 cc 95% alcohol). Allow the sections to remain here to complete differentiation. 6. When sections have reached the desired color transfer them to 95% alcohol. Transfer to fresh 95% for three additional times to fully remove the aniline. 7. Put through 1 change of 100% alcohol. 8. Take through oil of cajeputmfor clearing. 9. Take through 4 changes of xylol to remove cajeput oil. 10. Mount on slides and cover slip. Measurements (1) Cell Area Supraoptic nucleus: Cells of the N80 were magnified 2200 times through a Zeiss microscope with a photo-changer attachment (objective: x 40; eyepiece: x 12.5; distance from floor to lens: 37.5 cm.). The sample consisted of 50 randomly selected cells (five cells from each of five 58 sections through the left and right portions of the nucleus) for each nucleus, anterior and tuberal, in each animal, in both horizontal and sagittal planes.’ A cell's outline was traced when its nucleolus was in focus. The areas of the cells were obtained by measurement with a Keuffel and Esser compensating polar planimeter. Hippocampal cells: Cell area for hippocampal cells was obtained in the same manner described for the N80 cells, the only difference being the number of cells sampled. Five cells from each of two sections through the left and right portions of the hippocampus of each animal were measured. (2) Number of Nucleoli The sample consisted of 50 cells from each nucleus in each animal and plane. The number of nucleoli was recorded for each individual cell in the sample. (3) Number of Cells By placing a Whipple-Hauser disc in the eyepiece of the microscope, a grid could be superimposed on a section of the NSC. Number of cells contained within successive squares were tabulated to yield a total number of cells per section. Only cells with nucleoli visible were counted. In instances where there were two or more nucleoli per cell, only one was tabulated. A11 thionin stained sections through the nucleus were examined and counted in.this manner. 59 Since the thionin set of sections represented only every third section through the nucleus, the number of cells per section was multiplied by three in order to take into account the cells in the intervening sections. 60 APPENDIX C Raw Data N=50. Tuberal f NSO. try, for Horizontal Plane 10115 O 1me Anterior d,by plan Gerbil d tuberal port Tuberal 1ne Sagittal Plane 101' an ters as determ Anterior h plane, both anter time Tuberal 1 ~1n square cen Horizontal Plane »1n eac Anterior Kangaroo Rat Sagittal Plane Tuberal NSO cell areas each animal, Anterior mmmMOIjmvmmV-HhmHomacovoou—Ilnmo mmmxohhxoxoxomxoxohmbxovbuioowrxoxoewo HMI‘HQHHmLfich-IQ'NMLfimmooommmo ml‘l‘ONFKDl‘mOLnl‘ko\OmRDOFFOml‘l‘Lnkom I-l mHHmoooxoxNaooouobxomaomooooooNomm \ouoml‘mt‘vxoxot‘mcoxohxomxommxomvmm 7.0 mNmNmkonDKDOHle‘u-lml‘mmmwwkoo hmv'oomhcoaomoommmmmhmbxobmhmm 8.0 \ocohxommmooooomoomooamxocoommmxom O O C V‘MMNNQ‘MMQ‘MMMQ‘NMNNNMMMMMM mr-Iboomu—uomv'wmmxooomoommmvmmmh O O MMMMMQ‘MV'Q'V‘WV'MMVV‘NNV'MNMNM \ocoommmwooommxohxohoohmooooxoxooxcru—I MVMNMV‘MQ'MQ'MMNQ‘NMMVNMMMMNQ‘ wrowrw\oah409huwu1mcngwwuaorqhwo~cnundawouuoxmr~ouncqouwurv<3c>o O I O O O O O O O O O O O O O O O O uwvLn«amr~unnuwhwomnouamcnanor~mr~an\cam oucowooxoxooxo¢~ O O O O O O O O O O O O 0 O 0.. O O O O O O O O O r~mr~r~mxoannawmcounooxmb H mmhfl'mmmwwm o o o o o o o o o o \D comboooommoxo ,_.| momommmvommowm \ombmhxohooooxomxohxo 6.2 MWOOQHVQ'OH O \Ol‘l‘l‘lfil‘l‘l‘mo H Hooocov—Ihhcooooxooxm o 0 0'. o o o o o o o o o o Hl‘ml‘kol‘mml‘l‘bomm H H 6.1 62 NSO cell areas (cont'd.) S-Day Deprived Rat Sagittal Plane Normalffiat Horizontal Plane Anterior Horizontal Plane Sagittal Plane I J Tuberal If Tuberal Anterior Tuberal Anterlor Tuberal Anterior 9.7 13.2 8.7 10.3 8.7 10.5 13.5 8.9 8.4 10.3 9.8 1 2 12.2 9:3 8.5 . 7.7 . O 5.3 7.5 8.6 7.3 10.1 6.2 7.7 8.3 8.5 5.9 9.8 12.1 11.5 9.0 10.8 11.3 5.8 6.3 7.0 6.0 5.0 8.7 11.6 8.9 8.1 9.1 9 . 8.7 10.9 8.3 7.4 11.4 11.9 8.3 10.1 14.0 7.7 13.7 10.3 8.6 10.3 11.8 9.7 11.4 11.5 11.2 8.4 7.5 9.4 7.7 6.0 9.0 6.0 4.5 6.0 11.1 7.5 10.5 6.9 4.6 8.8 7.5 6.9 \DV‘ 8.6 8.8 5.7 5.6 9.6 6.9 7.2 5.5 6.0 5.3 5.3 7.7 10.1 8.7 7.9 7.2 7.2 7.5 8.2 4.7 5.7 9.5 11.1 6.7 7.7 7.3 6.2 6.4 6.7 6.3 9.5 10.6 10.5 7.0 8.9 7.7 8.5 5.3 4.8 10.1 10.3 12.1 8.2 8.0 . 7.6 8.9 7.1 6.7 6.1 5.6 5.6 6.5 8.9 8.5 6.7 5.4 4.3 6.7 4.7 63 8.0 5.0 6.9 6.0 7.1 5.7 5.9 8.1 6.5 7.9 0:400“) HHu—ll-ir-l V'mNONF O O O O O O‘GOHO‘ r—IH HO‘NMQ‘ o o o o o ommmw H LOO moo mm 9.9 com [‘l‘ H 12.2 8 9 9 9 10.9 9 9 8.5 O 0 «1o F4H 9.0 8.3 mm 7.7 8.2 8.0 00 moo com 7.3 6.6 4.6 vm 6.6 6.0 6.9 [‘0 r-iCh 6.7 mm cocoon-4m l‘KOfl'kab \OLnI-IFM \ov'xolnm O‘ONHO ooonnm H O‘N‘l‘ 8.9 7.9 12.2 10.7 1 11.6 10.3 11.8 9.3 8.2 10.6 10.3 9.5 10.0 9.0 10.8 MN com 8.3 10.8 7.0 10.4 6.6 7.7 7.4 6.3 8.7 5.0 8.7 5.7 5.0 7.2 6.8 8.7 10.1 14.3 1 6.8 12.5 5.2 . 6.3 6.1 8.0 6.7 Deprived Rat Normal Rat N=20. Gerbil Sagittal Horizontal Sagittal Horizontal Sagittal Horizontal Hippocampal cell areas in square centimeters as determined by planimetry, in both planes. for each animal, Kangaroo Rat Sagittal Horizontal \OmflmmmommmHF-IV'NN o o o o o o o o o o o a o o o \DkaOl‘l‘Lfile‘kaDFKOOSOKD l‘kDLfiKOOMONmml‘Hfl'F-ikom \OOWI‘OQWI‘LDLDWI‘FI‘I‘ MHMI‘NO\MMHNONMH mhxoxohxoxoxobxoxoxoxo mnxowlnoooooxooxmooo O \oxohxomxoxoxot‘hxohh 7.0 64 6.3 4.4 7.1 8.1 Number of cells for each 156 thick section through the- anterior and tuberal nuclei, in each animal and plane. Kangaroo Rat (Sagittal Plane) Anterior NSO Section Number Cell Count. Section Number Cell Count 27 5 225 42 30 54 228 38 33 82 231 35 36 75 234 39 39 80 237 36 42 65 240 39 45 54 243 33 48 41 246 35 51 49 249 29 54 70 252 30 57 72 255 19 60 82 258 8 63 74 261 6 66 74 69 74 72 50 75 48 78 35 81 30 84 20 87 10 90 8 93 2 174 1 177 2 180 4 183 10 186 12 189 21 192 30 195 41 198 40 201 47 204 59 207 56 210 52 213 42 216 43 219 41 222 35 65 Kangaroo Rat (Sagittal Plane) Tuberal NSO Section Number Cell Count Section Number Cell Count 45 1 171 18 48 0 174 16 51 3 177 16 54 9 180 31 57 23 183 33 60 33 186 29 63 48 189 34 66 83 192 48 69 83 195 42 72 84 198 . 36 75 79 201 50 78 62 204 54 81 57 207 42 84 74 210 45 87 57 213 24 90 59 216 31 93 54 219 29 96 44 ' 222 32 99 39 225 34 102 21 228 26 105 17 231 15 108 18 234 9 111 14 237 4 114 13 240 l 117 13 120 4 123 15 126 12 129 6 132 1 135 13 138 1 147 2 150 5 153 11 156 22 159 18 162 15 165 19 168 16 66 I" Kangaroo Rat (Horizontal Plane) Anterior NSO Tuberal NSO Section Number- Cell Count Section Number Cell Count 108 46 117 5 111 109 120 27 114 148 123 11 117 134 126 17 120 80 129 3 123 36 132 33 126 57 135 13 129 55 141 81 132 43 114 6 135 4 117 3 141 74 120 42 144 112 123 17 102 60 126 20 105 102 129 5 108 126 132 41 111 92 135 84 114 70 138 81 117 59 141 38 120 61 123 66 126 75 129 67 132 72 135 52 141 97 144 148 67 Gerbil (Sagittal Plane). Anterior NSO Tuberal NSO Section Number Cell Count Section Number. \Cell Count. 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 141 144 147 150 153 156 159 162 165 168 171 174 177 180 183 186 189 192 195 198 201 204 22 50 44 58 53 36 42 50 70 58 48 65 50 33 43 47 39 38 36 29 22 13 4 4 17 21 43 38 29 65 68 50 42 57 54 58 46 49 75 54 28 24 17 6 6 68 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 123 126 129 132 135 138 141 144 147 150 153 156 159 162 165 168 171 174 3 31 42 59 65 68 71 36 28 17 21 26 Gerbil (Horizontal Plane) Anterior NSO Tuberal NSO. Section Number Cell Count Section Number. .Cell Count 147 16 177 10 150 20 180 17 153 19 183 43 156 19 186 31 159 18 189 71 162 12 192 80 165 20 195 63 168 29 198 50 171 59 201 97 174 147 204 19 177 238 180 8 180 173 183 42 183 56 186 26 186 51 189 56 156 12 192 75 159 36 195 55 162 30 198 51 165 20 201 93 168 28 204 46 171 12 174 42 177 75 180 99 183 132 186 152 189 60 192 39 69 Deprived Albino Rat (Sagittal Plane) Anterior NSO Section Number Cell Count Section Number .Cell Count 16 15 ' 2 189 ‘ 68 18 65 192 103 21 17 195 122 24 99 198 90 27 62 201 112 30 91 204 117 33 135 207 121 36 171 210 109 39 112 213 129 42 129 216 127 45 134 219 100 48 143 222 113 51 125 225 113 54 139 228 118 57 136 231 113 60 129 234 120 63 108 237 88 66 108 240 96 69 100 243 99 72 90 246 74 75 82 249 6 78 69 252 16 81 45 255 47 84 44 87 26 90 11 93 13 96 9 99 5 156 7 159 7 162 5 165 11 168 27 171 29 174 43 177 39 180 49 183 73 186 70 70 Deprived Albino Rat (Sagittal Plane) Tuberal NSO 7Ce11 Count Section Number Cell Count Section Number 36 2 228 23 39 3 231 9 42 9 234 2 45 16 237 2 48 32 51 36 54 35 57 37 60 55 63 47 66 36 69 22 72 19 75 18 78 11 81 10 84 6 87 4 90 5 93 l 168 l 171 6 174 5 177 4 180 5 183 6 186 6 189 10 192 15 195 21 198 25 201 30 204 42 207 24 210 26 213 38 216 30 219 21 222 27 225 27 71 Deprived Albino Rat (Horizontal Plane) Anterior NSO Tuberal NSO Section Number Cell Count Section Number- Cell Count 189 6 204 2 192 5 213 8 195 1 216 31 201 28 219 46 204 217 222 ‘ 91 207 606 225 58 210 775 228 76 213 424 210 20 216 192 213 45 219 15 216 69 192 5 219 36 195 17 222 42 198 59 225 38 201 242 228 12 204 624 207 805 210 535 213 195 216 42 72 Weight of animals before perfusion Animal Weight in grams Kangaroo rat (#1) 49 Kangaroo rat (#2) 53 Gerbil (#1) 56 Gerbil (#2) 50 Normal Rat (#1) ' 371 Normal rat (#2) 405 Deprived rat (#1) 298 Deprived rat (#2) 286 73 Aug 6 1969 llHI"WWII!"IWJIWIIHHIlllllfllHlllHlllllflHll 50284 4503