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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 I ,l' ’7 J 6// .3 r’ .r n were brain about exists differences areas deposits the iron iron, ectomy determine fluctuate direct and distributed (pooled body. extracellular Although The made This Brain in and those iron (cortex) measurement and in the globus purposes in brain by iron if study (e.g. castration levels iron brain. the factors in in exogenous spectr0photometry. the many DISTRIBUTION, deficiency of pallidus the AND was rat has estrous iron brain of of the compartments BRAIN of rat which brain; EFFECTS aspects Sex localized determined natural Joanna this stores; on brain, iron brain; estrogen hormones contains and cycle ABSTRACT brain IRON alters affect study concentrations (2) Marie OF of substantia SEX events as (3) By by and IN occurs SEX determine iron that iron in were well alters DIFFERENCES, THE behavior, brain relatively histochemical are determine Hill pregnancy); different HORMONES metabolism brain levels. in RAT: responsible as to: in which nigra) iron the the different if (l) of iron: effects little the or a parts large serum high-iron sex localize and and sex methods effects throughout the is hormones for amounts difference and is of lower cellular (4) role unevenly of known sex the ovari- iron liver on areas and of iron of 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 com- ments 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 accumul- ations 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 counter- stain. 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 of Calleja and olfactory tubercle. Iron = brown. Perl's-DAB counterstained with thionin. Coronal view. Magnification X 82 ...... 90
l6. Photomicrograph of iron-filled cells and fibers in the globus pallidus. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 900 ...... 90
l7. Photomicrograph of iron-filled cells and fibers in the ventral pallidum. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 476 ...... 93
l8. Photomicrograph of iron-filled cells and fibers in the substantia nigra. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 476 ...... 93.
19. Photomicrograph of small neuron-like iron- filled cells in the globus pallidus. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 900 ...... 95
20. Dark-field photomicrograph of bundles of fine iron-filled fibers in the globus pallidus. Iron = white. Perl's-DAB counterstained with thionin. Magnification X 1000 ...... 95
21. Photomicrograph of "cascades" of iron—filled fibers seen in the ventral pallidum and olfactory tubercle. Iron a brown. Perl's-DAB counterstained with thionin. Magnification 9 X 476 ...... 7
22. Dark-Field photomicrograph of iron in bouton- like structures on or in lateral septal neurons. Iron a white. Perl's-DAB counterstained with thionin. Magnification X 226 ...... 97.
vii LIST OF FIGURES -- continued
FIGURE PAGE
23. Dark-field photomicrograph of iron in bouton-like structures on or in bed nucleus of the stria terminalis neurons. Iron = white. Perl's-DAB counterstained with thionin. Magnification X 226 ...... 100
24. Photomicrograph of iron in bouton-like structures on or in ventral pallidal neurons. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 900 ...... 100
25. Dark-field photomicrograph of fine grains of iron in cells of the supraOptic nucleus. Iron = white. Perl's-DAB counterstained with thionin. Magnification X 216 ...... 103
26. Dark-field photomicrograph of fine grains of iron in cells of the suprachiasmatic nucleus Iron = white. Perl's-DAB counterstained with thionin. Magnification X 200 ...... 103
27. Iron concentration of liver, pooled globus pallidus and substantia nigra, cortex and serum of control males and females in each of the four stages of the estrous cycle. Mean values i l S.E.M., see ANOVA tables for sample sizes ...... 110
28. Iron concentration of liver, pooled globus pallidus and substantia nigra, cortex and serum every four days throughout pregnancy and 15-24 hours postpartum. Mean values i l S.E.M., see ANOVA tables for sample sizes...115
29. Iron concentration of pooled globus pallidus and substantia nigra of control males (CONT d), castrated males (CAST 6), castrated males with estrogen implants (CAST 6+EST), females in estrus (ESTRUS 9), ovariectomized females (OVX Q), and ovariectomized females with estrogen implants (OVX o+EST). Mean valuesi l S.E.M., see ANOVA tables for sample sizes...121
viii LIST OF FIGURES -- continued
FIGURE PAGE
30. Iron concentration of cortex of control males (CONT 6), castrated males (CAST 6), castrated males with estrogen implants (CAST 6+EST), females in estrus (ESTRUS 9), ovariectomized females (OVX g), and ovariectomized females with estrogen implants (OVX 9+EST). Mean values i l S.E.M., see ANOVA tables for sample sizes ...... 125
31. Iron concentration of liver of control males (CONT 6), castrated males (CAST 6), castrated males with estrogen implants (CAST 6+EST), females in estrus (ESTRUS 9), ovariectomized females (OVX ), and ovariectomized females with estrogen implants (OVX 9+EST). Mean values $.1 S.E.M., see ANOVA tables for sample sizes ...... 128
32. Iron concentration of serum of control males (CONT 6), castrated males (CAST 6), castrated males with estrogen implants (CAST 64EST), females in estrus (ESTRUS 9), ovariectomized females (OVX 9), and ovariectomized females with estrogen implants (OVX 9+EST). Mean values.i.l S.E.M., see ANOVA tables for sample sizes ...... 130
33. Body weight of control males (CONT 6), castrated males (CAST 6), castrated males with estrogen implants (CAST 64EST), females in estrus (ESTRUS 9), ovariectomized females (OVX a), ovariestomized females with estrogen imp ants (OVX 9+EST). Mean values i 1 S.E.M., see ANOVA tables for sample sizes ...... 133
ix INTRODUCTION
Iron deficiency is the most prevalent nutritional
disorder faced by the p0pulations not only of "under-
developed" nations but also of nations, such as the United
States, which enjoy a varied and abundant food supply (WHO,
1968; Ten State Nutritional Survey, 1970; Nutrition Canada,
1973; Garby, 1973; Kessner and Kolk, 1973; HANES, 1974). An excessive accumulation of iron in the body tissues also afflicts a significant fraction of the human p0pulation
(Sheldon, 1927; MacDonald, 1964; Pollycove, 1972). Abnormal iron levels are accompanied by a variety of both physical and behavioral disorders (see Pollitt and Leibel, 1976;
Oski, 1979; Dooling et al., 1974; Goldberg and Allen, 1980).
It is known that iron is stored in relatively large amounts in the brain, chiefly in the basal ganglia (wuth,
1923; Tingey, 1937; Cumings, 1948, 1968; Hallgren and
Sourander, 1958; Musil, Haas and Waurschinek, 1962;
Harrison, Netsky and Brown, 1968) and that iron deficiency significantly alters the behavior of man and animals (Glover and Jacobs, 1972; Pollitt and Leibel, 1976; Oski, 1979;
Weinberg, Levine and Dallman, 1979; Williamson and Ng,
1980). However, there has been little work on those factors which directly affect iron metabolism of the brain either in iron deficiency or in the iron replete condition.
Sex hormones influence behavior, and these hormones, chiefly estrogen, are responsible for a sex difference in
l 2 iron metabolism with effects on the uptake, levels of storage, and the mobilization of iron (Steenbock, Semb and
Van Donk, 1936; Smith and Otis, 1937, Otis and Smith, 1940;
Widdowson and McCance, 1948; Kaldor and Powell, 1957;
Dubrunquez and Lederer, 1963; Murray and Stein, 1968;
Bj¢rk1id and Helgeland, 1970; Linder et a1., 1973; Cook,
Hershko and Finch, 1973; Hershko and Eilon, 1974; Planas,
1976). Sex hormones are known to act directly on hormone- sensitive areas of the brain regulating the secretion of gonadotrophins and thereby influencing behavior (see reviews by Young, 1961; Flerko, 1966; Lisk and Barfield, 1975;
McEwen, 1981). However, neither a sex difference in brain iron levels nor any effect of fluctuating levels of sex hormones on brain iron levels has been reported. Deter- mining if a sex hormone such as estrogen influences brain iron levels will provide suggestive evidence that this hormone plays a role in brain iron metabolism. Secondarily, this evidence will suggest either that brain iron is influ- enced by an internal environmental factor, related to sex difference, which constantly affects other iron storage areas, or that the brain is "spared" from such a sex dif- ference. Also, the potential that the availability of iron may fluctuate in the brain tissues in response to changing hormone levels, as in the female reproductive cycle, has significance in the study of any aspect of brain iron metabolism. 3
The purpose of this study is to: (1) localize iron
deposits in the rat brain, (2) determine if a sex difference
exists in brain iron stores both quantitatively and in
location, (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.
Both histochemical and spectrOphotometric determination of brain iron are made. Not only does the histochemical identification of iron in the brain function to localize iron deposits, but also, the intensity of the reaction can be used as an indication of the amount of iron present in different brain areas. Spectrophotometry provides an accurate quantitative measure of iron, and the tissues analyzed by this method include the serum, liver, high iron areas of the brain (pooled globus pallidus and substantia nigra), and a lower iron area (cortex). The spectrophoto- metric determination of serum and liver iron along with brain iron serves two purposes. It indicates not only those animals which may have anemia or some defect in iron metab- olism and should therefore be excluded from the study, but also demonstrates whether the experimental manipulations
influence iron levels in the brain in the same direction and magnitude as occurs in the serum and liver. LITERATURE REVIEW
Three hundred years ago, Syndenham recognized the use of iron as a specific remedy for chlorosis (Fairbanks et a1,
1971), now recognized as iron deficiency anemia, a common disorder among young women of menstrual and child—bearing age. Iron was thus the first trace element known to be essential. Since that time, the study of iron metabolism has been an active field of research. Much of the data has accumulated from the study of man as many aspects of iron exchange can be studied from blood and small samples of liver and bone marrow which can be collected without lethal effects. Next to man, the laboratory rat has been the animal upon which most iron metabolism research has been centered.
Despite the vast amount of research in the field of iron metabolism, in some areas our knowledge is incomplete:
- The mechanism of absorption of iron from food in the intestine is not completely understood. There is some dis- agreement as to the way in which the intestine regulates uptake of iron and what factors are important in the bio- availability of iron in food.
- The mechanisms of iron transfer from blood to cell, cell to cell, and between compartments within a cell are unclear. The realtive importance of various oxidizing and reducing agents in this process is disputed.
- Lastly, a question deserving more attention: what is 4 5 the function of the large amount of iron stored in the brain--what is its role in brain metabolism and behavior?
A review of the literature specifically dealing with iron in the brain will follow a brief outline of iron metabolism in mammals. Iron in Biological Tissues
Iron has two special properties which will make it a vital constituent of every mammalian cell as well as an essential element in biological processes throughout nature:
iron can exist in two stable oxidation states (Fe++, Fe+++), and it can form many complexes. In biological systems, almost all iron is involved in processes related to oxygen metabolism.
Iron is present in tissues in two forms, heme and non- heme compounds. The heme compounds include hemoglobin (O2 carrier), myoglobin (O2 carrier), the heme enzymes catalase and peroxidase (protect cells from the harmful by-products of oxygen, superoxide ion and hydrogen peroxide), the cyto- chromes (electron transport), cytochrome oxidase (terminal oxidase), xanthene oxidase (purine metabolism and iron mobilization) and tryptophan dehydrogenase (tryptophan oxidation). The non-heme compounds include transferrin
(iron transport), ferritin (iron storage), hemosiderin (iron storage), aldehyde oxidase (indole amine degradation), tyrosine dehydrogenase (catecholamine synthesis), succinic dehydrogenase (Kreb's citric acid cycle), NADH-dehydrogen- ase, monoamine oxidase, ribonucleotide reductase, aconitase, aldolase, lipoxidase, and others (Freiden, 1974; Subcommit- tee on Iron, 1979; Yasunobo, Mower, and Hayaishi, 1975).
In an iron replete man, iron is distributed in the following way: 65% total body iron in hemoglobin
3% total body iron in myoglobin
13% total iron in ferritin
12% total iron in hemosiderin
5% total body iron in unknown compounds (taken from Frieden and Osaki, 1974) At birth, the total body iron concentration of a mammal is increased by the elevated red blood cell mass and the presence of appreciable storage iron in the liver; however, during suckling, body iron reserves are depleted (Lintzel, Rickenberger and Schairer, 1944; Widdowson and McCance, 1948, 1951; Keen and Hurley, 1980). At weaning the iron stores begin to accumulate and by 16 weeks of age, the rat has a threefold increase in liver iron (Cook, Hershko and Finch, 1973). The rate of accumulation of iron begins to slow at about 32 weeks (Kaldor and Powell, 1957) and a low rate of increase is maintained throughout life (Bjorklid and Helgeland, 1970: Kaldor and Powell, 1957; Widdowson and McCance, 1948). Iron Exchange The general pathways of iron exhange are well known. External exchange occurs largely through the intestinal tract. Iron attached to chelates is absorbed by the mucosa of the duodenum and jejunum, where it is trapped by iron binding proteins and made available to the transfer system (Gitlin and Cruchaud, 1962; Charlton et a1., 1965). The binding capacity of the protein is regulated by the iron needs of the individual. Some absorption also occurs by diffusion, governed by the amount of iron in the gut lumen (Thompson and Valberg, 1980). Only a small portion of dietary iron is available to the mucosa, as much intestinal iron forms polymerized iron hydroxide which is not absorb- able (Huebers and Rummel, 1975; Thompson and Valberg, 1980). In rats, the dietary intake of iron is about 100 times that of man (when expressed on a per kilogram basis), making absorption the primary route for meeting increased iron requirements (Cook, Herschko and Finch, 1973). Iron excretion occurs primarily through the feces by exfoliation of the intestinal mucosa. In man, very little iron is lost this way (McCance and Widdowson, 1937); however, in the rat this represents an important route of excess iron excretion (Cheney et a1., 1967). Within the body, iron is transported by plasma trans- ferrin. About 90% of the body iron is in the blood, bone marrow, liver, and Spleen (the erythron). The group next highest in iron concentration includes the kidney, heart, 9 skeletal muscles, pancreas, and brain (Cheney et a1., 1967; Keen and Hurley, 1980). Newly absorbed iron is almost immediately taken up by bone marrow where it is incorporated into heme in the formation of red blood cells which soon after appear in the blood. In the reticuloendothelial cells, primarily in the spleen, iron from effete erythrocytes is released, stored in ferritin or hemosiderin, or transported by transferrin to be used in the formation of new red blood cells, to the liver to be stored in hepatocytes or to other body tissues to meet their individual needs (Cheney et a1., 1967; Cook, Hershko and Finch, 1973). In man, with a limited capacity for absorption and slight loss through excretion, much of the iron for heme formation comes from the recycling of iron released through the breakdown of senescent red blood cells (Noyes et a1., 1964). In the rat, however, absorption and excretion play a greater part in maintaining iron balance (Cheney et a1., 1967; Cook, Hershko and Finch, 1973) than in man . 10 Sex Differences in Iron Metabolism Sex differences brought about by gonadal hormones are apparent in several aspects of iron metabolism. As first reported by Steenbock et a1. (1936) and later confirmed by others (Widdowson and McCance, 1948; Kaldor and Powell, 1957; Bjorklid and Helgeland, 1970; Cook, Hershko and Finch, 1973; Linder et a1., 1973; Hershko and Eilon, 1974), female rats have liver iron stores two to three times greater than male rats. A similar sex difference also occurs in the total body iron (Otis and Smith, 1940; Kaldor and Powell, 1957), spleen iron (Widdowson and McCance, 1948; Kaldor and Powell, 1957), kidney iron (Linder et a1., 1973), serum iron (Cook, Hershko and Finch, 1973; Hershko and Eilon, 1974), but not heart iron (Linder et a1., 1973). Female rats have more liver ferritin (iron storage protein), more apoferritin, a faster rate of liver ferritin turnover (Bjorklid and Helgeland, 1970; Linder and Munro, 1973; Linder et a1., 1973; Hershko and Eilon, 1974), greater liver iron exchange (Hershko and Eilon, 1974), absorb more iron from their diet (Otis and Smith, 1940; Murray and Stein, 1968; Cook, Hershko and Finch, 1973: Hershko and Eilon, 1974), have a greater plasma iron turnover, and a larger percentage saturation of transferrin (Hershko and Eilon, 1974) than male rats. Iron stores are low at weaning and the above sex dif- ferences begin to emerge, under the influence of sex hormones, only after sexual maturity at 6 to 7 weeks of age ll (Widdowson and McCance, 1948; Kaldor and Powell, 1957; Bjorklid and Helgeland, 1970; Cook, Hershko and Finch, 1973). Ovariectomy causes reductions in liver iron (Steenbock et a1., 1936; Widdowson and McCance, 1948; Dubrunquez and Lederer, 1963; Hershko and Eilon, 1974), plasma iron turn- over, and serum iron (Hershko and Eilon, 1974). Castration results in increases in liver iron (Widdowson and McCance, 1948; Dubrunquez and Lederer, 1963; Hershko and Eilon, 1974), plasma iron turnover, and serum iron (Hershko and Eilon, 1974). The replacement of the appropriate sex hormones to gonadectomized rats results in a return to the values found in normal intact animals (Hershko and Eilon, 1974). It is clear from the above studies that both testicular and ovarian hormones influence iron exchange. These hormones exert their effects through erythrOpoiesis, fer- ritin synthesis, ferroxidase activity of ceruloplasmin, and iron absorption by the intestinal mucosa. Erythropoiesis is enhanced by testosterone and repressed by estrogen (Kennedy and Gilbertson, 1957). The higher serum and liver iron values of the female could, in part, be explained by the inhibition of erythropoiesis. With less iron involved in hemoglobin formation, more is available for serum and storage. Although iron can induce the synthesis of the iron storage protein ferritin (Linder et a1., 1973), estrogen can directly stimulate ferritin synthesis and therefore increase 12 iron storage (Bjorklid and Helgeland, 1970). Estrogens have a stimulatory effect on the concentration of ceruloplasmin, a ferroxidase, which brings about mobilization of iron from iron stores (see Planas, 1973). Elevated levels of cerulo- plasmin could thus cause a rise in serum iron. The influ- ence of estrogen on the rate of absorption of dietary iron by females is probably the most important factor in the establishment of greater iron reserves. Erythropoiesis, ferritin synthesis, and ceruloplasmin have their primary affect in altering the internal distribution of iron in storage and erythroid compartments. 13 Iron in Pregnancy During pregnancy and lactation, increased iron stores in the female may be a means of providing iron to offspring. During pregnancy, not only do sex hormones change, but also the iron needs of the female are greatly increased. Preg- nancy depletes iron stores (Steenbock et a1., 1936; Widdow- son and McCance, 1948; Hershko, Cohen and Zajicek, 1976), causes an increase in iron absorption (Manis and Schacter, 1962; Hershko, Cohen and Zajicek, 1976), and increases plasma iron turnover (Hershko, Cohen and Zajicek, 1976). However, Hershko, Cohen and Zajicek (1976) report that the depletion of liver iron occurs only in the last 5 to 6 days of pregnancy in the rat, and that there is actually a 24% increase in hepatic iron stores the first 15 days of preg- nancy. In the rat, progesterone increases until day 15 of pregnancy (Sato and Henkin, 1973); the increase in iron absorption and hepatic iron storage coincides with the high levels of this ovarian hormone. The precipitous drop in storage iron during the last few days of pregnancy could be from a combined effect of a drop in sex hormone stimulated absorption and the vastly increased needs for iron for growth of the young. On day 21 of pregnancy, only 21% of the initial storage iron remains (Hershko, Cohen and Zajicek, 1976). Liver iron stores remain low during lactation (Widdowson and McCance, 1948), and even 12 weeks after weaning her young, a mother rat's hepatic iron stores still do not match that of an unmated female of the same age l4 (Widdowson and McCance, 1948). Several authors caution against extrapolating the sex difference in iron metabolism found in the rat to other species. Their reason stems from the observations of Widdowson and McCance (1948). In this study, a sex dif- ference in liver iron was found in poultry, mice, and rats, but not in guinea pigs or rabbits. The authors, however, do not report the age since sexual maturity of the guinea pig nor the reproductive condition of the rabbits. Since age and reproductive condition can influence sex differences in liver iron storage levels, the reported lack of a sex difference in the guinea pigs or rabbits must remain in question. In humans a sex difference in serum and liver occurs, but it is in the opposite direction to that found in the rat: females have lower levels than do males. The monthly loss of iron through menstruation (averaging about 40 ml of blood and thus 20 mg of iron, Beaton, 1974) and the iron stress of pregnancy combined with the rather low intestinal absorption of iron in humans probably accounts for the fact that premenopausal women have lower iron stores than men (Charlton et a1., 1970). It has been estimated that in some populations, 50% of the women of reproductive age have no iron stores (see Frieden and Osaki, 1974). 15 Regional Distribution and Quantification of Brain Iron in the Human Brain The presence of iron in the brain, detected by histo- chemical means, was first reported by Zaleski (1886), followed in 1915 by Guizzetti. Guizzetti recognized a characteristic pattern of iron distribution in the brains of various mammals and humans. The globus pallidus and sub- stantia nigra were consistently found to have stainable iron depostis although the iron reaction was not equally intense in all species. The first systematic study of the distribution of iron in the brain was conducted by Spatz (l922a,b). The immer- sion of thick macroscopic sections of human brain tissue into potassium ferricyanide (Turnbull's blue) revealed a characteristic pattern of iron distribution and allowed Spatz to subdivide the centers of the central nervous sys- tem into four groups according to the amount of iron present.- Group one, having the most intense iron reaction, included the globus pallidus and substantia nigra. The red nucleus, caudate, putamen, subthalamus, and dentate nucleus formed the second group. The third group included the cerebral cortex, anterior thalamus, mammillary body, midbrain tectum, cerebellar cortex, and central grey matter of the third ventricle and gave a week iron reaction. The fourth group, in which no staining was observed, was comprised of the medulla oblongata, grey matter of the Spinal cord, spinal and sympathetic ganglia and the white l6 matter of the central nervous system. In microscOpic sections, iron appears as a fine granu- lar deposit in the neuropil, oligodendrocytes, and nerve cells, mainly located in the globus pallidus and substantia nigra (Spatz, 1922a; Muller, 1922). WOllemann (1951) measured the intensity of the iron reactions with photometric means and obtained results in agreement with Spatz's observations. As it is not known whether the histochemical methods stain all the iron present in tissue, quantification of iron by chemical means has been made on human brain tissue by several workers (WUth, 1923; Tingey, 1937; Cumings, 1948; Hallgren and Sourander, 1958; Sundermann and Kempf, 1961; Musil et a1., 1962; Courville et a1., 1963; Harrison et a1., 1968). Although the results from these studies show that a definite parallelism exists between chemical and histochemical measurements (see Table 1 below), the actual amounts reported vary considerably. Differences in extraction techniques, analytical methods, iron contamination, presence of blood in the tissue, and state of health and age of the individuals account for some of this variation. Hallgren and Sourander (1958) report that the iron content of the globus pallidus and substantia nigra is of the same magnitude as that of the liver, but that the amount of iron in the whole brain equals about one- fifth the content of an iron replete liver. That iron increases with age has been shown histochem- ically (Guizzetti, 1886; Spatz, 1922a) and biochemically 17 Table l. The Distribution of Non-haemin Iron in Different Parts of the Human Brain Autopsy Cases, 30-100 Years of Age mg iron/100 g fresh weight Globus pallidus 21.30i3.49 Red nucleus 19.48i6.86 Substantia nigra 18.4616.52 Putamen .32i3.43 Dentate nucleus .35:4.86 Caudate nucleus 9.28:2.14 Thalamus 4.76:1.16 Cerebellar cortex 3.35i0.87 Motor cortex* 5.03i0.88 Occipital cortex 4.55i0.67 Sensory cortex 4.32:0.58 Parietal cortex 3.81:0.67 Temporal cortex 3.13:0.57 Prefrontal cortex 2.92i0.41 Frontal white matter 4.24i0.88 Medulla oblongata 1.40:1.16 Meninges 1.02:0.29 Liver 13.44i9.36 9540-100 years of age (from Hallgren and Sourander, 1958) l8 (Tingey, 1937; Hallgren and Sourander, 1958; Sundermann and Kempf, 1961). Hallgren and Sourander measured the iron in 81 human brains of various ages and showed that different centers of the central nervous system accumulate iron at different rates. Some areas reach maximal levels that are maintained, and others accumulate iron throughout life. They report that the iron content of the globus pallidus and substantia nigra increases rapidly the first 2 decades of life and no further increase occurs after age 30, whereas the caudate and putamen do not reach maximal levels until age 50—60. Within the cerebral cortex, motor cortex has the most iron, followed by occipital, sensory, and parietal cortex. Lower values are found in the prefrontal and temporal areas. Only the medulla oblongata showed no increase of iron content with age. These authors also found that, except for cases of anemia when brain and liver iron values were both depressed, the brain iron levels do not correlate with liver iron values. Contrary to Hallgren and Sourander's results, Sunder- mann and Kempf (1961) report that the iron content of the globus pallidus, caudate putamen, and red nucleus increases throughout life, whereas a maximum is reached by 30-40 years in the cortex and the thalamus. 19 Brain Iron and Neurological Disorders The excess accumulation of iron in the body tissues in hemochromatosis is found also in brain iron stores (Sheldon, 1927; Cammermeyer, 1947). Increased brain iron in the globus pallidus also has been reported in Hallervorden-Spatz disease (Spatz and Metz, 1926; Meyer, 1958; Dooling et a1., 1974) accompanied by disturbances in gait. General iron metabolism appears normal; however, brain iron shows a slower than normal turnover (Szanto and Gallijas, 1966). Goldberg and Allen (1980) have shown that brain iron accumulation is not a specific effect, for Hallervorden- Spatz patients also have increased brain copper, zinc, calcium, and manganese. Although iron is greatly increased in the globus pallidus, non-pallidal areas (caudate, putamen, substantia nigra, cortex, and hippocampus) actually show a decrease in iron compared to normal brains. Histochemically demonstrable iron is evident in the plaques and areas of neurofibrillary change seen in the cortex of Alzheimer's patients (Goodman, 1953). However, Hallgren and Sourander (1960) were unable to find an increase in total iron levels in the affected cortical areas. Wilson's disease is a disorder of c0pper metabolism, and slightly increased brain iron has been reported (Cumings, 1948). Although Spatz and Metz (1926) report increases in basal ganglion iron in cases of Huntington's chorea, Courville et al. (1963) were unable to find a consistent difference in the brain iron levels between normal and Huntington's patients. Increased brain 20 iron is reported in cases of Kaschin-Beck's disease, a disorder caused by increased dietary iron (Hsaing, 1941); however, the relative distribution follows normal patterns (Spatz, 1922a). In dementia paralytica, brain iron depostis are increased (Spatz and Metz, 1926; Tingey, 1937). Freeman (1930) has reported a slight decrease in the brain iron of patients“ with schizophrenia, and increased amounts of brain iron have been demonstrated in the globus pallidus of psychotic patients (Spatz and Metz, 1926; Strassman, 1945). The epileptiform discharges occurring after head trauma and hemorrhagic infarction have been traced to the deposition of hemoglobin iron in the neuropil (Rubin and Willmore, 1980). The damaging effects of iron are due to its catalytic role in lipid peroxidation. The peroxidation of lipids is a chain reaction caused by the hydroxyl radical rearranging lipid bonds and destroying membranes. The superoxide radical (027) formed from the reaction of ferrous iron (Fe2)and molecular oxygen (02) reacts with H202 to produce the very reactive hydroxyl radicals (OH’). Many in 2329 systems are present to prevent lipid peroxidation either by preventing the reduction of iron (e.g., ceru- loplasmin, transferrin), trapping or breaking down peroxides GX-toc0pherol, catalase and peroxidase), or dismutating the superoxide radical (superoxide dismutase) (Halliwell, 1979). Regardless of these preventative systems, any neurological disorder in which iron and oxygen can interact the 21 destruction of biological membranes by lipid peroxidation can result. 22 Brain Iron in Laboratory Animals There are no reports in the literature of the histo- chemical localization of iron in the brains of non-human mammals except for the reference to a "weak iron reaction" in the brains of animals by Guizzetti (1886) and Spatz (1922a). According to these authors, animal brain tissue contains less iron than human tissue, and the quantity parallels the evolutionary stage of the animal. The mouse brain reportedly gives no iron reaction, whereas in the rabbit, cat, dog, and monkey, a positive reaction of in- creasing intensity is found in the globus pallidus and substantia nigra. In the monkey brain, iron is visible in the dentate nucleus but not the red nucleus. An increase in iron with age in mammal brains in also reported by these authors. Reports of the chemical determination of brain iron concentration in non-human mammals are also sparse. The studies of Dallman and coworkers on the effects of iron deficiency on brain iron of the developing rat (Dallman, Siimes and Manies, 1975; Dallman and Spirito, 1977; Weinberg, Dallman and Levine, 1979; Weinberg, Levine and Dallman, 1979) provide some information. Included in these studies are measurements of control animal non-heme iron concentration in whole rat brain from birth to about 11 weeks of age. The values given should represent "normal“ brain iron concentrations for the Sprague-Dawley rat. After the removal of the blood from 23 brain tissue, total non-heme iron is only slightly less than total iron; only a small percentage of the brain iron is in heme compounds (Rafaelson and Kofod, 1969). Total non-heme iron in the whole brain is reported to be: age (wks) ugFejg wet wt. female rats: (Dallman, Siimes and Manies, 1975) 3 9 4 9.3 6 9.8 10 10 ll 11 male rats: (Dallman and Spirito, 1977) birth 5 1 5.3 2 5.13 3 6.11 5 7.0 8 6.4 male and female rats: (Weinberg, Dallman and Levine, 1979) (Weinberg, Levine and Dallman, 1979) 4 6-7 (f) 6-6.5 (m) 8 7.39 (f) 7.09 (m) Investigation of the development of brain iron in the male rat shows that brain iron concentration is low at birth and increases only slightly during development from 5 ug/g wet weight at birth to about 6 ug/g wet weight at 8 weeks. However, as the brain is in a period of rapid growth during this time, the total iron in the brain is actually increas- ing. A fourfold increase in non-heme iron occurs between 3 days and 3 weeks of age, and a 40% increase occurs from 3 to 8 weeks, the peak uptake of iron by the brain occurring between days 12 and 18. The iron compounds in the brain of the adult male have an extremely slow rate of turnover 24 (Dallman and Spirito, 1977). The values obtained from the measurement of iron in male brains is fairly consistent from experiment to exper- iment; however, the amounts of iron reported for the female brain are higher in the Dallman, Siimes and Manies (1975) study than in the others. Slight changes in the procedures of iron measurement, diet, source or strain of experimental animal may explain this difference. There was no statis- tically significant sex difference reported in the brain iron of control animals in any of the studies; however, in all cases, the female brain had a higher concentration of iron/g wet weight than the male. As most data were from young animals, a statistically significant sex difference may not yet have developed. In one study group in which the diet of pregnant rats was supplemented with iron from 18 days of pregnancy through lactation, 4 week old female pups had accumulated significantly more brain iron than male littermates (Weinberg, Dallman and Levine, 1979). Other reports (Rafaelson and Kofod, 1969; Williamson and Ng, 1980) give values of between 10 and 11 ugFe/g wet weight for the whole brain non-heme iron of adult rats--age and sex un- reported. Unperfused rabbit brain contains 20 ugFe/g wet weight non-heme iron (Hanig and Aprison, 1967). The pattern of development of brain iron in the mouse (Keen and Hurley, 1980) differs considerably from the devel- 0pment of brain iron in the rat reported by Dallman and Spirito (1977). At birth, the mouse brain total iron 25 concentration is about 17 ugFe/g wet weight, drops by day 5 to about 7 ugFe/g wet weight, remains constant until about 3 weeks of age, and then increases until at 8 weeks the level at birth, 17 ugFe/g wet weight, is reached (Keen and Hurley, 1980). Although a systematic study of the iron concentration in separate areas of the brain has not been performed on non-human mammals, the following values have been reported in the adult rat--age and sex unreported (Youdim and Green, 1977): cerebellum .075i.008 ug/mg protein brain stem .052:.008 ug/mg protein caudate .092:.008 ug/mg protein amygdala .058i.005 ug/mg protein cortex .050i.003 ug/mg protein hypothalamus .076 ug/mg protein Fractionation of brain tissue in 0.32 M sucrose yields a nuclear fraction Pl’ a crude mitochondrial fraction P2, and a microsomal supernatant fraction. The percentage of total non-heme iron in each fraction as reported for the rat (Youdim and Green, 1977) and for the dog (Hallgren and Sourander, 1958) is as follows: rat dog Nuclear fraction P1 8% 14% Crude mitochondrial fraction P2 55% 46% Microsomal and supernatant P3 37% 40% 26 Rajan, Colburn and Davis (1976) resuspended the crude mitochondrial fraction, separated it into bands on Ficoll gradients, and identified the components with electron microscopy as well as measured the total iron in each. In whole brain of the 6 week old male rat analyzed in this manner, 82% of the iron was a myelin fraction, 6% in mito- chondrial fraction, and 12% in synaptosomal fraction. Similar subcellular fractionation of various areas of the rat brain (see Table 2) show no consistent pattern in the distribution of iron among different compartments. 27 Table 2. Determination of Iron in the Subcellular Fractions of Different Brain Areas Subcellular Brain Areas Fractions ug/mg Protein Brain Stem Myelin 0.15:0.02 Synaptosome 2.03il.92 Mitochondria 0.40i0.04 Synaptic vesicles 1.79:1.09 Membrane 4.70:0.86 Striatum Myelin 2.17i2.0 Synaptosome 1.30:0.04 Mitochondria 0.87:0.15 Membrane 8.0 i2.l Cerebral Cortex Myelin 5.7312.01 Mitochondria 6.52il.50 Membrane 0.18i0.05 Synaptic vesicles 1.4liO.32 Hypothalamus Myelin 1.19:0.35 Synaptosome 2.76:0.80 Mitochondria 2.09i1.33 Cerebellum Myelin 5.81:3.39 Mitochondria ----- Note: The values shown in the Table represent each of the average of four determinations of the tissue samples prepared from different rat brain at various times. (from Rajan, Colburn and Davis, 1976) 28 Forms of Iron in the Brain As in the other tissues of the body, brain iron occurs in both heme and non-heme compounds. When blood, and there- fore hemoglobin, is removed from the brain, the non-heme component is practically equal to total iron (Rafaelson and Kofod, 1969). Ferritin and the known iron enzymes comprise only a portion of the measurable iron in the brain; the greater part of the brain iron occurs in yet unidentified form(s). Ferritin is a specialized tissue protein designed for iron storage (Harrison et a1., 1974) in which iron is stored as a hydrous ferric oxide phosphate complex within the protein shell (Drysdale, 1976). Ferritin stains positively with iron stains (Strassman, 1945; Deizel, 1955), appears dis- persed within the cell (Westcott et a1., 1953), is water soluble, and is heat stable to 70°C (Weinfeld, 1964). Only about one-third of the human brain non-heme iron is in this form (Hallgren and Sourander, 1958) and only 15-25% in whole rat brain (Dallman, Siimes and Manies, 1974), whereas 60—70% of liver non-heme iron is stored in ferritin (Kaldor, 1958; Cook, Hershko and Finch, 1973). Sundermann and Kempf (1961) report nearly identical patterns of distribution and change with age for brain total iron and ferritin. Hemosiderin, the other iron storage compound of the liver, spleen, and bone marrow has been identified histo- chemically in damaged brain tissue (Strassman, 1945). Hemosiderin appears as yellow-brown deposits in unstained 29 tissue and stains positively for iron (Strassman, 1945; Subcommittee on Iron, 1979). It is considered to be an aggregate form of ferritin, is insoluble in water, and contains lipids and carbohydrates as well as protein (Deizel, 1955; Weinfeld, 1964; Subcommittee on Iron, 1979). The intracellular granular iron deposits of the globus pallidus and substantia nigra do not contain lipids or carbohydrates, as determined histochemically, and therefore apparently do not contain hemosiderin (Deizel, 1955). The remaining known iron compounds are enzymes. Although not present in large quantities, the following enzymes are of importance in brain chemistry because of involvement either directly or indirectly in neurotrans- mitter metabolism. The iron containing enzymes aconitase and succinic dehydrogenase are related to the metabolism of four of the eight most abundant compounds in the brain (occurring in concentrations > 3 mM). The pathway for the synthesis of these compounds--glutamate, y-aminobutyric acid (GABA), glutamine, and glutathione--is derived directly from Kreb's cycle via a shunt, sometimes called the GABA shunt, occur- ring between a-ketoglutarate and succinate (see Cooper, Bloom and Roth, 1978). Aconitase catalyzes reactions required for the synthesis of‘a—ketoglutarate, and the conversion of succinate to fumarate is catalyzed by suc— cinate dehydrogenase. It has been estimated that the GABA shunt may account for 10-40% of the total brain metabolism 30 (see Cooper, Bloom and Roth, 1978). The peroxides formed from the oxidation of monoamine neurotransmitters are degraded by catalase, an iron contain- ing enzyme. The greatest concentrations of catalase are found in the norepinephrine-rich hypothalamus and the dopamine-rich substantia nigra (Brannan et a1., 1981). Tyrosine hydroxylase is the initial and rate limiting enzyme in the synthesis of the catecholamine neurotrans- mitters norepinephrine (NE) and dopamine (DA) (Sourkes, 1972). In the brain, the enzyme is associated with the synaptosome fraction (Cooper, Bloom and Roth, 1978). The exact relation of iron to the activity of monoamine oxidase (MAO) is not clearly understood; however, the maintenance of normal MAO levels requires iron (Symes, Missala and Sourkes, 1971). MAO is an important enzyme in the degradation of NE, DA, and serotonin. Iron is essential for the catalytic activity of trypto- phan hydroxylase, the initial and rate limiting enzyme in the synthesis of serotonin (Kuhn et a1., 1980). Aldehyde oxidase, a key enzyme in the degradation of serotonin, is also an iron requiring enzyme (Bray, 1975). As well as having a role in the synthesis and degrada- tion of biogenic amines, iron may be an important factor in chelate formation in the storage and transport of catechola- mines. Rajan, Colburn and Davis (1971) have reported significant amounts of iron, and other metals, in the sub- cellular fractions of rat brain which are known to have high 31 concentration of catecholamines. The molecular structure and functional groups associated with the catecholamines is compatible with these molecules having strong metal chela- tion potentials. Ferrous iron specifically enhances the binding of serotonin to serotonin-binding protein in synap- tosomes and serotonergic tracts (Tamir, Klein and Rapport, 1976) and is involved in serotonin receptor mechanisms (Lehmann, personal communication). The fact that iron is involved in so many different aspects of many different neurotransmitters probably ac- counts for the fact that the distribution of iron does not correspond exactly to the known distribution of any neuro- transmitter. Rafaelson and Kofod (1969) claim a small amount of iron is likely in a complex attachment to lipids in the brain. Perhaps this is in relation to iron's structural role in many membranes and membrane bound proteins (Jacobs and Worwood, 1974). In brain tissue treated with prussian blue, iron appears in the neuropil (Spatz, 1922a), perhaps in association with myelin. In addition, Rajan, Colburn and Davis (1976) found that over 80% of the iron in resuspended crude mitochondrial fraction from whole rat brain is in the myelin subfraction. In this regard, it is noteworthy that (l) the peak uptake of radio iron by the rat brain-—days 12 to 18 postnatally (Dallman and Spirito, l977)--coincides with the beginning of CNS myelination--10 to 12 days post- natally (Norton and Poduslo, 1973); (2) both myelin 32 formation and iron accumulation continue late into adulthood (Hallgren and Sourander, 1958; Norton and Poduslo, 1973); (3) both iron in the male rat brain (Dallman and Spirito, 1977) and myelin (Davison and Peters, 1970) are stable, showing very slow turnover rates; and (4) in Dallman, Siimes and Manies' (1974) study in which a persistent depression of brain iron occurred following short-term iron deficiency in the rat pup (see below), the iron deprivation occurred during postnatal days 10 to 28, the period of the initiation of myelin formation and its rapid proliferation. 33 Iron Deficiency, the Brain and Behavior Iron deficiency is characterized by anemia, stunted growth, epithelial changes, gastrointestinal abnormalities, abnormal lipid metabolism, weakness, increased susceptibil- ity to infection, anorexia, amenorrhea, menstrual irreg- ularity, and alterations in behavior (Sherman, 1978; Sub- committee on Iron, 1979; Oski, 1979). The behavioral changes in patients with iron deficiency include irritability, apathy, listleness, fatigue, lack of ability to concentrate, pagophagia (pathological craving for ice), and in children, inattention, hyperactivity, and decreased scholastic performance (Webb and Oski, l973a,b; Cantwell, 1974; Pollitt and Leibel, 1976). In studies of laboratory animals, the behavioral effects of iron deficiency include decreases in total activity, a reversal of diurnal rhythm (Glover and Jacobs, 1972), and changes in maze learning and forced exercise performance (Edgerton et a1., 1972; Pollitt and Leibel, 1976), and general responsiveness to environmental stimuli and learned task performance (Weinberg, Levine and Dallman, 1979; Weinberg, Dallman and Levine, 1979). The behavioral effects of iron deficiency are thought to result from altered metabolism due to decreased amounts of iron available for the brain iron enzymes involved in cellular oxidative functions and neurotransmitter metabolism (Pollitt and Leibel, 1976; Mackler et a1., 1978; Youdim et a1., 1980). 34 Following a brief period of iron deficiency in the young rat (days 10 to 28), a deficit in brain iron persists even to the adult stage (Dallman, Siimes and Manies, 1974). The behavioral changes in general responsiveness, reac- tivity, and avoidance learning seen in these animals also persist in rehabilitated adult rats (weinberg, Dallman and Levine, 1979). These experiments suggest that severe iron deficiency during development induces irreversible changes in the brain. Although in iron deficiency the activity of the iron enzymes cytochrome C, cytochrome oxidase, succinic dehydro- genase, and monoamine oxidase are reduced in various body tissues (Dallman and Schwartz, 1968; Symes, Missala and Sourke, 1971; Youdim et a1., 1980), in the brain, neither the ability to carry out oxidative phosphorylation nor the levels of these enzymes appear to be affected (Mackler et a1., 1978; Youdim et a1., 1980). However, the activity of aldehyde oxidase, a key enzyme in serotonin degradation, is significantly reduced in the brain of iron deficient rats, and concentrations of serotonin and total 5-hydroxyindole compounds are reported to be elevated (Mackler et a1., 1978). The increased concentration of serotonin can cause drowsiness, inattentiveness, and a decreased ability to learn (Douglas, l974)-—symptoms often associated with iron deficiency. Youdim and associates found that, except for a decrease in serotonin, no changes occurred in the activities of 35 enzymes related to catechole or indole amine metabolism nor in the level of dopamine in the brain tissue of iron defi- cient rats (Youdim et a1., 1980). However, the hyperac- tivity caused by drug induced increases in serotonin and dopamine was depressed in iron deficient animals even though, under these conditions, the amounts of these puta- tive neurotransmitters were alike in normal and iron- deficient rats. That the decreased activity was due to a decreased post-synaptic response (i.e., at the receptor level) is supported by the fact that putative agonists to dopamine and serotonin also cause a depressed behavioral response (Youdim et a1., 1980), and a further study (Ash- kenazi, Ben Shachar and Youdim, 1980) has demonstrated a 50% decrease in dopamine receptors in the caudate of iron deficient rats. Iron is important in many aspects of brain function, with involvement in the catecholamine and serotonin neuro- transmitter systems especially evident. Catecholamine and serotonin systems are sex hormone sensitive (Kalra et a1., 1972; Kalra and McCann, 1973) and are involved in the sexual differentiation of the brain (Arai and Gorski, 1968) and in the central nervous system regulation of gonado- trophin secretion (Ganong, 1975; McCann and Moss, 1975). The availability of iron could thus affect the central nervous system regulation and coordination of reproductive functions. 36 In view of the prevalence of iron deficiency in the world and its possible long term effects on brain neuro- transmitter systems, the effects of hormones and other factors on the iron accumulating abilities of the brain merit investigation. It is the purpose of this study to (1) 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. MATERIALS AND METHODS Both histochemical and spectrophotometric evaluation of brain iron are made. The histochemical techniques provide qualitative determinations of brain iron by localizing deposits and indicate by the intensity of the stain the amount of iron present. SpectroPhotometry yields a quanti- tative measure of iron within specific areas of the brain. HISTOCHEMISTRY Animals Intact Sprague-Dawley (SD) rats were obtained from the Animal Breeding Center, National Institutes of Health, Beth- esda MD; castrated and ovariectomized animals purchased from Taconic Farms, Germantown, NY, were delivered the day after surgery. 'The animals were kept in groups of two to four in standard plastic laboratory cages and provided with water and Purina Rat Chow ad libitum. The light cycle was 8 hrs light and 16 hrs dark, the dark period beginning at 1330 hrs. All animals were allowed to adjust to our labaoratory conditions for at least 3 weeks before being experimentally utilized. Treatment Groups The histochemical investigation was composed of four separate studies, each designed to answer specific questions about brain iron deposits. Below is an outline of each of 37 38 the studies with a description of the purpose and the number, age, and sex of the animals. Any special treatments are included with the description of the group concerned. Study 1 - to determine (1) location of iron in the rat brain, (2) if brain iron increases with age, and (3) if there is a sex difference in brain iron. 4. 12-18 wk old virgin females 4. 12—18 wk old virgin males 4. 32 wk old retired breeder females 4. 32 wk old retired breeder males 3. 43 wk old retired breeder females l 3 52 wk old retired breeder female Study 2 - to determine if pregnancy causes visible changes in brain iron either in location or amount. These animals can be compared with the control animals of the same age in Group 1. 2, 18 wk old 7 days pregnant females 4, 18 wk old postpartum females 3, 32 wk old 16 days pregnant females About 1 hr before the dark period of the light cycle, a female was placed in a cage with a sexually mature male. If lordosis and mating were observed, the pair remained togeth- er for 1 to 2 hrs, after which the female was removed. 39 Thus, the time of insemination is known within about 2 hrs. Study 3 - to determine if ovariectomy causes visible changes in brain iron either in location or amount and to determine if estrogen causes a visible change in brain iron compared with untreated ovariectomized animals. 2, 18 wk old females, ovariectomized at 10 wks 2, 32 wk old females ovariectomized at 28 wks 2, 18 wk old females, ovariectomized at 10 wks, received estrogen implant at 17 wks 3, 32 wk old females ovariectomized at 28 wks, received estrogen implant at 30 wks Estrogen implants were prepared by heating a few grams of B-estradiol 3-benzoate (Sigma Chemical Company, St. Louis, MO) in a small beaker. When the powder melted, a thin wire was dipped into the liquid, removed, and the coating allowed to air dry. This process was repeated several times until a pellet about the size of a grain of rice formed. Any wire protruding beyond the hormone “grain" was then removed. The animal was anesthetized with an intraperitoneal injection (0.05 cc/100 g body weight) of Nembutal (sodium pentobarbital 60 mg/ml, Abbott Laboratories, N. Chicago, IL). The back of the neck was shaved and then washed with a 1:750 solution of Zephiran (Winthrop Laboratories, New York, NY). A 3 cm incision was made longitudinally through the 40 skin on the back of the neck. The implant was broken in half to remove the wire and then inserted under the skin. Before closing the incision with two or three clips, the wound was packed with Furacin (Eaton Veterinary Labora- tories, Norwick, NY). Study 4 - to determine if castration causes any visible change in brain iron, either in location or amount and to determine the effect of estro- gen on the brain iron of castrated males. These animals can also be compared to the females of similar age in Group 3. 2, 32 wk old males castrated at 30 wks 2, 32 wk old males castrated at 28 wks, received estrogen implant at 30 wks The estrogen implant was made and inserted in the same manner as in Study 3 animals above. Preparation of Tissue All animals were sacrificed between 0800 and 1300 hrs. The animals were first weighed and then anesthetized with an intraperitoneal injection of Nembutal (0.1 cc/100 g body weight). The chest cavity was opened and the animal per- fused intracardially with 50 ml 0.9% saline, followed by 250 ml of 10% formalin in 0.9% saline. Immediately upon comple- tion of perfusion, the brain was removed from the skull and placed in a solution of 10% formalin in 10% alcohol. The 41 tissue remained in this solution until cut. To prevent any possible metal contamination of the tissue, great care was exercised to avoid touching the brain with surgical instru- ments. Brains were cut frozen, with a sliding microtome. Most were cut at 50 pm in the coronal plane; however, a few were cut in the sagittal or horizontal plane and a few cut at 100 or 150 pm. The knife was coated with silicone stopcock grease (Dow Corning, Midland, MI) to prevent metal contami- nation from the knife. Although many procedures are available for the local- ization of iron in biological tissues (e.g., Pearse, 1961; Humason, 1979), Perl's reaction (prussian blue) is found to be most effective on the rat brain tissue used here. A modification of a recently discovered intensification of the Perl's reaction with diamino benzidine (DAB) was used on some tissue (Nguyen-Legros, et a1., 1980). Perl's reaction localizes ferric iron (Fe+++) by forming blue-colored ferric ferrocyanide when tissue is acidified in the presence of potassium ferrocyanide. The acid (HCl) serves to remove some of the Fe+++ from the proteins to which it is bound. The Turnbull blue reaction for ferrous iron (Fe++) (Humason, 1979) produces no visible reaction in rat brain tissue suggesting that most iron in the rat brain is in the ferric form. The intensification of the Perl's reaction with DAB is based on the oxidation of benzidine by H202 in the presence 42 of a suitable catalyst (Nguyen-Legros et a1., 1980). Oxidized benzidine compounds form blue or brown colored precipitates. Many compounds can catalyze the oxidation including hemoglobin, myoglobin, peroxide (Tietz, 1976), and ferric ferrocyanide (Nguyen-Legros et a1., 1980). Here the ferric ferrocyanide of the Perl's reaction catalyzes the oxidation of benzidine by H202, forming a brown precipitate. Since Perl's reaction is blue, Neutral Red was used as a counterstain (only sections 50 pm or less were counter- stained). However, when the intensification step with DAB was used, thionin was the counterstain. 43 Staining Techniques Perl's stain for ferric iron Acid wash all glassware; avoid the use of metal instruments. Solutions 2% Hydrochloric acid (HCl): To 2 m1 concentrated HCl add deionized water to the 100 m1 mark. 2% Potassium ferrocyanide (K4Fe(CN)6): To 2 g potassium ferrocyanide add deionized water to the 100 m1 mark. Neutral Red 7.5 g Neutral Red (Chroma-Gesellschraft) 2.5 g Safranin O (Chroma-Gesellschraft) Add deionized water to the 1000 m1 mark. Staining;procedure 1. Rinse tissue 10-20 min in deionized H20. 2. Mix 1:1 freshly prepared 2% H01 and 2% potassium ferrocyanide; heat quickly to 56°C. Immerse tissue and agitate for 3 min. Rinse tissue in deionized H20 - 10 min. Mount on subbed microscope slides. When slides are dry, immerse in 70% alcohol C‘U‘l-l-‘UO 30 sec. 7. Distilled H20 30 sec. 44 8. Immerse in Neutral Red 1 min. 9. Rinse in H20 1 min. 10. 70% alcohol 30 sec. ll. 95% alcohol - 2 changes 15 sec each. 12. Absolute alcohol - 2 changes 30 sec each. 13. Xylene - 2 changes at least 3 min each. 14. Mount with Permount. Iron - blue, Background - pink 45 Perl's DAB for ferric iron Solutions 2% HCl and 2% Potassium ferrocyanide are made the same as in Perl's reaction. 0.2 M monobasic sodium phosphate To 27.6 g monobasic sodium phosphate add deionized water to the 1000 m1 mark. 0.2 M dibasic sodium phosphate Add 28.4 g dibasic sodium phosphate (rapidly stirring with a magnetic stirrer) to 800 m1 deionized water; when dissolved add deionized water to the 1000 m1 mark. 0.1 M phosphate buffer pH 7.4 To 190 ml of 0.2 M monobasic sodium phosphate add 180 m1 of 0.2 dibasic sodium phosphate; add deionized water to the 2000 m1 mark. Diamino benzidine DAB (Sigma Chemical Company, St. Louis, MO) Dissolve 0.05 g DAB in 100 ml 0.1 M phosphate buffer pH 7.4; filter. 1% Hydrogen Peroxide (H202) To 3.3 ml of 30% H202 add deionized water to the 100 m1 mark. DAB is a possible carcinogen; it is handled with care: used glassware, leftover solution, and other material can apparently be decontaminated by immersion in a solution of laundryibleach. 46 Staining procedure 1. React tissue as in Perl's stain steps 1 through 4 (heating of Perl's solution to 56°C can be omitted as satisfactory reaction is obtained with solutions at room temperature; this also results in less tissue damage). Immerse tissue in DAB solution kept cool with ice on rotary table 20 min. 3 Add 1 ml of 1% solution of H202 20 min. 4. Rinse in deionized water 10 min. 5 Mount on subbed slides. 6 When dry, counterstain with thionin pH 4.5. The above DAB procedure was performed on tissue without pretreatment with Perl's and also on tissue in which the ferric iron had been reduced to the ferrous form with mercap toacetic acid (1.0%)., In neither of these situations was DAB staining apparent. 47 * Thionin pH 4.5 Solutions Formalin alcohol To 40 m1 commercial formaldehyde add 460 ml 95% ethanol. Chloroform ether alcohol Mix together 25 ml 95% ethanol 25 ml ether 200 ml chloroform Thionin pH 4.5 Mix together 12.5 ml of 1% aqueous thionin solution (Fisher Scientific Co., Fair Lawn, NJ). 56.5 ml 1.0 M acetic acid 42.5 ml 1.0 ml sodium acetate 138.5 ml deionized water Acid alcohol To 5 m1 concentrated HCl add 295 ml 70% ethanol. Acetic acid alcohol Add 1 m1 glacial acetic acid to 500 ml 95% ethanol. Staininggprocedure l. 95% alcohol 10 min. 2 Formalin alcohol, 5 min. 3. 95% alcohol 3 min. 4 Chloroform, ether, alcohol 10 min. 48 95% alcohol 3 min. Acid alcohol 5 min. 70% alcohol 3 min. Deionized H20 2 min \OWVO‘UI Thionin stain (longer as stain ages) 3 min. 10. Deionized H20 wash. ll. Deionized H20 2 min. 12. 70% alcohol 3 min. l3. 95% alcohol 3 min. 14. Acetic acid alcohol, watch closely; check every 2-3 min. 15. 95% alcohol 3 min. 16. 100% alcohol 3 min. 17. 100% alcohol 3 min. 18. Xylene 3 min. 19. Xylene 3 min. 20. Mount with Permount. Iron - brown/black, Background - blue * R.C. Switzer, unpublished technique. 49 Analysis of Data The histochemical localiZation of iron is useful in determining the distribution of iron in the brain and permits within-section determinations of the relative concentration of iron among different areas of the brain. However, most of the tissue in this study was stained with Perl's method alone as the intensification procedure was discovered only after most of the material had been pro- cessed. At best, the Perl's method produces a light stain in rat brain tissue and accurate determinations of small between-section differences are difficult to make. Slight differences in the thickness of the section or in the processing of tissue could result in greater differences than the treatments themselves. The judgement of differ- ences in intensity of stain in the histochemistry results section include only large relative differences and have the above mentioned limitations. The determination of quantitative differences in brain iron among treatment groups in this study is based primarily on spectrophotometry because it is an accurate, reproducible and sensitive method of measuring iron concentration. 50 SPECTROPHOTOMETRY Animals The Sprague-Dawley (SD) rats used in the pregnancy study were obtained from the National Institutes of Health laboratories. They arrived 11 to 14 wks of age and were allowed to adjust to our laboratory conditions for at least 3 wks before mating. All other rats were bred in our own facility, the Laboratory of Brain Evolution and Behavior, Poolesville, MD, from breeding stock purchased from the National Institutes of Health. The animals were kept in groups of two to four in laboratory cages and provided with water and Purina Rat Chow ad_libitum. The light cycle was 8 hrs light and 16 hrs dark, the dark period beginning at 1330 hrs. Treatment Groups The spectrOphotometric determination of iron was performed on the serum, liver, and both high iron (globus pallidus + substantia nigra) and lower iron (cortex) areas of the brain. The investigation was organized into three studies each designed to answer specific questions about brain iron. The outline below describes the purpose of the study and the number, age, and sex of the animals. Any special treatment is included in the description. 51 Study 1 - to determine (1) if the levels of brain iron change throughout the estrous cycle when endogenous levels of sex hormones fluctuate, and (2) if a sex difference occurs in brain iron levels. The stage of estrus is determined by vaginal smear. 10, 12 wk old virgin female rats is estrus 10, 12 wk old virgin female rats in metestrus 10, 12 wk old virgin female rats in diestrus 10, 12 wk old virgin female rats in proestrus 10, 12 wk old virgin male rats Sexual maturity in rats is reached between 6 and 8 wks; delaying sacrifice until 12 wks of age should allow sex hormone related changes to occur. The stage of estrus of the females was determined by vaginal smear. Vaginal Smear A cotton-tipped applicator was inserted into the vagina and then rolled onto a subbed microsc0pe slide, air dried for 10 sec, and then immersed into a 10% solution of for- malin. The smear was stained with Harris' Hematoxylin (see below). Vaginal smears were analyzed and the stage of estrus determined according to the description by Turner (1961). Vaginal smears contain polymorphonuclear leukocytes and epithelial cells. In the proestrus stage of the estrus cycle, smears contain nucleated epithelial cells, singly or in sheets. Estrus smears are characterized by masses of 52 cornified epithelial cells. Metestrus follows estrus and in vaginal smears some leukocytes are found among the epi- thelial cells. Diestrus smears contain large numbers of leukocytes and few epithelial cells. Study 2 - to determine if brain iron levels change during pregnancy, a period when liver iron is raised initially, then depleted, and maternal sex hormone levels change. 7, 19 wk old female rats, 4 days pregnant, lst pregnancy 7, 19 wk old female rats, 8 days pregnant, lst pregnancy 7, 19 wk old female rats, 12 days pregnant, lst pregnancy 7, 19 wk old female rats, 16 days pregnant, lst pregnancy 7, 19 wk old female rats, 20 days pregnant, lst pregnancy 7, 19 wk old female rats, 12-20 hrs post partum, lst litter Pregnant females were obtained by placing a female in a cage with a sexually mature male at about 1 hr before the dark period of the light cycle. If lordosis and mating were observed, the pair remain caged together for l to 2 hrs, after which the female was removed. Thus, the time of insemination was known within about 2 hrs. 53 As much as possible, the pregnancies were allowed to occur so that at the day of perfusion, all animals were 19 wks i 6 days of age. Study 3 - to determine (1) if ovariectomy or castration affects brain iron levels, and (2) if 3 wks treatment with estrogen increases or de- creases iron levels compared with control animals and ovariectomized or castrated animals. 10, 12 wk old female rats ovariectomized at 4 wks of age 10, 12 wk old female rats ovariectomized at 4 wks of age and implanted with estrogen at 9 wks of age 10, 12 wk old male rats castrated at 4 wks of age: 10, 12 wk old male rats castrated at 4 wks of age; and implanted with estrogen at 9 wks of age The intact males and females from Study 1 are the control animals to which the above treatments can be com— pared. Gonadectomy occurred at 4 wks of age, before the onset of sexual maturity. The estrogen implants were left in for 3 wks prior to sacrifice in the expectation that 3 wks is long enough to effect estrogen—dependent changes in iron. 54 Ovariectomy Four week old female rats were anesthetized with an intraperitoneal injection of Nembutal (0.05 cc/100 g body weight). The lower back was shaved and then washed with a 1:750 solution of Zephiran. A 3 cm incision was made longitudinally through the skin of the back with a scalpel and the incision was kept open with a retractor. The Opening was moved over an ovary and a small (0.7 mm) inci- sion was made through the body wall at the level of the ovary. The ovary was picked up with forceps and pulled through the opening. The blood vessels were cauterized (Codman neurocoagulator) and the ovaries cut from the uterus with fine scissors. The incision in the body wall was closed with one or two stitches. The retractor was moved over the other ovary which was removed in a similar fashion. Before closing the midline incision with clips, the wound was packed with powdered Furacin. Castration Four week old male rats were anesthetized with an intraperitoneal injection of Nembutal (0.05 cc/100 g body weight). The scrotum was shaved and then washed with a 1:750 solution of Zephiran. A 1 cm incision was made along the base of the scrotum through the scrotal skin and a small opening made at the base of each cremasteric pouch with fine scissors. By exerting a slight pressure on the upper scrotum, the testes were pushed out of the openings of the 55 cremasteric pouches. The blood vessels were cauterized and the testis cut from the vas deferens with scissors. The wound was packed with powdered Furacin and the incision closed with two or three stitches. Estrogen implant preparation Silastic tubing (Dow Corning, Midland, MI) .078 in ID x .125 in OD was cut into 10mm lengths and one end closed off with a dab of Silastic Medical Adhesive (Dow Corning, Midland, MI). When the adhesive was dry the tube was filled with beta-estradiol 3 benzoate - about 0.01 g (Sigma Chemical Co., St. Louis, MO) - and the other end of the tubing closed off with adhesive. The implant was soaked in a 0.9% saline solution for 24 hrs before being placed in the animal. Implants prepared in this manner permit a relatively steady rate of diffusion of hormone (Ciaccio, L.A., personal communication). Insertion of implant Nine week old ovariectomized females and castrated males were anesthetized with intraperitoneal injection of Nembutal (0.05 cc/100 g body weight), The back of the neck was shaved and then washed with a 1:750 solution of Zephi- ran. A 3 cm incision was made longitudinally through the skin on the back of the neck. The implant was inserted under the skin. Before closing the incision with two or three clips, the wound was packed with Furacin. 56 Harris' Hematoxylin Solutions Hematoxylin Hematoxylin crystals 5.0 gm Alcohol, 95% 50.0 cc Ammonium or potassium alum 100.0 gm Distilled water 1000.0 cc Mercuric oxide 2.5 gm Dissolve the hematoxylin in the alcohol, the alum in the water by the aid of heat. Mix the two solutions. Bring the mixture to a boil as rapidly as possible and then remove from the heat and add the mercuric oxide. Reheat the solution until it becomes a dark purple, about 1 min, and promptly remove the container from the heat and plunge it into a basin of cold water. The solution is ready to use when cool. Add 2-4 cc of glacial acetic acid to 100 cc of solution if desired. Acid Water Distilled water 1000 cc Hydrochloric acid, concentrated 10 cc Eosin-Phloxine Solution Stock Eosin Eosin Y, water soluble 1.0 gm Distilled water 100.0 ml Stock Phloxine Phloxine B 1.0 gm Distilled water 100.0 ml Working Solution Stock Eosin 100.0 m1 Stock Phloxine 10.0 ml Alcohol, 95% 780.0 ml Glacial acetic acid 4.0 m1 57 Make up working solution as needed. Working solution should be changed at least once a week. Staining Procedure l. Wash with tap water. 2 Harris' hematoxylin for 10 min. 3. Rinse in tap water. 4 Differentiate in acid water - 3 to 10 quick dips. Check the differentiation with the microscope-- nuclei should be distinct and the background very light or colorless. Wash in running tap water for 5 min. Stain with eosin from 15 sec to 2 min depending on the age of the eosin and the depth of counterstain desired. 70% alcohol. 95% alcohol. Absolute alcohol - at least 2 changes. 10. Xylene - 2 changes 11. Mount in Permount. Results: cornified epithelial cells - pink all other cells - blue 58 Table 3. Summary of Iron Measurement Study number age in of wks at animals sacrifice Females intact proestrus 10 12 estrus 10 12 metestrus 10 12 diestrus 10 12 day of pregnancy at sacrifice pregnant 7 4 l9 7 8 19 7 12 19 7 16 19 7 20 19 7 postpartum 19 age in age in wks of wks of gonadectomy implant ovariectomized (OVX) 10 4 12 OVX + estrogen implant 10 4 9 12 Males intact 10 12 castrated (CAST) 10 4 12 CAST + estrogen implant 10 4 9 12 59 Collection of TisSue for the Spectr0photometric Measurement of Iron All animals were sacrificed between 0800 and 1300 hrs. They were first weighed and then anesthetized with an intraperitoneal injection of Nembutal (0.1 cc/100 g body weight). The chest cavity was opened and a 3 cc syringe equipped with a 20 gauge needle was filled with blood from the base of the heart. The needle was removed from the syringe and the blood poured into an acid-cleaned test tube and refrigerated, undisturbed, until the following day. After the blood sample was taken, the animal was perfused intracardially with 200 ml of saline to remove blood from the tissues. The brain and a portion of the liver were removed, placed in separate plastic weighing dishes, and frozen on a block of dry ice. When frozen, the dish was covered over with aluminum foil, sealed in a plastic bag, and kept frozen (-20°C) for up to 12 wks. During perfusion, the uterus was removed from intact, ovariectomized, and estrogen treated females, its diameter measured, and, where present, the ovaries were inspected for the presence of follicles. In pregnant animals, the number of embryos was recorded except at 4 days of pregnancy at which they were too small to be seen. However, the uterus size was recorded for the 4 day pregnant rats. 60 The morning following perfusion, the blood sample, now clotted, was spun for 10 min at 3000 RPM and the serum pipetted off into acid-cleaned screw-top vials and frozen for up to 12 wks. 61 Requirements for the Spectrophotometric Measurement of Iron Before the iron present in biological tissues can be measured by spectrophotometric means the cells must be separated and broken open; the iron is then removed from proteins; the proteins are precipitated; ferric iron is reduced to the ferrous form; copper is complexed to prevent interference with iron measurements; and ferrous iron is reacted with a chromogen to form a colored complex. The procedure used here is based on the serum iron method of Zak, Baginski and Epstein (1980), modified for measurement of tissue extracts. The extraction of iron from tissue is based on the method of Weinfeld (1964) in which only the storage iron compartment, non-heme iron, is ex- tracted, the heme compounds being resistant to acid hydro- lysis. In the rat brain the non-heme iron is practically identical to total iron when the blood has been removed from the tissue (Rafaelsen and Kofod, 1969). The tissue was broken up by homogenization, and a cool, heat, cool cycle promoted cell lysis. It has been deter- mined by Weinfeld (1964) that complete extraction of non- heme iron from tissue occurs by hydrolysis in a 2.8N HCl solution at 90°C for 60 min. For this reason, here, to two parts tissue homogenized in iron-free water, one part of 8.5N HCl was added, giving a strength of 2.8N. In the determination of serum iron, the removal of iron from the transferrin was accomplished by mercaptoacetic acid 62 (Fielding and Ryall, 1970; Zak, Baginski and Epstein, 1980) and trichloracetic acid (Tietz, 1976). Trichloroacetic acid has the added advantage of precipitating proteins (Tietz, 1976; Zak, Baginski and Epstein, 1980). Since most chromogens react with ferrous ions, ferric iron must be reduced. Of available reductants, mercapto- acetic acid, used here, has the added advantage of com- plexing copper, thus eliminating the possibility of a copper-chromogen complex interfering with iron-chromogen measurements. The chromogen 2,4-bis(5,6-diphenyl-l,2,4-triazin-3—y1) pyridine tetrasulfonate (BDTPS), was chosen because it is very sensitive and stable. At a pH between 2 and 6, ferrous iron forms a magenta—colored complex with a peak maximum at 565nm where it has a molar absorptivity of 32,000 (Zak, Baginski and Epstein, 1980; G. Frederick Smith, 1980). The pH of the final solution can be adjusted by using a 30% sodium acetate solution in serum sample determinations. However, in the determination of brain and liver iron, BDTPS was dissolved in a saturated sodium acetate solution and the pH of the brain extract further adjusted by the addition of saturated sodium acetate to the test solution. The intensity of the colored complex was measured with a Beckman DU spectrophotometer. The sample was measured against a reagent blank and the amount of iron determined by comparing the reading to that of a known concentration of iron, the iron standard. 63 Solutions for Iron Spectrophotometry To prevent iron contamination, acid wash all glassware with nitric or hydrochloric acid, avoid the use of metal instruments, use only distilled or deionized water for making solutions and rinsing glassware, and purchase metal free reagents. Iron Standard Solution 0.0010 mg Fe/ml (1.0 ug/ml) purchased from G. Frederick Smith Chemical Co., Columbus, Ohio. Trichloroacetic Acid - CC13COOH To 1.1 lb metal—free trichloroacetic acid (G. Frederick Smith Chemical Co., Columbus, OH) add metal—free water to the 500 ml mark. This makes a 100% solution. Mercaptoacetic Acid - (Thioglycolic acid) HSCHZCOOH 98% solution purchased from.A1drich Chemical Co., Milwaukee, Wisconsin. Precipitating/Reducing Solution To 20 ml of trichloroacetic acid solution add 1 ml mercaptoacetic acid and dilute to the 100 ml mark with metal-free water. Saturated Sodium Acetate - NaCZH302'3H20 To metal—free water stirred with a magnetic stirrer add sodium acetate (G. Frederick Smith Chemical Co., Columbus, OH) until it no longer goes into solution. Decant off the saturated 64 solution. 30% Sodium Acetate - NaC2H302- 3H2 O To 30 gms sodium acetate add metal-free water to the 100 ml mark. Color Reagent in Saturated Sodium Acetate - 2,4- bis(5,5-diphenyl,1,2,4-Triazin-3-yl)pyridine tetrasulfonic acid, tetra sodium salt (DBTPS) Dissolve 200 mg DBTPS (G. Frederick Smith Chemical Co., Columbus, OH) in 5 ml metal-free water; add saturated sodium acetate solution to the 100 m1 mark. Color Reagent in 30% Sodium Acetate - DBTPS Dissolve 200 mg DBTPS in 5 m1 metal—free water; add 30% sodium acetate solution to the 100 m1 mark. 65 Determination of Serum Iron* Serum samples were brought to room temperature. 500 pl of serum sample, 500 pl of metal-free water, and 1 m1 of precipitating/reducing agent were pipetted into an acid- cleaned test tube, mixed well by inversion, and allowed to stand 10 minutes. The sample was spun at 3000 RPM for 10 minutes, and a 1 m1 aliquot of the supernatant pipetted into a 2 m1 autoanalyzer cup. (These cups have been found to be quite free of iron.) Standard and blank solutions were prepared by pipetting 500 pl each of metal-free water and iron standard into separate cups and adding 500 pl preci- pitating/reducing agent to each. The serum sample, reagent blank, and iron standard solution were each treated with 300 pl BDTPS in 30% sodium acetate. After 5 minutes, the absor- bance of the standard and of the sample were determined at 565 nm against the reagent blank solution. Serum iron in ugFe/ml = Absorbance of sample ABSOrbance of standard X dilution factor Dilution factor = 2 because serum was diluted in an equal volume of metal-free water. Example: Absorbance of sample = 0.30 Absorbance of standard (1 ul/ml) = 0.22 :19 X 2 = 2.73 ugFe/ml .22 *(Zak, Baginski and Epstein, 1980) 66 Preparation of Liver Extract The liver sample was thawed, weighed, and placed in an acid-cleaned homogenizing tube to which was added an equal weight of iron-free water. The liver sample was homogenized by a motor-driven teflon homogenizer until a fine suspension was formed. A 0.2 gm sample of the homogenate was placed into a preweighed acid-cleaned test tube, and 0.8 gms of iron-free water were added. The tube was then corked and refrigerated for at least 30 minutes. To promote cell lysis, the tissue was heated and then recooled. The cork was first replaced with a wad of clean cotton wool, and the sample heated in a dry bath 10 minutes at 90°C and then cooled in an ice bath 10 minutes. The sample was reweighed and metal-free water added to correct for any water loss through evaporation. 500 pl of 8.4 N HCl were added, the weight recorded, and the sample heat ex- tracted in a dry bath at 90°C for 60 minutes. After cooling to room temperature, the sample was weighed and metal-free water added to correct for any water loss through evapor- ation. The sample was then spun 10 minutes at 3000 RPM and filtered. Filters were prepared by placing a plug of Whatman #2 filter paper in the tapered tip of a Pasteur pipette. The sample was drawn up through the filter with a pipet aid. The tip below the plug of filter paper can be broken off if desired. 67 The sample was transferred to an acid-cleaned test tube, corked, and refrigerated until analyzed. 68 Determination of Liver Iron 50 p1 of liver extract, 450 p1 metal-free water, and 500 pl precipitating/reducing agent were pipetted into an autoanalyzer cup. Blank and standard solutions were pre- pared by pipetting 500 pl metal-free water and 500 pl iron standard into separate autoanalyzer cups and adding 500 p1 of precipitating/reducing solution to each. All solutions were allowed to sit for 10 minutes after which 300 pl BDTPS in saturated sodium acetate were added. After 10 minutes the absorbances of the standard and sample were determined at 565 nm against the reagent blank solution. Liver iron in pg/gm wet weight: Absorbance of sample X extraction X sample dilution dilution Absorbance of standard factor factor Extraction dilution factor = 15 because the iron from 0.1 gm of liver was diluted in 1.5 gms of solution. Sample dilution factor = 10 because 50 pl of sample was diluted in 450 p1 metal-free water. Example: Absorbance of sample = .40 Absorbance of standard = .22 (1 pl/ml) ——- .40 X 15 X 10 = 272.7 nge/g wet weight .22 69 Preparation of Brain Extract Each frozen brain was cut coronally on a cryostat into 270 pm slabs which were placed on clean microscope slides, warmed only long enough to promote adherence and them refrozen. The knife was coated with silicone stopcock grease and the excess wiped off. This prevented metal contamination of the tissue from the blade. While viewing the slabs with a dissecting microsc0pe, the desired areas, globus pallidus, substantia nigra, and cortex, were removed with a microcoring device (Palkovits, 1973) which removed cylinders of tissue approximately 0.7 mm in diameter and 270 pm thick. The microcoring device was prepared from a 19 gauge hypodermic needle from which the pointed end was ground off making a flat-ended cylinder. The tip of the cylinder was sharpened on a grinding stone and wiped clean. To prevent metal contamination of the sample, the needle was coated with silicone stopcock grease and the excess wiped off. The needle was attatched to a micrOpipetting tube with a mouthpiece. A small wad of disposable wiping tissue was placed in the tubing to prevent saliva from entering the sample; this plug was changed frequently. The desired sample was cored out of the slab of tissue on the microscope slide with the sharpened tip of the needle and then blown out of the microcoring needle into an acid- cleaned homogenizing tube. 70 The microscope slide was kept frozen during coring by placing it on a metal block kept cold in an alcohol/dry ice bath. The cores were collected in preweighed acid-cleaned homogenizing tubes and cores from the globus pallidus and substantia nigra of a brain placed in one tube, the cortex sample into another. It was necessary to pool the globus pallidus and substantia nigra in order to obtain a suf- ficiently large tissue sample. The boundaries of the globus pallidus and substantia nigra were determined using as a reference and guide a set of Perl's-DAB stained, coronally cut sections of rat brain. The cortex sample came from the area dorsal to the globus pallidus. The homogenizing tube was reweighed after coring and the weight of tissue collected determined. 150 pl of metal-free water were pipetted into the homogenizing tube and the spherical ground glass homogenizer rotated by hand until the tissue was finely ground. Another 150 pl of metal-free water were pipetted down the walls of the tube and the homogenizer to wash adhering tissue into the solution. The sample was transferred into an acid- cleaned test tube, weighed, corked, and refrigerated for at. least 30 minutes. To promote cell lysis, the tissue was heated then recooled. The cork was first replaced with a wad of clean cotton wool and the sample heated in a dry bath at 90°C for 10 min then cooled 10 min in an ice bath. The sample was 71 reweighed and metal-free water added to correct for any water loss through evaporation. 150 pl of 8.4 N HCl were added; the weight recorded; and the sample heat extracted in a dry bath at 90°C for 60 min. After cooling to room temperature, the sample was weighed and metal-free water was added to correct for any water loss through evaporation. The sample was spun 10 min at 1000 RPM and filtered. The extract was placed in an acid-cleaned test tube and refrig- erated until analyzed. 72 Determination of Brain Iron 100 p1 of brain extract, 100 pl saturated sodium acetate, 300 p1 metal-free water, and 500 pl precipitat- ing/reducing agent were pipetted into an autoanalyzer cup. Blank and standard solutions were prepared by pipetting 500 pl metal-free water and 500 p1 iron standard into separate autoanalyzer cups and adding 500 p1 of precipitating/re- ducing solution to each. All solutions were allowed to sit for 10 min after which 300 pl BDTPS in saturated sodium acetate are added. After 10 min the absorbances of the standard and the sample were determined at 565 nm against the reagent blank solution. Brain iron in pg/gm wet weight: Absorbance of sample X extraction sample of standard dilution diIUtion Absorbance factor factor The extraction dilution factor of a sample is the number of times the iron in a sample is diluted by the added iron-free water and HCl. Tissue collected usually weighs between 0.010 and 0.012 gms and the iron in this tissue was diluted in 450 pl of liquid. For 0.012 gms of tissue, the extrac- tion dilution factor is 37.5. Sample dilution factor equals 5 because 100 pl of extract was diluted with 100 p1 of saturated sodium acetate and 300 pl metal-free water. 73 Example: Absorbance of brain sample = 0.06 Absorbance of iron standard (1.0 pl/ml) = 0.22 Lgé X 37.5 x 5 = 51.1 nge/g wet weight .22 74 Analysis of Data Analysis of variance was used to compare the means of estrous cycle, sex difference, pregnancy, effects of gonad- ectomy, and effects of estrogen implants data. All data met the assumption of variance homogeneity, as tested by maximum variance divided by minimum variance. Tukey's test was performed on the combined estrous cycle and sex difference data in order to compare all possible pairs of estrous cycle stages and to compare the male with each stage of the estrous cycle. Regression analysis coupled with analysis of variance was done on pregnancy data. Designed contrasts, comparing day 4 of pregnancy to the other days of pregnancy, were also performed. Orthogonal contrasts tested the effects of gonadectomy and estrogen implants data. For ease of computation, contrasts were performed among treatment groups with equal replication. Excess replicates were deleted on a random basis. In those instances where samples were deleted because of unequal replication, analysis of variance was performed on the total sample size, and it was found that there was little or no change in the means or standard errors of the groups, nor in the F values and associated probabilities in the analysis of variance. Statistical methods are those of Gill (1978) and Bliss (1967). RESULTS HISTOCHEMISTRY The Perl's-DAB treated tissue was chosed for the photography in this section because it intensified the Perl's reaction manyfold and permits the use of thin sec- tions. A 40 um section stained with DAB demonstrates the presence of iron, even in areas of the brain in which the iron concentration is low, and gives a detailed resolution ‘of the location of iron at the cellular level. DAB has the added advantage of being highlighted by dark-field micros- copy. Thus, we have a method which not only yields a clear view of iron-containing structures for light-field micro- scopy, but also in which interference from counterstaining can be removed with dark-field microSCOpy. The photo- graphs presented here were taken with both dark-field and light-field illumination. The intensification step does not demonstrate the presence of iron in any area in which the Perl's method alone does not indicate that iron is present and no staining was apparent with DAB without pretreatment with ferrocyanide. Study 1 a. Localization of iron.in the rat brain. Iron is unevenly distributed in the brain, occurring in many different areas and in differing amounts (See Figures 1 through 8), and is present in different types of cells and structures in these areas. 75 76 nucleus tract tubercle bulb pallidum 52 pallidus a of nucleus Caudate-putamen Olfactory Olfactory Olfactory Globus EntOpeduncular Ventral view counterstain. thalamus no Ventral iron, Parasagittal for 4.5. nigra X iron. Cortex stain of Substantia Perl's-DAB Magnification Hippocampus accumulations iron. rat. colliculus are / of g___ _-.~— - female Superior / areas old nucleus nucleus‘N Stained Distribution week nucleus colliculus olivary 1. trigeminal nucleus~\\\\\\\\\\~ Inferior Cerebellar Spinal Superior Facial Figure 77 Figure 2. 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. Figure 3. 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 accumulations of iron. Magnification X 5.4. Figure 4. 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. Figure 5. 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 Figure 2. . ' FEMALE lCortex Cingulate cortex Lateral septal nucleus Caudate—putamen nucleus Bed nucleus of the stria terminalis Anterior commissure Ventral pallidum Organum vasculosum of the lemina terminalis Island of Calleja Olfactory tubercle Figure 3. MALE Anterior commissure Organum vasculosum of the lamina terminalis Ventral pallidum Island of calleja Olfactory tubercle Figure 4. FEMALE Cortex Cingulate cortex Choroid plexus Caudate-putamen nucleus Globus pallidus Figure 5. MALE Globus pallidus 79 Figure 6. 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. Figure 7. 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. Figure 8. 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 Figure 6. FEMALE . Cortex Cingulate cortex Dentate gyrus Hippocampus Lateral habenular nucleus Ventral thalamus Caudate—putamen nucleus Central amygdalar nucleus Entopeduncular nucleus Ventro-medial hypothalamus Figure 7. FEMALE Cortex Superior colliculus Central grey Reticular formation Medial geniculate nucleus Dentate gyrus Hippocampus Oculomotor nucleus Substantia nigra Interpeduncular nucleus Figure 8. MALE Superior colliculus Dentate gyrus Oculomotor nucleus Substantia nigra Interpeduncular nucleus 81 In brain tissue iron occurs in granules, singly or in clumps; amorphous accumulations; branching fiber-like pro- cesses; fibers; and on or in glial cells and neurons. Whether the iron is within the axoplasm or within the myelin sheath of the fibers cannot be determined from this mater— ial. When iron is found in association with cell bodies, it either 1) completely fills the cytoplasm of the cells (com- monly glial cells), 2) occurs as bouton-like structures which appear to encrust the surface of the perikaryon and neuronal processes of nerve cells, or 3) appears as a fine dusting of small grains within the perikaryon of neurons. In the following description, the iron accumulating areas will be organized more with respect to the type of cellular organization of the iron-containing structures than to the subdivision of the brain to which the site belongs. Iron is present in those parts of the brain in which the blood-brain barrier is absent. Iron-appears here as extracellular granules andamorphous accumulations, fre- quently in association with blood-vessels, and also in branched angular figer-like processes. Sometimes iron is so highly concentrated that determining its structural localization is difficult. These areas include the pineal, the pituitary, area postrema (Figure 9), and the choroid plexus (Figures 4 and 10). The circumventricular areas and some peripheral areas of the brain, some of which are not protected by the blood- brain barrier, also accumulate iron in granules. Figure 9. 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. Figure 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 Figure 9. Area postrema Iron accumulations Central canal. Vagus nucleus Hypoglossal nucleus Figure 10. Subfornical organ Choroid plexus 84 amorphous accumulations and/or fiber-like extensions. These areas include the subfornical organ (Figure 10), the epen- dyma of the ventricles of the brain, the lateral edge of the Optic tract (Figure 11), the organum vasculosum of the lamina terminalis (Figures 2 and 3), and in many areas of the ventro-medial hypothalamus (Figure 6). Within the ventro-medial hypothalamus and median eminence, iron occurs in the tanycytes (Figures 12, 13 and 14) and in granules, clumps of granules, and fibers (Figures 12, 13 and 14). The granules are similar in size and distribution to the gran- ules stained by the Bargmann modification of the Chrome- Hematoxylin method for neurosecretion (Pearse, 1961). Iron also may be seen outlining the capillaries of the median eminence and lateral hypothalamus. The rest of the brain is protected by the blood-brain barrier, and in these areas the sites which accumulate the most iron are the globus pallidus including the entopedun- cular nucleus (Figures 1, 2, 4 and 5), ventral pallidum (Figures 1, 2 and 3), islands of Calleja (Figures 2 and 3), substantia nigra (reticular zone) (Figures 1, 7 and 8), the interpeduncular nucleus (Figures 7 and 8), and the deep nuclei of the cerebellum (Figure 1). By the ventral pal- lidum, I refer to the precommissural ventral anterior extension of pallidal tissue into the olfactory tubercle which is broken up into finger-like extensions by the medial forebrain bundle and which ends in the islands of Calleja (see Figures 1, 2, 3 and 15). Even in those 85 Figure 11. Photomicrograph of iron—filled fibers in the lateral edge of the Optic tract. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 200. Figure 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 Figure 11. Iron—filled fibers Figure 12. Third ventricle :rgzr Iron-filled tanycytes , Ventro—medial hypothalamus 1}, Clumps of iron—filled '.? granules 87 Figure 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. Figure 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 Figure 13. Iron—filled tanycytes Third ventricle Clumps of iron—filled granules Ventro—medial hypothalamus Arcuate nucleus Iron—filled fibers Figure 14. Third ventricle Iron—filled tanycytes Iron—filled fibers Median eminence Iron—filled granules Iron-filled tanycytes 89 Figure 15. Photomicrograph of iron distribution in the ventral pallidum, islands of Calleja and Olfactory tubercle. Iron = brown. Perl's-DAB counterstained with thionin. Coronal view. Magnification X 82. Figure 16. Photomicrograph of iron-filled cells and fibers in the globus pallidus. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 900. 90 Figure 15. Ventral pallidum Island of Calleja Olfactory tubercle: _Polymorph zone Pyramidal zone Figure 16. Iron—filled glial cells Iron-filled fine fibers 91 conditions in which the iron concentration Of the brain is low, iron is usually visible in the above areas of the brain. In all of these areas, iron is localized in glial cells and in fibers--see globus pallidus (Figure 16), ventral pallidum (Figure 17), and substantia nigra (retic- ulata) (Figure 18):' The glial cells are like oligodendro- cytes in appearance, and iron fills the cytoplasm obliter- ating internal cellular detail. Some iron-filled cells are larger than typical glial cells and may be small neurons (Figure 19). In Perl's stained sections of these areas, the background neurOpil is a pale blue wash. The DAB intensification demonstrates that iron in the neuropil is present in fibers (see Figures 15 through 21). In the globus pallidus and continuing into the adjacent striatum, iron is seen in bundles of fine fibers (Figures 1,2 and 4 - low magnification, Figure 20 - high magnification. In the ventral pallidum, thick fibers are more common, and "cascades" of thick fibers are seen running in a dorso- ventral direction in the plane of frontal sections (Figure~ 21). In the substantia nigra (reticulata), the iron—filled fibers are of medium size, and in the cerebellum, the fibers are from medium to large in size. Except for the most medial Olfactory tubercle near the islands Of Calleja, iron-containing granules are rarely found in these areas. Within the islands of Calleja, iron is within fibers in the core and both in and around the granule cells (Figure 15). Whether the iron is within the 92 Figure 17. Photomicrograph of iron-filled cells and fibers in the ventral pallidum. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 476. Figure 18. Photomicrograph of iron-filled cells and fibers in the substantia nigra. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 476. Figure 17. Iron—filled glial cells -’Varicose fiber Iron—filled fibers Figure 18. 'Iron—filled glial cells \ Iron—filled fibers. 94 Figure 19. Photomicrograph of small neuron-like iron- filled cells in the globus pallidus. Iron = brown. Perl's-DAB counterstained with thionin. Magnification X 900. Figure 20. Dark-field photomicrograph of bundles of fine iron-filled fibers in the globus pallidus. Iron = white. Perl's-DAB counterstained with thionin. Magnification X 1000. 95 Figure 19. Small neuron—like iron—filled cells Figure 20. éé. a: .1 Bundles of fine iron—filled fibers Figure 21. Photomicrograph of 'cascades" of iron-filled fibers seen in the ventral pallidum and olfactory tubercle. Iron = brown. Perl‘s-DAB counterstained with thionin. Magnification X 476. Figure 22. Dark-field photomicrograph Of iron in bouton-like structures on or in lateral septal neurons. Iron = white. Perl's-DAB counterstained 96 with thionin. Magnification X 226. Figure 21 Figure 22. 97 ”Cascades' of iron—filled fibers Iron in bouton—like structures on or in lateral septal neurons 98 thin rim of cytoplasm of these cells or on the surface cannot be determined from this material. A similar distribution of iron in glia and fibers is seen in many other parts Of the rat brain. However, iron occurs in a much lower concentration, is frequently present in the walls of blood vessels, and is apparent in Perl's stained sections only under Optimal conditions. These areas include the olfactory bulb where iron occurs in periglomer- ular areas, in olfactory tracts, and in glial cells among the granular cells (Figure l), the caudate, putamen, and accumbens nuclei (Figures 1, 2, 4 and 6), the ventral tier of thalamic nuclei (Figure 6), the lateral habenular nucleus (Figure 6), inferior colliculus (Figures 1 and 7), and, to a lesser degree, superior colliculus (Figures 1 and 7), reticular formation (Figures 1 and 7), medial geniculate nucleus (Figure 7), brachium of the inferior colliculus, lateral lemniscus, oculomotor nucleus (Figures 7 and 8), facial nucleus (Figure 1), and superior Olive (Figure 1). In the midbrain central grey, iron is localized in glia, blood vessels, and clumps of granules (Figure 7). In other low iron areas, iron is not present in glia and fibers but within bouton-like encrustations which appear to be on the surface of the perikaryon and neuronal proces— ses of nerve cells. This type of iron accumulation occurs frequently in the lateral septum (Figure 22, see also Figure 2), bed nucleus of the stria terminalis (Figure 23, see also Figure 2), and occasionally is seen in the ventral pallidum Figure 23. Dark-field photomicrograph of iron in bouton-like structures on or in bed nucleus of the stria terminalis neurons. Iron = white. Perl's-DAB counterstained with thionin. Magnification X 226. Figure 24. Photomicrograph of iron in bouton-like structures on or in 99 ventral pallidal neurons. Iron = brown. Perl's—DAB counterstained with thionin. Magnification X 900. 100 neurons structures pallidal bouton—like ventral in in or Iron on 24. Figure in neurons or on terminalis structures stria the of 23. bouton—like in nucleus Figure Iron bed 101 (Figure 24), diagonal band of Broca, lateral to the organum vasculosum of the lamina terminalis in the medial preoptic areas, lateral to the anterior hippocampus, and in the cortex layers 2, 3 and 5 (Figures 2, 4, 6 and 7). In some low-iron areas of the brain, a fine dusting of iron-containing grains, apparently distributed within the cytoplasm of neurons, is visible. This type of distribution is sometimes seen in the paraventricular nucleus, supraoptic nucleus (Figure 25), suprachiasmatic nucleus (Figure 26), dentate gyrus of the hippocampus (Figure 1, 6, 7 and 8) cingulate cortex (Figures 2, 4 and 6), pyramidal cells of the olfactory tubercle (Figures 2 and 3), and in the central amygdalar nucleus (Figure 6). Iron is found in the hippocampus adjacent to the cells of CA 3 where the granule cells of the dentate gyrus terminate in the mossy fibers (Figure 6). Here iron is present in stellate-shaped structures. Study 1 b. Brain iron and age. The Perl's reaction is mild to absent in the brain tissue from rats 12 weeks of age or younger. Whether the DAB step will permit the localization of iron in the brains of younger animals is not yet known. The intensity of the iron stain increases with increasing age of the subjects, and the oldest animals have the darkest stain. Although the iron concentration increases with age in the rat brain, the relative intensity among brain areas and the distribution of 102 Figure 25. Dark-field photomicrograph of fine grains of iron in cells of the supraoptic nucleus. Iron = white. Perl's-DAB counterstained with thionin. Magnification X 216. Figure 26. Dark-field photomicrograph of fine grains of iron in cells of the suprachiasmatic nucleus. Iron = white. Perl's—DAB counterstained with thionin. Magnification X 200. 103 ’Figure 25. Iron in supraoptic nucleus neurons Figure 26. Iron in suprachiasmatic nucleus neurons 104 iron does not appear to change; as animals get older, the presence of iron in low iron areas becomes evident. Due to a sex difference, which will be discussed in the following section, only within sex comparisons of iron con- centration and age will be made. A quantitative, spectro- photometric determination of age effects on iron concentra- tion is the object of a future study. Study 1 c. Sex difference in brain iron. The iron reaction in the brains of female rats is darker than the reaction in the brains of male rats. Figures 2 and 3, 4 and 5, and 7 and 8 compare the iron concentration of female and male rats 32 weeks of age in the high-iron areas of the telencephalon and mesencephalon. These are: globus pallidus, ventral pallidum, substantia nigra, and interpeduncular nucleus. Whereas the female brain produces a more intense iron reaction that the male, the pattern and distribution of iron appears to be the same in both sexes. Male siblings raised under the same con- ditions and sacrificed at the same age will have very similar reactions. However, female siblings raised under the same conditions and sacrificed at the same age will have a range of reaction intensities. Among a group of rats 12- 18 weeks of age, the strongest reactions are from.female tissue; however, the milder reactions of the females are only as intense as the male reaction. Among 32 and 43 week old rats, variability also occurs in the intensity of the 105 stain of the female brain tissue; however, the tissue from the older animal stains darker than the tissue from the younger animal (as described above in the effects of age). Vaginal smears were not taken at sacrifice so that it could not be determined if the variability among the females was correlated with the estrous cycle. Study 2. Brain iron and pregnancy. The treatments are ranked below in order of increasing intensity of the Perl's reaction. 1. 18 week Old postpartum females. 2. 32 week old 16 days pregnant females. 3. 18 week old 7 days pregnant females. The reaction Of the last group in the globus pallidus and substantia nigra is the most intense reaction observed in any age or treatment group as stained by the Perl's method alone. Brain iron apparently increases early in pregnancy and then decreases during the term of pregnancy. No obvious differences occur in the distribution of iron in the brain or in the relative intensities among brain areas during pregnancy. Study 3. Ovariectomy and ovariectomy plus estrogen treat- ment effects on brain iron. The treatments are ranked below in order of increasing intensity of the Perl's reaction. 106 1. 18 week Old females ovariectomized at 10 weeks, and 18 week Old females ovariectomized at 10 weeks and received an estrogen implant at 17 weeks. 2. 32 week old females ovariectomized at 28 weeks. 3. 32 week Old females ovariectomized at 8 weeks and received an estrogen implant at 30 weeks. The 18 week Old females which had been ovariectomized at 10 weeks of age have a brain iron concentration about the same as a male or lightly-staining female of the same age. Ovariectomized females of the same age which had received an estrogen implant at 17 weeks of age do not appear to differ from any of the above groups. Thirty-two week old females which had been ovariectomized at 28 weeks stain about as darkly as males and lightly-staining females of the same age. However, the 32 week old ovariectomized females which had received an estrogen implant at 30 weeks are visibly darker than any Of the groups mentioned here and nearer in intensity to that reached by 18 week old rats in the first week of pregnancy. Neither ovariectomy nor ovariectomy plus estrogen implant visibly changes the distribution or relative inten- sity of brain iron between the high iron concentrating areas within each treatment group: no area or areas in the brain are affected to any greater degree by these treatments as can be determined using the Perl's method. 107 Study 4. Castration and castration plus estrogen treatment effects on brain iron. No differences in the intensity of the Perl's reaction are visible between castrated males and intact males of the same age or between the castrated males and castrated males which had received the estrogen implant. There is also no apparent difference in the patterns of distribution or the relative intensities between the various high iron areas of the brain. 108 SPECTROPHOTOMETRY Study 1 - to determine (1) if the levels of brain iron change throughout the estrous cycle and (2) if a sex difference occurs in brain iron levels. The iron concentration of the pooled globus pallidus and substantia nigra (GP+SN) fluctuates throughout the estrous cycle with the highest concentration of iron oc- curring at proestrus (Figure 27). The lowest concentration of iron occurs at metestrus with estrus and diestrus having intermediate levels. Proestrus is significantly greater than diestrus, estrus, or metestrus and diestrus, estrus and metestrus do not differ significantly from each other (Table 4). The control male (GP+SN) iron level falls between than of control females in metestrus and estrus and differs significantly only from females in proestrus. The iron concentration of the cortex does not vary significantly thrOughout the estrous cycle (Figure 27 and Table 4), and the cortex iron concentration of the control male does not differ from the females in any stage of estrous. Although the liver iron concentration fluctuates throughout the estrous cycle (Figure 27), the pattern is not the same as in the GP+SN. The diestrus liver iron concen- tration is as high as the proestrus iron level with met- estrus levels lowest and estrus levels intermediate: none of these differences are significant. The control male liver iron concentration is significantly lower than the 109 Figure 27. Iron concentration of liver, pooled globus pallidus and substantia nigra, cortex and serum of control males and females in each of the four stages of the estrous cycle. Mean values 1 l S.E.M., see ANOVA tables for sample sizes. 110 Figure 27 22 24o~ 0‘“. mm E'- 230- 2.3 Liver lJ-‘I 3.30 C235 220'- 2?. :gw 210—45 E '5 a. Globus Pallidus + f—\ Substantia Nigra g zoo-35 25 15 o w L l l 1 nge/mliS.E.M. Control Diestrus Proestrus Estrus Metestrus d 9 9 Q 9 111 Table 4. Analysis of Variance and Tukey's Test of Iron Measures of Estrous Cycle and Sex Difference Data Source DF ‘MS F P GP+SN Total 34 Between 4 768.17 6.74 <0.01 Within 30 113.99 CORTEX Total 33 Between 4 16.91 0.44 NS Within 29 38.36 LIVER Total 39 Between 4 34,142.49 99.51 <0.001 Within 35 343.12 SERUM Total 34 Between 4 0.83 1.60 NS Within 30 0.52 Tukey's Test* GP+SN METESTRUS MALE ESTRUS DIESTRUS PROESTRUS 15.40i2.49 20.14i1.60 20.85:2.6l 24.71i5.10 42.50i6.31 LIVER MALE METESTRUS ESTRUS DIESTRUS PROESTRUS 75.46i5.36 205.15i7.39 215.27i7.67 228.55i6.11 230.98i5.09 *The means i l S.E. subtended by the same line do not differ from each other at p=0.05. 112 females in every stage of estrus: the level is only about 1/3 that of the females (i.e., 75.46 pg Fe/g wet weight) and has not been included in Figure 27 because of the magnitude of the difference. Although the serum iron concentration fluctuates slightly throughout the estrous cycle, with the highest level occurring at estrus (Figure 27), none of the dif- ferences are significant. The control male serum iron level, although lower, does not differ significantly from the females in any stage of estrus. 113 Study 2 - to determine if brain iron levels change during pregnancy. A significant rise in the iron concentration of GP+SN occurs from day 4 to day 8 of pregnancy (Figure 28 and Table 5). By day 12, the iron concentration of GP+SN has fallen to about day 4 levels where it is maintained, with slight but not significant decreases, until day 20 of pregnancy. At 15 to 24 hours postpartum (PP), the iron level is slight- ly, but not significantly, increased. Fluctuations in the iron concentration of the cortex follow a similar pattern (Figure 28); however, the changes are not significant (Table 5). Liver iron concentration decreases throughout pregnancy (Figure 28 and Table 5). Regression analysis demonstrates that the curve has a significant negative slope with a significant non-linear component, a variation in the rate of decrease (prediction equation, Y= 7.85X + 355.63). Liver iron concentration is greater at day 4 of pregnancy than on any other day of pregnancy tested. At PP, the iron concen— tration of the liver is even less than at 20 days, dropping to about 1/2 of the concentration of day 4 of pregnancy. The pattern of liver iron change during pregnancy differs from that seen in GP+SN where an initial rise is followed by a drop after which levels do not change until PP. 114 Figure 28. Iron concentration of liver, pooled globus pallidus and substantia nigra, cortex and serum every four days throughout pregnancy and 15—24 hours postpartum. Mean values i l S.E.M., see ANOVA tables for sample sizes. 115 Figure 28 T USLIJ 300 IRON IE 250 S2 LIJ 3 200 "'45 NON-HEME I- Globus § Pallidus-+- L 35 Substantia 2’ 150 nigra 0| MTfiOTAL 3. 100 -25 . Cortex S 15., u! 41 (D 3 W E! Serum E 2 B 1 U. U) 3. o' 4 8‘ 12 16 20 15-24 m. post partum DAY OF PREGNANCY 116 Table 5. Analysis of Variance with Regression of Iron Measures of Pregnancy Data Source DF MS F P GP+SN Total 35 Between 5 129.67 1.99 NS Regression l 86.50 1.33 NS Residual 4 140.46 2.16 NS Q 1. day 4 vs day 8 1 407.75 6.27 <0.05 Q 2. day 4 vs day 12 l 32.37 0.49 NS Q 3. day 4 vs day 16 l 4.85 0.07 NS Q 4. day 4 vs day 20 1 6.97 0.10 NS Q 5. day 4 vs PP l 13.37 0.20 NS Within 30 65.01 CORTEX Total 35 Between 5 90.79 2.08 NS Regression l 17.50 0.40 NS Residual 4 109.11 2.50 NS Q 1. day 4 vs day 8 1 38.05 0.87 NS Q 2. day 4 vs day 12 1 82.42 1.88 NS Q 3. day 4 vs day 16 1 65.66 1.50 NS Q 4. day 4 vs day 20 l 60.66 1.39 NS Q 5. day 4 vs PP l 16.42 0.38 NS Within 30 43.66 LIVER Total 35 Between 5 20,237.97 9.41 <0.001 Regression l 69,384.30 32.26 <0.001 Residual 4 7,951.31 3.70 <0.05 Q 1. day 4 vs day 8 1 10,103.02 4.70 <0.05 Q 2. day 4 vs day 12 1 9,610.11 4.47 <0.05 Q 3. day 4 vs day 16 l 36,695.97 17.06 <0.001 Q 4. day 4 vs day 20 l 45,660.47 21.23 <0.001 Q 5. day 4 vs PF 1 79,059.58 36.76 <0.001 Within 30 2,150.39 117 Table 5 continued. Source DF MS P SERUM Total 35 Between 5 2.87 11.96 <0.001 Regression 1 2.85 11.87 <0.001 Residual 4 2.87 11.97 <0.001 Q 1. day 4 vs day 8 l 0.24 1.00 NS Q 2. day 4 vs day 12 1 3.16 13.17 <0.005 Q 3. day 4 vs day 16 l 0.00 0.00 NS Q 4. day 4 vs day 20 1 9.36 38.92 <0.001 Q 5. day 4 vs PP 1 0.34 1.42 NS Within 30 0.24 118 Regression analysis of the serum iron concentration demonstrates a slight but significant negative slope. On days 12 and 20, a dip in serum iron concentration is ob- served; both are significantly different from the day 4 iron concentration, but the other days of pregnancy do not differ significantly from day 4. The pattern of change of serum iron concentration differs from both the GP+SN and the liver. 119 Study 3 - to determine (1) if gonadectomy affects brain iron levels and (2) if 3 weeks estrogen treatment in- creases or decreases iron levels compared with control and gonadectomized rats. Gonadectomy at 4 weeks of age has different effects on the iron concentration of the GP+SN in males than in females (Figure 29 and Table 6). Whereas ovariectomy causes no change in GP+SN iron concentration compared with control females in estrus, castration causes a significant increase in GP+SN iron compared with control males. Three weeks of estrogen treatment of gonadectomized males and females causes a slight, but not significant, decrease in the GP+SN iron levels compared with gonadectomized males and females. The GP+SN iron concentration of both castrated males and castrated males with estrogen implants are significantly greater than the control male level. Among the females, neither ovariectomy nor ovariectomy with estrogen implant has significantly different GP+SN iron levels than the control female in estrus. The iron concentration of the cortex with gonadectomy and gonadectomy with estrogen treatment differs from that of the GP+SN, and females respond differently than males (Figure 30 and Table 6). Castration causes a slight, but not significant, increase in cortex iron compared with control males, but ovariectomy causes a significant decrease in cortex iron compared with females in estrus. Castration plus estrogen treatment causes a significant drop in cortex 120 of with nigra males ANOVA with 9), see females (ESTRUS castrated substantia S.E.M., 1 d). and i estrus (CAST in values ovariectomized pallidus males females and Mean globus 9), castrated 6+EST), (OVX 9+EST). pooled 6), of (CAST (OVX sizes. females (CONT sample implants implants males for concentration Iron control tables estrogen estrogen ovariectomized 29. Figure l2! é XAO + .183 915!N + S"P!I|9d . (‘5 eg1ue1sqn3 XAO anolE) 6 808.183 P 183 .LSVO + P lSVO P .LNOO 9 0 92 DZ 08 98 l l sz ambu w a s FlHDIElM 13M 6 /5" NOHI awaH-NON 1v101 122 Table 6. Analysis of Variance with Orthogonal Contrasts of Iron Measures of Control Males (CM), Castrated 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) Source DF MS F P GP+SN Total 35 Between 5 191.56 2.24 NS Q 1. Males vs Females 1 510.31 5.96 <0.05 Q 2. CM vs CAST, CAST+EST 1 409.32 4.78 <0.05 Q 3. CAST vs CAST+EST 1 5.45 0.06 NS Q 4. ESTF vs ovx, 0VX+EST 1 8.43 0.10 NS Q 5. OVX vs 0VX+EST 1 24.31 0.28 NS Within 30 85.57 CORTEX Total 35 Between 5 52.97 2.79 <0.05 Q 1. Males vs Females 1 141.69 7.48 <0.025 Q 2. CM vs CAST, CAST+EST 1 0.67 0.04 NS Q 3. CAST vs CAST+EST 1 41.85 2.21 NS Q 4. ESTF vs OVX, 0VX+EST 1 77.12 4.07 <0.10 Q 5. OVX vs 0VX+EST 1 3.55 0.19 NS Within 30 18.94 LIVER Total 47 Between 5 21,670.92 71.85 <0.001 Q l. Males vs Females l 55,027.24 182.45 <0.001 Q 2. CM vs CAST, CAST+EST l 23,787.48 78.87 <0.001 Q 3. CAST vs CAST+EST l 15,345.64 50.88 <0.001 Q 4. ESTF vs OVX, 0VX+EST 1 9,121.26 30.24 <0.001 Q 5. OVX vs 0VX+EST 1 5,073.00 16.82 <0.001 Within 42 301.61 123 Table 6 continued. Source DF MS F P SERUM Total 53 Between 5 6.61 16.95 <0.001 Q 1. Males vs Females l 1.65 4.23 <0.05 Q 2. CM vs CAST, CAST+EST 1 0.73 1.87 NS Q 3. CAST vs CAST+EST l 15.40 39.49 <0.001 Q 4. ESTF vs OVX, 0VX+EST 1 0.00 0.00 NS Q 5. OVX vs 0VX+EST l 15.25 39.10 <0.001 Within 48 0.39 BODY WEIGHT Total 47 Between 5 38,914.27 78.38 <0.001 Q 1. Males vs Females 1 110,688.02 222.94 <0.001 Q 2. CM vs CAST, CAST+EST l 39,963.02 80.49 <0.001 Q 3. CAST vs CAST+EST 1 12,376.56 24.93 <0.001 Q 4. ESTF vs OVX, 0VX+EST 1 31,518.75 63.48 <0.001 Q 5. OVX vs 0VX+EST l 25.00 0.05 NS Within 42 496.50 124 males females S.E.M.. 1 i ovariectomized castrated +EST), and values (CASTG Q), (CONTd‘), Mean (OVX implants males +EST). 9 females control (OVX estrogen of sizes. with implants cortex sample males of 9),ovariectomized for estrogen (ESTRUS castrated tables with d), concentration ANOVA estrus (CAST females in Iron see 30. Figure [25 Q + EST OVX Q OVX Cortex Q 0.0.0 030?. ESTRUS d + CAST d v v f o? 0%. ' .0V . 0 50° O 02020! 0. 0? o? CAST ' v v v O O O... . O O . . 3030 O 0? CONTd 15 10 was LHHoIaM 13M 6 30 /6r' NOHI awaH-NON 1v101 Figure 126 iron compared with both control males and castrated males but, in females, ovariectomy plus estrogen treatment causes a slight, but not significant, rise in cortex iron compared with ovariectomy. The iron level in the cortex of the ovariectomy plus estrogen treatment group remains less than that of females in estrus. The liver iron concentrations of the male and female treatment groups differ (Figure 31 and Table 6). Whereas castration causes a significant increase in liver iron concentration compared with control males, ovariectomy causes a decrease compared with females in estrus. Both gonadectomized groups respond the same way to estrogen treatment, however, with a significant rise in liver iron values. Even with gonadectomy occurring as young as 4 weeks of age, the liver iron concentration of the ovariectomized females still exceeds that of the castrated males. The serum iron concentration of the male and female treatment groups differs significantly (Figure 32 and Table 6). Castration has no effect on serum iron level compared with the control males; however, ovariectomy causes a drop in iron concentration compared with females in estrus. Estrogen treatment of 3 weeks significantly increases serum iron concentration in both castration plus estrogen and ovariectomy plus estrogen groups compared with castrated males and ovariectomized females. This response is similar to the response of liver iron concentration to estrogen treatment . 127 males. females S.E.M., ovariectomized i1 castrated d+EST), and d), 9), values (CAST (CONT (OVX Mean implants males females 9+EST). (OVX control estrogen sizes. of with implants liver ovariectomized sample males of 9), for estrogen (ESTRUS tables castrated with 5), concentration ANOVA estrus (CAST see in females Iron 31. Figure l28 Q ' O. 0 0 .0 .0 .' V ' v v V v v 0 ..0 0 0 0 0 '0 0 0 0 0 .0 0 0 O 0 0 2020 A 0 .0 ..0 0 0 ’0 0 ’0 0 0 0 0'0 .0 0 0° .0 .0 ’0 0 ’0 .0 0 0 0 0 ’0 ’0 0.0.0 :0 O O 0 ’0 0 ’0 0 ’0 0 0 :0° .0 0 0 0 0 0 0 0 0 0 0’0 0: 0 0 0.0 0 .0 .0 .0 0 0 0 0 0 0 ’0 0 0 0 0.0.0 .0 A ..O 020 0 .0 0 .0 ’0 .0 0 0 .0 0 ’0 0 0 0 0 A . .. O. 0.. O A O 0 0 0 0 .0 .0 0 + A O O O . O . 0 O O O O OVX EST Q ... D O 0 .0 O 0 O O .0 '0 0 ’0 0 .0 0'0 0.0. 0 OOO 0 0 O O .0 0 .0 0 .0 .0 ’0 0 0 0 .0 ’0 0 O 0 :0 0 0 0 0 ’0 0 0 0 0 0 0 ’0 .0 0 0. .0 0 0 0 .0 0 0 VX 0 0 0 0 ’0 0 0 °. 0 0 0 0 0 0 0 ’3 0 f0 .0 '0A 0 .0 20 .0 0 .0 .0 0.0 0 0 0 0 0 0 .0 0 0 A ..A. A ‘ A A O A A Liver 9 O 0 0 0 0 0 0' 0 0 0 .0 O 0 0 0 O 0 0 0 00 O 0 .O .O O O .0 .0° 0 '0 0 0 0 0 0 0 O 0 0 O 0 00 0 .O '0 0 0 0’ 0’ O 0 0 .0 .0 0 0.:.0 .0 0 0 0.0 .0 0.0.0 0 0 0 ’0 0 .0 0 0 0 ’0 ’0 0 0 0 00 .0 :0 0 .0 0 0 0 0 0:0.0'0 0 .O 0 O 0 .O 0 O 0 0 0 O . .... 02°: 0202 0.0. A 0 .0 0.0 0.0 .0 0 .0 0 .0 0 .0 .0 020 A A ... A A A’. A 000 ESTRUS d .. 0 0 0 0 0 0’0 0 0 .0 0 0 0 0 0 0 .0 0 0 .0 0 0 0 0 ’0 0 + .0 .0. 0?? 0 0 ’0. 0 CAST d O v 0 0 0 0 0 0 0 .‘0 O 0 0 0 :0 0 ’0 0 0:0 0 0 0 0 O 0 0 0 0 0 0 ’0 0 0 0 0 0 0 0 0! . .. A A A O OOO 0 CAST d CONT - '- *- - 190 150- 170 130 110- 210 was L-HHDEIM 13M 6 /5fi NOHI awaH-NON 1v101 3| Figure 129 males females S.E.M, l ovariectomized i castrated dWEST), and 6), 9), values (CAST (OVX (CONT Mean implants males females 9+EST). (OVX control estrogen sizes. of with implants serum ovariectomized sample males of o), for estrogen (ESTRUS tables castrated with d), concentration ANOVA estrus (CAST females in see Iron 32. Figure I30 o XAO 183 + O XAO 6 808183 P 183 lSVO + PlSVO P .LNOO as was l-T-IUJ/NOHI 6" canola 131 The body weights of the male and female treatment groups differ significantly (Figure 33 and Table 6). Castration causes a drop in body weight compared with control males, but ovariectomy causes an increase in body weight compared with females in estrus. Estrogen treatment of 3 weeks causes a further drop in both the castrated plus estrogen and ovariectomy plus estrogen groups compared with castrated males and ovariectomized females. Estrogen treatment has the opposite effect on body weight compared to that on liver and serum iron concentration. 132 estrus females see in 6), (CAST females S.E.M., l i males ovariectomized d+EST), and values 9), (CAST castrated Mean (OVX 6), implants (CONT 9+EST). females sizes. (OVX males estrogen sample with control implants for of ovariectomized males g), tables estrogen weight (ESTRUS castrated Body with ANOVA 33. Figure I33 o XAO 183 + lufileM <5 XAO Apes <5 808183 9 .183 .LSVO + P lSVO P lNOO 00l 002 008 'W'3'8 l-T-SWVHE) Q; unby DISCUSSION Details of the localization of iron in the rat brain made possible by the DAB intensification of the Perl's method, the fluctuation of brain iron during the estrous cycle, the changes during pregnancy, the effects of gonad- ectomy, and the effects of estrogen treatment, discussed below, are reported here for the first time. I propose that this new information, when considered together, suggests: (l) a role for the presence of iron in the brain, and (2) an interpretation of its pattern of distribution. The pattern of distribution of iron in the rat brain reported here is similar to the distribution which occurs in the human brain (see literature review). The presence and extent of accumulation of iron appears to be a character- istic feature of each brain area. Although iron is required for the metabolism of every cell, the distribution and concentration of iron in discrete areas and structures of the brain suggest that its accumulation occurs either as a requirement or as a consequence of some specific metabolic process or processes. Since iron is frequently associated with large molecules and since most of the brain is pro- tected by the blood-brain barrier, special mechanisms transport iron and associated large molecules (e.g., trans- ferrin) across the basement membranes of capillary walls and into cells. Such an active uptake further suggests that iron is an important requirement in some neural metabolic 134 135 process. The uptake of iron in brain areas not protected by the blood-brain barrier, however, could be considered a more passive accumulation. Myelin The localization of iron in fibers and oligodencrocytes in many of the high iron areas of the brain suggests that the iron might be associated with a particular myelin prod- uced by iron-containing glial cells. In the brain subcel- lular fractionation studies of Rajan et a1. (1976), a large portion of brain iron occurs in the myelin fraction. Also, the persistent deficiency in brain iron following an iron deficient diet in the studies of Dallman et a1,(l975) occurs when iron deficiency overlaps with the periOd of myelination of the central nervous system. Also, in the male rat, there is a suggestion of an association of myelin and iron since both myelin (see Norton, 1976) and brain iron Dallman and Spirito, 1977) are found to have very low turn- over rates. The association of iron with myelin, however, does not explain the presence of iron in the brain where it is not within glial cells but in granules or on or in the perikarya of nerve cells. Monoamines Monoamines, such as dOpamine, norepinephrine, and serotOnin, require iron for many aspects of their normal metabolism (see literature review). Although the monoamines occur in some high-iron areas (Table 7), the distribution and relatvie abundance of a monoamine and iron in the rat 136 GABA GABA GABA GABA GABA GABA GABA the of Serotonin Serotonin Serotonin Serotonin Serotonin Serotonin Serotonin Serotonin Areas processes fiber-like Concentrating and Norepinephrine Norepinephrine Norepinephrine Norepinephrine Norepinephrine Norepinephrine Iron in GABA* Dopamine Dopamine accumulations. and amorphous fibers fibers Monoamines and of and granules, glia tract glia in in in (reticulata) grey areas Optic nuclei nucleus Distribution hypothalamus of eminence organ present present Calleja nigra present - - formation The bulb - central edge thalamus of quadrigemina plexus pallidum habenula pallidus 7. iron iron cerebellar postrema Tanycytes Arcuate Median Brain. iron Subfornical Islands Interpeduncular Substantia Choroid Striatum High Periventricular Table Rat Corpora OVLT Globus High Olfactory Deep Ventro-medial Reticular Area Lateral Ventral Ventral Low Lateral Midbrain 137 and high Bloom to Serotonin Serotonin Serotonin Serotonin Serotonin Cooper, moderate 1977; Moore, containing Norepinephrine Norepinephrine Norepinephrine Norepinephrine Norepinephrine Norepinephrine Norepinephrine and areas Jones 1981. Dopamine D0pamine Dopamine 1971; includes neurons of Beaudet, GABA areas) and and system Ungerstedt, perikarya in fiber from: or terminalis Descarries monoamines on is mossy the (periventricular nucleus nucleus stria and and of Parent, of present nucleus cortex - hippocampus continued. septum amygdala gyrus 1978; preOptic 7 iron nucleus concentration Roth, Suprachiasmatic Supraoptic Cortex Table Bed Cingulate Central Paraventricular Low Lateral *Distribution Dentate Anterior Medial 138 brain are not highly correlated, and monoamines are con- centrated in many sites where iron is not. U-aminobutyric acid i-aminobutyric acid (GABA) is concentrated in those areas of the brain in which iron is abundant and localized in glia and fibers (Tab1e7). Further, a glutamic acid decarboxylase (GAD), although different from the neuronal CAD, is present in glial cells in the brain (see Cooper, Bloom and Roth, 1978). However, the corpora quadrigemina, containing only moderate amounts of iron, are among the regions of the rat brain containing the highest concentra- tion of GABA (see Cooper, Bloom and Roth, 1978). Although the presence of iron in glia and fibers, as occurs in the globus pallidus and substantia nigra (reticulata), may be related to the metabolism of GABA, this relationship does not exPlain the presence of iron in the other high-iron areas of the brain. Peptides There is a correlation between the distribution of iron in the brain and the known neuroactive peptides. This correspondence is especially evident in the highAironwareas of the brain (Table 8), and concentrations of known peptides occur only where iron is present. Except for the deep cerebellar nuclei and lateral islands of Calleja, all high- iron regions of the brain are associated with at least one of the known peptides (Table 8). -Enkepha1in is found‘in the highest concentration in the globus pallidus (Hong et a1., 139 Hormones Brain. Enkephalin Rat P the Releasing accumulations Enkephalin of P Substance P Areas 1&0 amorphous Enkephalin Enkephalin AV, lateralis) Substance and n.) n., Substance Neurotensin (pars Oxytocin B-Endorphin layer. P Concentrating Somatostatin processes Enkephalin Iron Somatostatin LHRH granular Enkephalin Enkephalin and Enkephalin Substance (parafascicular Somatostatin Somatostatin Oxytocin Enkephalin fibers TRH TRH Vasopressin (parafascicular in fibers Neurotensin P P P fiber-like and and and Somatostatin Enkephalin Peptides* cells cells of Substance Substance Substance Vasopressin Enkephalin Enkephalin Enkephalin Enkephalin Enkephalin Angiotensin Enkephalin Enkephalin Enkephalin VasoPressin Angiotensin Angiotensin Angiotensin LHRH Angiotensin LHRH LHRH LHRH Angiotensin Angiotensin granules glial tract glial in in in grey areas optic nuclei nucleus Distribution hypothalamus of organ eminence present present Calleja nigra - The - formation bulb eApresent central edge plexus thalamus quadrigemina of pallidum habenula pallidus 8. iron iron cerebellar postrema Tanycytes Arcuate Median iron Subfornical Interpeduncular Choroid Straitum Table Islands Substantia Periventricular Corpora OVLT Olfactory Lateral High Globus Deep High Area Ventral Ventro-medial Low Ventral Lateral Reticular Midbrain 140 is Ganten 1980; and 1978; a1., Neurotensin et concentration Hfikfelt, Enkephalin and Moscowitz high P to cortex) Elde 1979; 1978; Enkephalin Substance Enkephalin Somatostatin moderate (frontal P Childer, Enkephalin a1., TRH TRH TRH et and Substance containing neurons Enkephalin Enkephalin Snyder Oxytocin Oxytocin Oxytocin Oxytocin of Dogerom 1981. Enkephalin P areas 1979; 1978; a1., LHRH et a1., perikarya Substance Somatostatin Enkephalin Vasopressin Enkephalin Enkephalin Vasopressin TRH, Vasopressin Vasopressin Angiotensin includes Roth, in et King or and on 1980; mossy peptides Bloom nucleus nucleus stria Silverman regions) of and a1., of nucleus present cortex et - 1978; hippocampus continued. septum system amygdala COOper, gyrus preoptic 8 iron a1., nucleus (ventricular terminalis fiber from: et Phillips Supraoptic Suprachiasmatic Table Central Paraventricular Bed Cortex Cingulate *Distribution Dentate Low Lateral Anterior Medial 141 1977), the brain region with the highest concentrations of iron as measured by Hallgren and Sourander (1958). Enkeph- alin and opiate binding sites have similar distribution patterns in the rat but have different relative concentra— tions (Atweh, 1977a,b,c); both occur in areas in which iron has been localized in the present study. Substance P is abundant in the iron rich substantia nigra (reticulata) and interpeduncular nucleus (see Cooper, Bloom and Roth, 1978; Elde and Hkaelt, 1978 for review). The hypophyseal hormone releasing factors and hypothalamic neurohypophyseal hormones are present in abundance in the circumventricular. and ventro-medial hypothalamic areas of the brain. These are regions in which the blood-brain barrier is absent and much iron accumulates. The association of iron with peptides is further evidenced by the eXistence of several known sites in which a peptide and iron are not only localized in the same general region but appear to be within the same cytoarchi- tecturally distinct structures. The presence of enkephalin within fine fibers of the globus pallidus has been deter— mines with immunocytochemical methods (Sar et al.,1978; Jacobowitz et a1.,1979. Although, as mentioned in the Results section, it cannot be determined from the present material whether the iron is within the axoplasm or myelin of iron-filled fibers, iron is present in fine fibers in the globus pallidus (Figure 20). Also, Jacobowitz et a1., describe large enkephalinergic axons seen ventral to the 142 anterior commissure projecting rostrally towards the olfac- tory tubercle. These large enkephalinergic fibers are similar in both appearance and distribution to the iron- filled "cascades" of large fibers found in the ventral pallidum and around the islands of Calleja of the olfactory tubercle (Figure 21). The localiZation of enkephalin within the granule cells of the dentate gyrus and continuing to the mossy fiber terminations adjacent to the pyramidal cells of the hippo- campal region CA3 and CA4 (Gall et a1., 1981), corresponds exactly to the localization of iron within this region in the rat (Figure 6), and applies equally well to the local- ization of zinc (McLardy, 1962). The substance identified by neurosecretory stains, and more recently with immunocytochemistry for neurOphysin (Elde and Hkaelt, 1978), has been localized in cells of the supraoptic nucleus, paraventricular nucleus, and to a lesser extent, the suprachiasmatic nucleus. This subs- tance is also seen as beaded fibers in the hypothalamic- hypophyseal tract and in clumps of granules in the ventro- medial hypothalamus, arcuate area, median eminence, and in periventricular areas. Iron is found within the supra- Optic, paraventricular, and suprachiasmatic nuclei and in fibers, clumps of granules in the ventro-medial hypo- thalamus, arcuate area, and the median eminence. Further- more, I have observed that the iron-containing granules are similar in size and distribution to the neurosecretory 143 granules as seen with the classic neurosecretory stains (unpublished observations). Although the distribution of luteinizing hormone releasing hormone (LHRH) overlaps with the presence of many other peptides in the iron-rich areas of the organum vasculosum of the lamina terminalis (OVLT), ventro-medial hypothalamic, and periventricular areas, LHRH is the only peptide localized within the tanycytes of the third ventricle in the rodent brain (Zimmerman et a1., 1974; King et a1., 1981). This study shows that the tanycytes of the third ventricle of the rat are iron-filled (Figures 12,13 and 14). The association of iron with the peptides of the nervous system suggests that iron might function in some capacity in the metabolism, transport, and/or storage of peptides, and the further association of iron with LHRH suggests a role for iron in neuroendocrine regulation. Iron occurs in a variety of structures such as in glial cells and in or on neurons, as well as within fibers, fiber-like processes, and granules. The presence of iron in these different structures could be related to its association with different neurologically active molecules or in different aspects of the metabolism of a single substance. Iron within the cytOplasm of neurons may be associated with the synthesis of a peptide or other sub— stance. However, since peptides are believed to be formed from larger molecules during passage along the axon 144 (Marks, 1978), it is interesting that iron is frequently in high concentration in fibers but only seldom, and at low concentration in perikarya. This suggests that the presence of iron in fibers could be related to either a carrier molecule or to a peptidase., The iron in bouton- 1ike structures could be related to peptides in terminals, either involved with the storage of the peptide or with a peptidase. Sullivan et a1, (1979) have recently described an enkephalinase which is specific for enkephalin and has a regional distribution similar to the opiate receptor binding sites and therefore to iron. Enkephalinase is a metalloenzyme which is inhibited by the iron chelators 1,10 phenanthroline and ethylenediaminetetraacetic acid (EDTA). It appears likely that the metal in the enkephalin- ase is iron and therefore that the distribution pattern of iron is related, at least in part, to the distribution of enkephalinase. Iron in Neuroendocrine Function Estrous cycle and pregnancy VThe suggestion that iron is involved in neuroendocrine functions is further supported by the results of this study which demonstrate that brain iron levels vary in conjunction with changes in ovarian hormone concentrations. During proestrus, when pooled globus pallidus plus substantia nigra (GP+SN) iron concentration is highest, plasma con- centrations of estrogen and progeSterone are at their peak 145 (Butcher et a1.,1974). Although estrogen levels remain low after day 4 of pregnancy, progesterone increases until about day 14, after which levels drop (Sato and Henkin, 1973). Iron levels in the GP+SN also increase during the first half of pregnancy. Although the brain iron level drops slightly toward the end of pregnancy, at least 12-24 hours post partum the GP+SN concentration is actually raised, representing, perhaps, the proestrus rise in iron preceeding a post partum estrus. Cortex iron concentrations do not change significantly either during the estrous cycle or during pregnancy; however, the pattern of change of the cortex during pregnancy is similar to that of the GP+SN. Iron is localized in glial cells and fibers in the GP+SN, whereas in the cortex iron is localized in the bouton-like structures on or in neurons. Perhaps the presence of iron in different structures reflects a different metabolic role and thus a different response to hormones. Sex difference There is a sex difference in the iron concentration of the GP+SN of male and proestrus female rats. During the other stages of the estrous cycle, the female GP+SN iron levels are within the male range. The iron concentration of the male GP+SN and the level to which the female GP+SN returns after a proestrus rise could represent a "baseline" iron concentration which is required for the optimal func- tioning of these areas in a 12 week old rat. The baseline level for older animals of either sex is not known. In the 146 cortex of 12 week old rats, iron concentration is the same for both sexes and during all stages of estrus. However, the histochemical demonstration of iron in the brain tissue of 32 week old rats demonstrates that the female has more cOrtex iron than the male of the same age (Figures 2,3,4, 5,7 and 8). Thus, with increasing age, male and female brain iron concentrations may differ even during non-pro- estrus stages. Since in the rat the percentage of total brain iron in the storage form, ferritin, increases with age (Dallman et a1., 1975), the increase in histochemically identifiable iron with age seen in this study may reflect an increase in storage iron only and not in non-ferritin forms of iron. Although the accumulation of iron in the brain appears to be influenced by ovarian hormones, the brain does not respond the same way as the liver. A rise and fall in liver iron concentration occurs during the estrous cycle, but liver iron rises during diestrus, before GP+SN levels. In this study, liver iron values did not increase through the first third of pregnancy, as do the GP+SN iron levels, but dropped continuously from 4 days pregnant through to 12-24 hours post partum. Also, the sex difference in liver iron concentration is much more pronounced than the sex difference in brain iron. Whereas the GP+SN iron dif- feredonly between proestrus females and males of the same age, the liver iron of the male is only about 1/3 that of 147 of the concentration of the female at any estrous stage. This difference in response between the brain and the liver could be due to a difference in the forms of iron present in these tissues fCabout 60-70% of the liver iron is in association with ferritin, whereas only about 15-25% of brain iron can be shown to be in this form; see liter- ature review), or the iron-accumulating tissues are respond- ing to different hormones. The estrogen rise during the estrous cycle occurs about 12 hours before progesterone (Butcher, et a1.,1974); perhaps the liver is responding to estrogen and the brain to progesterone or to some temp- oral or concentration relationship between the two ovarian hormones. During pregnancy, the GP+SN iron levels, as shown here, more closely match the pattern of the plasma progesterone concentration (Sato and Henkin, 1973) than do the liver iron values (Figure 28). Ovariectomy, castration and estrogen implantation Ovariectomy does not change the iron concentration of the GP+SN compared with that of non-proestrus females. The iron concentration of this part of the brain remains at the "baseline" level, the maintenance of which, evidently, is not dependant upon the presence of ovarian hormones. In the spectrophotometric study, estrogen implants also have no significant effect on the iron concentrations of the GP+SN. In the histochemical study, when ovariectomy was performed at a young age, estrogen treatment had no visible effect on the brain iron levels. Only when 148 ovariectomy ocCured in an adult, followed shortly by , estrogen treatment, did the hormone appear to increase GP+SN iron. Either estrogen alone does not increase brain iron, or ovariectomy after a long period of time affects the iron-accumulating abilities of the GP+SN. Cortex iron, however, decreases after ovariectomy-and does rise slightly, but not significantly, with estrogen treatment which Sug- gests that the maintenance of cortex iron concentration may be influenced by ovarian hormones in a manner differ- ent from that which influences the GP+SN. Testicular hormones apparently suppress the iron- accumulating abilities of the GP+SN since an increase in brain iron is seen in those areas with castration. Iron levels in the cortex are apparently unaffected by castra- tion. The "baseline" GP+SN iron level in the normal male is apparently meaintained by suppressing iron accumulation. Estrogen treatment had no significant effect on the iron concentration of the GP+SN in the castrated male. Both ovariectomy and castration have profound effects on the iron accumulation of the liver, which suggests that the ovarian hormones are necessary in order to maintain the high liver iron concentrations in the female and that testicular hormones suppress liver iron accumulation in the male. Estrogen implants caused significant increases in liver iron in both sexes. Thus, both male and female liver tissues were able to respond to estrogen administration, and the hormone had similar effects in both sexes. 149 Estrogen stimulated ferritin synthesis (Bj¢rklid and Helgeland, 1970) which results in the increase in iron concentration seen in the liver of estrogen treated males and females in this study. However, if the brain is also accumulating iron in the form of ferritin, one would expect a similar response. Estrogen treatment also stimulates the synthesis of ceruloplasmin (Planas, 1973), a ferroxidase. Increase cerulOplasmin may act to move iron from tissues into the blood. This might explain the slight, but not significant, decreases in brain iron concentration seen with estrogen treatment. The rise in GP+SN iron concentra- tion during proestrus may be due to progesterone alone or to some temporal or concentration relationship between est- rogen and progesterone as noted above.' Even though gonadectomy was performed at four weeks of age in this study, well before sexual maturity in the rat, the male and female brain tissues responded differently to gonadectomy. Thus, by 4 weeks of age, a sex difference in the brain iron accumulating abilities had already occurred. Perhaps this sex difference in the brain is another central nervous system function subject to the organizational effects of early gonadal hormone exposure (MacLusky and Naftolin, 1981). Globus Pallidus and NeuroendocrinezFunctiOn, The globus pallidus and substantia nigra are generally thought to be part of the motor systems of the brain. Neither of these regions are loci for estrogen receptors 150 nor are they generally included in the group of brain regions thought to be involved in reproductive functions. Only two studies were found that link the globus pallidus to sex differences or sexual behavior. There is a sex difference in the effects of globus pallidus lesions on weight gain in the rat (Hahn and Lenard, 1977), and vagino- cervical stimulation incudes an increase in 2-deoxy-D-glu- cose utilization in the globus pallidus (Allen et a1., 1981). Vaginocervical stimulation affects a number of physiological processes involved with reproductive events including pro- gesterone secretion, sperm transport, sexual receptivity, locomotion, and perception of pain in female rats (see Allen et at., 1981). Brain areas showing increased meta- bolism following vaginocervical stimulation that have been implicated in the control of reproduction include: globus pallidus, medial preOptic areas, mesencephalic reticular formation, bed nucleus of the stria terminalis, and dorsal raphe. Iron in the Medial Preoptic Area Electrochemical stimulation with stainless steel elec- trodes or the application of iron salts to the medial preoptic area of the brain induces ovulation (Everett and Radford, 1961; Dyer and Burnet, 1976; van der Schoot et al., 1978; Columbo and Saporta, 1980). These procedures cause irritation and/or lesioning in the site of the iron deposi- tion. Recently, Columbo and Saporta (1980) have demonstr- ated that the iron deposition causes increased 2-deoxy-D- 151 glucose uptake and suggest that the increased metabolic rate produced by iron is the ovulation-inducing stimulus. Interpretation of Iron Fluctuation in the Brain The fact that endogenous iron in the medial preoptic area can induce ovulation suggests that the naturally occur- ring proestrus peak in the iron concentration of the globus pallidus and ventral pallidum, areas immediately adjacent to the preoptic region might somehow be involved in the normal ovulatory stimulus. The distribution of iron described here suggests an association of iron with peptide metabolism and, in some areas, specifically with enkephalinase.‘ Because endogenous opioid peptides have been implicated in the regulation of the proestrus luteinizing surge of the rat (Pang et a1., 1977; Muraki et a1., 1979) and met-enkephalin in the hypothalamus fluctuates during the rat estrous cycle (Kuman et al.,1979), the activity of the associated enkephalinases might also be expected to fluctuate with changing events in the estrous cycle. Perhaps the iron accumulation in the globus pallidus during proestrus is related to a change in peptide metabolism which, in turn, influences reproductive functions. The fluctuations of iron seen in the high-iron areas of the GP+SN may also reflect a similar change occurring in the high-iron ventro-medial hypothalamic areas, thus influencing LHRH directly through the association of iron with LHRH in the organum vasculosum of the lamina terminalis 152 tanycytes, and arcuate and median eminence areas. The monoamines, involved in gonadotropin release (McCann and Ross, 1975) and sexual behavior (Meyerson et a1., 1973) are regulated by iron-containing enzymes (see literature review). This represents another route through which changes in iron availability can affect reproductive functions. The results of this study suggest, firstly, that iron in involved in neuroendocrine regulation and reprod- uctive functions and secondly, that the distribution pattern of iron in the brain may be related to the associa- tion of iron with neuroactive peptides. These results empnasize the importance of iron for the normal metabolism of the brain and behavior. Iron deficiency, a common world-wide nutritional disorder, has effects on reproduct- ive physiology. Perhaps the amenorrhea and menstrual irregularity associated with iron deficiency anemia in women is due to insufficient iron present to permit adequ— ate functioning of the gonadotrOpin release mechanism rat- her than being due to anemia per se. 153 Why the Brain has so much Iron. Prefaced with a reminder that very little is known about the metabolism of iron in the brain, the following is an outline of how the available facts can be interpreted in order to explain how it is that the brain accumulates so.much iron. Brain iron can be subdivided into three compartments: myelin iron, non-myelin non-ferritin iron, and ferritin iron. The first compartment, myelin iron, increases in total amount during the myelination period of brain growth. However, the amount of iron in this form will level off- as myelination slows down and will remain at a steady level after about 6 month of age in the rat. The second compartment, non-myelin non-ferritin iron, includes all the iron enzymes and those forms of iron involved in the dynamics of neuronal and glial metabolism. This compartment could remain at a relatively constant amount after brain development ceases, however, fluctuations in amounts of iron could take place as increased accumu- lation or turnover of specific iron molecules occurs in response to specific metabolic needs. The third compartment, ferritin iron, is likely the compartment of brain iron which increases with the increas- ing age of the animal. In the liver, ferritin iron in- creases with the age of the animal (see literature review). Also, Dallman et al.(1974), report that the brain tissue 154 from older rats has a higher percentage of ferritin than that found in the tissue of young rats. The accumulation of iron in ferritin is not necessarily a direct function of the immediate needs of the brain for it is not likely that the older rat brain requires more iron in order to function. Perhaps those parts of the brain which have the facility to store iron, and other metals (e.g. globus pallidus and substantia nigra) accumulate metals without a mechanism for regulating the amounts of metal taken up. Metals remain stored because there is no ready mechanism for their removal. Thus, the increase in iron in the brain with age can be explained during develoPment by the increases in myelin and nonémyelin non-ferritin iron. After the brain ceases to grow the ferritin iron compartment continues to increase as is seen in the liver. 155 Future Research Two main hypotheses developed from this study: (1) the distribution of iron is related to the metabolism of peptides, and (2) iron is involved in neuroendocrine regulation. Further research with a view to demonstrating the validity of these hypotheses is outlined below. A relationship of iron and peptides can be better understood by answering the following questions: - Do both iron and a peptide occur in the same structure? Do, for example, both iron and enkephalin occur in the same fibers within the globus pallidus and ventral pallidum? Do both iron and neurophysin occur in the same granules in the ventro-medial hypothalamus? - During the estrous cycle do the levels of enkephalin change, or does the activity of enkephalinase change with the fluctuations of iron concentration? Does an iron chelator (e.g. Desferal) in the globus pallidus effect the amount of enkephalin or the activity of enkephalinase? Does the inhibition of enkephalinase effect the concentra- tion of iron? A relationship of iron to neuroendocrine regulation would be better understood by answering the following questions: - Does iron concentration fluctuate during the estrous cycle in the medial preoptic area, organum vasculosum of the lamina terminalis, ventro-medial 156 hypothalamus and median eminence? - Does an iron chelator in the third ventricle, medial preoptic area or globus pallidus prevent or in any measur- able way effect ovulation? Also, a role for the proestrus rise in iron concent- ration in the globus pallidus in the ovulatory process could be demonstrated if : (l) electrochemical stimulation of the globus pallidus caused ovulation, or if (2) an iron chelator in the globus pallidus prevented ovulation. The techniques required to answer the above questions are presently available. 157 SUMMARY The results of the histochemical localization of iron in the rat brain and the spectrophotometric measurement of iron concentration in the pooled globus pallidus and sub- stantia nigra and the cortex as well as in samples of the serum and liver have demonstrated that: - Brain iron is unevenly distributed in the rat brain in a pattern similar to that found in the human brain. - Brain iron occurs in different structures in different parts of the brain. In the high-iron areas of globus pallidus, ventral pallidum, substantia nigra (reticulata) and dentate nucleus iron occurs in glial cells and fibers; in the circumventricular organs iron occurs in granules, tanycytes, fiber-like processes and amorphous accumulations. In lower iron areas such as the lateral septum, bed nucleus of the stria terminalis and cortex iron occurs in bouton-like structures on or in the perikarya of neurons. In the supraoptic, paraventricular and suprachiasmatic nuclei iron occurs as a fine dusting of grains within the perikarya of cells. - The distribution of iron in the rat brain correlates to some extent with the distribution of neuroactive peptides; in come cases, iron and a peptide appear to be localized in the same structure. 158 - Brain iron increases with age in the rat brain. - Brain iron fluctuates during the estrous cycle in the iron-rich areas, rising to the highest levels during proestrus. - Brain iron concentration increases during the first third of pregnancy and then falls but is not depleted by pregnancy. - There is a sex difference in iron content in the high-iron areas of the brain between intact males and females in proestrus. - Ovariectomy and castration have different effects on brain iron levels. 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