Michigan State University

..____ LIEMRY Michigan State University ———— OVERDUE FINES: 25¢ per day per ite- RETURNING LIBRARY MATERIALS: Place in book return to remove charge from c1 rcuht1on records BRAIN IRON'IN THE RAT: DISTRIBUTION, SEX DIFFERENCES, AND EFFECTS OF SEX HORMONES By Joanna Marie Hill A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1981 ABSTRACT BRAIN IRON IN THE RAT: DISTRIBUTION, SEX DIFFERENCES, AND EFFECTS OF SEX HORMONES By Joanna Marie Hill Although the brain contains relatively large amounts of iron, and iron deficiency alters behavior, little is known about those factors which affect brain iron or the role of n J .r iron in the brain. Sex hormones are responsible for sex r’ ’7 differences in many aspects of iron metabolism throughout ,l' .3 I the body. 6// The purposes of this study were to: (l) localize iron deposits in the rat brain; (2) determine if a sex difference exists in brain iron stores; (3) determine the effects on brain iron levels of natural events in which sex hormones fluctuate (e.g. estrous cycle and pregnancy); and (4) determine if exogenous estrogen alters the effects of ovariectomy and castration on brain iron levels. Brain iron was localized by histochemical methods and direct measurement of iron concentrations of high-iron areas (pooled globus pallidus and substantia nigra) and lower iron areas (cortex) of the brain, as well as the serum and liver were made by spectr0photometry. This study has determined that brain iron: is unevenly distributed in the rat brain; occurs in different cellular and extracellular compartments in different parts of the Joanna Marie Hill brain; and increases with age. Brain iron fluctuates during the estrous cycle, rising to the highest levels during proestrus. During the first third of pregnancy brain iron concentration rises and although the level falls later, pregnancy does not deplete brain iron. There is a sex difference in the brain iron concentration between males and females in the proestrus stage of the estrous cycle, and ovariectomy and castration have different effects on brain iron levels. The results of this study suggest that: the pattern of iron distribution may be related to the participation of iron in the metabolism of peptides; brain iron accumulation is influenced by ovarian hormones; and iron plays a role in neuroendocrine regulation. ACKNOWLEDGEMENTS Grateful acknowledgment is made to my coadvisers Dr. J.I. Johnson and Dr. R.C. Switzer for their interest, guidance and constructive criticisms throughout all stages of this study. Also, the helpful comments of my committee members Dr. M. Balaban, Dr. C.D. Tweedle and Dr. S.T. Kitai are sincerely appreciated. It is a pleasure to acknowledge Dr. P.D. MacLean, Chief, Laboratory of Brain Evolution and Behavior, NIMH not only for his enthusiasitc support but also for the laboratory space, equipment and supplies he made available to me. I wish to thank Mrs. J. Bupp for her editorial comments and competent typing of the dissertation and Mr. R. Harbaugh for invaluable technical assistance with surgical procedures and the care and breeding of animals. I thank also my husband, Jim for his assistance with the statistical analysis and for the patience, understand- ing and support I received from him and my daughters, Andrea and Katherine, without which this study would not have been possible. ii TABLE OF CONTENTS PAGE LIST OF TABLES ........................................ iv LIST OF FIGURES ........................................ v INTRODUCTION ........................................... 1 LITERATURE REVIEW ...................................... 4 MATERIALS AND METHODS ................................. 37 HISTOCHEMISTRY ...................................... 37 Animals ............................................ 37 Treatment Groups ................................... 37 Preparation of Tissue .............................. 40 Staining techniques ................................ 43 Analysis of Data ........... , ....................... 49 SPECTROPHOTOMETRY ................................... 50 Animals ............................................ 50 Treatment Groups ................................... 50 Collection of tissue for the Spectrophotometric Measurement of Iron ................................ 59 Requirements for the Spectrophotometric Measurement of Iron ................................ 61 Solutions for Iron Spectrophotometry ............... 63 Determination of Serum Iron ........................ 65 Preparation of Liver Extract ....................... 66 Determination of Liver Iron ........................ '68 Preparation of Brain Extract ....................... 69 Determination of Brain Iron ........................ 72 Analysis of Data ................................... 74 RESULTS ............................................... 75 HISTOCHEMISTRY ...................................... 75 SPECTROPHOTOMETRY .................................. 108 DISCUSSION ........................................... 134 SUMMARY .............................................. ‘157 BIBLIOGRAPHY ........................................ 160 iii LIST OF TABLES TABLE PAGE The Distribution of Non-haemin Iron in Different Parts of the Human Brain Autopsy Cases, 30-100 Years of Age ....................... 17 Determination of Iron in the Subcellular Fractions of Different Brain Areas ............... 27 Summary of Iron Measurement Study ................ 58 Analysis of Variance and Tukey's Test of Iron Measures of Estrous Cycle and Sex Difference Data ............................................ 111 Analysis of Variance with Regression of Iron Measures of Pregnancy Data ...................... 116 Analysis of Variance with Orthogonal Contrasts of Iron Measures of Control Males (CM), Cast- rated Males (CAST), Castrated Males with Estrogen Implants (CAST+EST), Intact Females in Estrus (ESTF), Ovariectomized Females (OVX) and Ovariectomized Females with Estrogen Implants (0VX+EST) .............................. 122 The Distribution of Monoamines and GABA in Iron Concentrating Areas of the Rat Brain ............ 136 The Distribution of Peptides and Iron Concent- rating Areas of the Rat Brain ................... 139 iv LIST OF FIGURES FIGURE PAGE 1. Distribution of iron. Perl's-DAB stain for iron, no counterstain. Stained areas are accumulations of iron. Parasagittal view of a 52 week old female rat. Magnification X 4.5 ........................................ ..76 Distribution of iron in the forebrain at the level of the anterior commissure in a 32 week old female rat. Perl's-DAB, no counterstain. Stained areas are accumulations of iron. Magnification X 5.4 ......... .78 Distribution of iron in the forebrain at the level of the anterior commissure in a 32 week old male rat. Perl's-DAB, no counterstain. Stained areas are accumula— tions of iron. Magnification X 5.4 .......... ..78 Distribution of iron in the forebrain at the level of the globus pallidus in a 32 week old female rat. Perl's—DAB, no counterstain. Stained areas are accumulations of iron. Magnification X 5.4 ................... ..78 Distribution of iron in the forebrain at the level of the globus pallidus in a 32 week old male rat. Perl's-DAB, no counterstain. Stained areas are accumulations of iron. Magnification X 5.4 .......................... ..78 Distribution of iron in the forebrain at the level of the thalamus in a 32 week old female rat. Perl's-DAB, no counterstain. Stained areas are accumulations of iron. Magnification X 5.4 .......................... ..80 Distribution of iron in the midbrain at the level of the substantia nigra in a 32 week old female rat. Perl's-DAB, no counterstain. Stained areas are accumulations of iron. Magnification X 5.4 .......................... ..80 V LIST OF FIGURES -— continued FIGURE PAGE Distribution of iron in the midbrain at the level of the substantia nigra in a 32 week old male rat. Perl's-DAB, no counterstain. Stained areas are accumulations of iron. Magnification X 5.4 ............................. 80. Photomicrograph of iron accumulation in area postrema and dorsal to the central canal Darker staining areas are accumulations of iron. Perl's-DAB counterstained with thionin. Magnification X 100 ............................. 83 10. Photomicrograph of iron accumulation in the subfornical organ and choroid plexus. Darker staining areas are accumulations of iron. Perl's-DAB counterstained with thionin. Magnification X 100 ............................. 83 ll. Photomicrograph of iron-filled fibers in the lateral edge of the optic tract. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 200 ............................. 86 12. Dark-field photomicrograph of iron—filled tanycytes and clumps of granules in the ventro-medial hypothalamus. Iron = white. Perl's-DAB counterstained with thionin. Magnification X 120 ............................. 86 13. Dark-field photomicrograph of iron-filled structures in the ventro-medial hypothalamus and arcuate area. Iron = white. Perl's-DAB counterstained with thionin. Magnification X 120 ........................................... 88 14. Dark-field photomicrograph of iron—filled structures in the ventro-medial hypothalamus and median eminence. Iron = white. Perl's-DAB counterstained with thionin. Magnification X 120 ........................................... 88 vi LIST OF FIGURES -- continued FIGURE PAGE 15. Photomicrograph of iron distribution in the ventral pallidum, islands

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