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Hormonal effects on the development of cerebral lateralization
Psychonem~endocrinalogy.Vol. 16. No. 1-3. pp. 121-129. 1991 Printed in Great Britain
0306-4530/91 $3.00 + 0.00 ©1991 Pergamon Press plc
H O R M O N A L EFFECTS O N THE DEVELOPMENT OF CEREBRAL LATERALIZATION MARIAN CLEEVES DIAMOND Department of Integrative Biology, University of California, Berkeley, California, U.S.A.
(Received 10 October 1989; in final form 13 April 1990)
SUMMARY The morphology of cerebral cortical laterality patterns differs between the sexes. In the male Long-Evans rat, the thickness of the cerebral cortex is, in general, greater on the fight side than on the left, with many areas showing statistically significant differences. In the female Long-Evans rat, the left side is thicker more often than the fight, but the differences, in general, are not statisticaily significant. These laterality patterns are maintained throughout the lifetime of the animal with few variations. Some of the male and female laterality patterns reverse with old age. The numbers of neurons and glial ceils in the area sampled, area 39, support the direction of cortical thickness measurements in male and female rats. Removal of the testes or ovaries at birth alters the usual cortical lateraiity patterns, illustrating that the sex steroid hormones play some role in determining laterality. In the neonates of both sexes, estrogen receptors are found in the cerebrai cortex, but the concentration is greater in the left male cortex than in the fight, the opposite being true for the female. Factors other than the sex steroid hormones, such as stress, can alter cortical laterality. Many studies indicate that plasticity of laterality is a factor to be considered when dealing with cortical morphology and, in turn, behavior.
INTRODUCTION "May knowledge of the brain provide people of all nations with greater tolerance, empathy, and appreciation of human behavior." IF THIS STATEMENT is s a t i s f a c t o r y f o r the p r e f a c e o f o u r " H u m a n B r a i n C o l o r i n g B o o k " (Diamond et al., 1985), then I think it might also serve as an appropriate introduction for an article on the role o f the sex steroid hormones on lateralization o f the cerebral hemispheres. One studies the similarities and differences in the structure o f male and female brains to gain a greater understanding o f the foundations of our complex behavior. By obtaining such knowledge we eventually will develop greater tolerance and more patience and respect, not only for those o f the opposite sex, but for those of the same sex. K n o w l e d g e o f b r a i n structure and function is one step toward peace, not only amongst nations in general, but also specifically between and within the sexes. Address correspondence and reprint requests to: Prof. Marian C. Diamond, 345 Mulford Hail, Department of Integrative Biology, University of California, Berkeley CA 94720, USA. 121
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Shakespeare has given us one lifetime sequence by defining the seven stages of man. Is there an equally suitable overall pattem which defines the various stages of woman? Perhaps her numerous life phases offer even greater complexity, because, with her menstrual cycle providing different ovarian hormonal levels daily throughout the month, her life stages might be considered monthly as well as during a complete lifetime. In other words, one discrete cycle of hormonal events continuously needs to fit into the larger developmental and aging circle of her total body, for neither process is independent of the other. This type of interaction typifies the complexity of problems involved in attempting to understand the role of hormones in establishing laterality in the cerebral cortex, that part of the brain which demonstrates such marked plasticity during a lifetime. Some of the many factors which need to be considered in studying laterality of the cortex are: age, gender, living conditions (including situations contributing to emotional status and stability), concentration of hormones (not just the sex hormones), and many others. Like most of life, the body is an ever-changing entity, responding constantly to its internal and extemal environment. A specific condition producing a discrete effect at one time may produce a quite different effect at another. Having thus hinted at the densely interconnected framework for the subject at hand, one now can begin to fill in some of the accumulated data which have become available as this field of brain laterality begins to unfold. CEREBRAL LATERALIZATION IN NORMAL MALE LONG-EVANS RATS In 1985 Robinson et al. presented a review table of examples of sex difference in neuroanatomical forebrain asymmetries in non-human mammals. They showed that sex differences in structural asymmetry were reported in three of the four studies existing at that time. In the same collection of review articles, Denenberg (1985) concluded that brain behavioral lateralization was a rather general property of all vertebrates, offering functional significance to the anatomical findings. However, observations in the literature on general cerebral cortical structural laterality were reported earlier. I am not aware of any reports on sex differences in rat cerebral cortical structural laterality before 1975. Prior to this time, for over a decade, we had been examining the effects of environmental conditions on the structure of the cerebral cortex, but we always pooled the data from the two hemispheres, assuming they were similar if not identical. In other investigations, we had been studying the normally developing and aging Long-Evans rat cortex, again combining the results from both hemispheres. However, with these latter data, we decided to separate the cortical thickness results from the fight and left hemispheres. Unexpectedly, we found that, in the male, Long-Evans rats, the right cerebral cortex was thicker than the left (Diamond et al., 1975). The areas measured included 10, 4, 3, 2, 18, 17, 18a, 39, according to Krieg's designations (Krieg, 1946). Out of 49 cortical areas measured, in male rats from 6 to 900 days of age, the differences, ranging on the average from 2% to 7%, between the thickness of the fight and left cortices were significant in 31 comparisons, or 63% (Diamond, 1988). Seventeen of the other cortical areas also showed right dominant asymmetry, but these differences were nonsignificant. Thus, 48 out of 49 areas (98%) measured in the male cortex of the Long-Evans strain were thicker on the fight side than on the left. These differences were present in the newborn and continued in varying degrees throughout the lifetime of the male rats, at least until 900 days of age, the last age we measured. However, by 900 days of age, none of the differences was significant. Is this lack of significant laterality in very old age responsible for some of the more docile, less aggressive, characteristics noted in aged males?
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The pattern of asymmetry was not uniform throughout the cortex. Some areas, such as 3 and 17, displayed strongly significant differences at every age (Diamond, 1988). This rat study was useful in that it inspired an investigation in humans which showed that the volume of area 17 was 5% greater in the right human cortex than in the left (Murphy, 1984). Though, in rats, areas 10, 3, 17 and 18 retained consistent, highly significant right-dominant patterns throughout most of life, area 2 showed no significant laterality differences during early life, i.e., 6, 14, 20, and 90 days of age. But, by 185 and 400 days of age, the right-left differences in area 2 became significant. Apparently the input to area 2 is symmetrical in each hemisphere until about 185 days of age, and then either the left hemisphere input decreases or the right increases as the right dominant asymmetrical pattern develops. The concentration of hormonal receptors in area 2 over the life span needs to be established and compared with other cortical areas. It also is necessary to consider some other factors that affect the development of the cortex, such as direct thalamic input or input from cortical areas on the same or opposite side. At present, it is not clear what factors might be causing these inputs to change at different ages in the separate hemispheres. Not only was the male right cortex thicker than the left in the Long-Evans strain of rats, but a similar pattern was found in the S1 strain, as well. This strain was developed in the University of California at Berkeley Psychology Department, where it was designated the "maze-bright" strain, in contrast to the S3s, the "maze-dull" strain. It is a pity that the $3 strain no longer exists (it was too expensive to maintain), for it would be intriguing to learn whether the asymmetry pattern was the same in the "maze-dull" as in the "maze-bright" animals. The greater thickness in the male right cortex compared to the left was supported by cell counts (McShane et al., 1988). Both neuron and glia counts indicated that more cells were present in the right cortex than in the left. Equal-sized samples of area 39 from right and left, male, cerebral cortical, thionine-stained, histological preparations were photographed to allow for neuron and glial counts on the same samples by two investigators. Final enlargement of the prints was 648×. The right sample had 10% (p<0.05) more neurons and 14% (p<0.05) more glial cells than the left. Thus, neurons and glial cell numbers account for some of the structural asymmetrical differences
Hormonal Factors Responsible for Male Cortical Asymmetry Several factors may account for cortical laterality. One is the role of the sex steroid hormones, previously mentioned by several investigators (e.g., Galaburda & Kemper, 1979; Denenberg, 1984; Geschwind & Behan, 1982). It is apparent that testosterone and stressrelated hormones are capable of altering cortical asymmetry in male rats. In an attempt to understand the role of testosterone in developing cortical asymmetry, we removed the testes on day 1 and measured cortical thickness at 90 days of age (Diamond, 1984). On histological sections of frontal, somatosensory and occipital cortices, in five out of seven areas measured (10, 4, 3, 2, 18, 17, 18a), the usual right-greater-than-left pattern of cortical dominance was reversed. Only areas 17 and 18a retained a significant right-greater-than-left pattern. With large samples of rats (N = 15 for intact males and N= 21 for gonadectomized males), these reversals in laterality were clearly evident, supporting the hypothesis that testosterone plays a role in determining cortical laterality in some cortical regions. It is known that, in order for testosterone to act on brain tissue, it is converted to estrogen by an aromatase enzyme. Though initially there was difficulty locating the aromatase enzyme in rat cortex, small amounts have been found in the cingulate cortex. Both male and female newborn rats possess estrogen receptors in their cortices for a least one month after birth
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(MacLusky et al., 1979a; 1979b). The concentration of these receptors is much less in later life. However, before we had this information, we administered estrogen (ethinylestradiol, 1 mg/kg) for 50 days, from 40 to 90 days of age, to day-1 ovariectomized rats and studied the effect on cortical thickness (Pappas et al., 1978). Areas 3, 4, 17, and 18 were significantly thinner ( 4 - 6 % ) in the rats receiving the estrogen, compared with sham-ovariectomized controls. These were adult rats, and estrogen receptors are found in greater concentration in the cerebral cortex of neonates. Nonetheless, these results helped form our hypothesis that the hemisphere with the greater concentration of estrogen receptors would be thinner than its counterpart. This prediction was supported by the study of Sandhu et al. (1968), with radioactive estradiol, showing that in the neonatal male rat (first postnatal month), the concentration of estrogen receptors was greater in the left cortex sample than in the fight. In a study of the role of estrogen in developing laterality, Lewis (1989) hypothesized that if testosterone is partially responsible for determining laterality in the male cerebral cortex, then blockade of the conversion of testosterone to estrogen might prevent the establishment of right dominance in the male cortex. He blocked aromatase action with ATD (1,4,6-androstatriene3,17-dione), which selectively inhibits aromatase during late prenatal and early postnatal life. ATD was given to pregnant females, and brain laterality patterns in the neonatal male pups were determined by examination of the size and structure of the cerebral cortex as it developed in the absence of active estrogen, as well as any effect ATD might have had on the female pups. In the third week of pregnancy, the females were given daily subcutaneous injections of ATD (5 mg in 0.1 ml sesame oil). At birth, the pups received 0.5 mg ATD in 0.1 ml of sesame oil. The intraperitoneal injections continued until postnatal day 14. The pups were weaned at 21 days of age and separated, three of like sex to each standard colony cage, until autopsy at postnatal day 41. The cerebral cortical laterality patterns were compared with age-matched, uninjected and sesame oil-injected controls. In the males, treatment with ATD caused a shift in laterality toward the left in seven of nine regions measured. This finding was particularly consistent and statistically significant in the somatosensory region (areas 4, 3, and 2) and in the hippocampus. The oil-treated males showed a smaller, statistically nonsignificant shift to the left. The ATD-treated females demonstrated a similar pattern favoring left laterality, generally not statistically significant and less robust than in the males. This study offers more consistent evidence in males of the role of testosterone in determining cerebral cortical laterality. Fleming et al. (1986) demonstrated that factors other than testosterone can alter cortical laterality. They showed that if the pregnant female was stressed during her last trimester (both temporarily confined and briefly exposed to increased temperature), her male pups no longer exhibited the dominant right cortical pattern. Behaviorally, these male pups later simulated female behavior during the sexual act by displaying lordosis and being submissive to other males. It also has been shown that prenatal stress reduces (feminizes) the size of the sexual dimorphic nucleus in the hypothalamus, an area directly involved in sexual behavior. Both these findings suggest that a biological basis for some forms of homosexuality may exist and underscore the importance of the relationship of brain structure with behavior. It is not clear what factors were involved in causing this reversal of cortical laterality in the male pups from stressed mothers. Fleming et al. (1986) reported that the left hemisphere increased in dimensions, and the right did not decrease. It has been reported that exposure of the fetus to an increase in temperature by 3°-4 ° C for 1 hr in the latter part of gestation will reduce fetal brain weight by 10%. These results suggest that it was not the exposure to the increased temperature that caused the laterality reversal but possibly the stress due to confinement. Could the maternal corticosterone released in response to stress be a determining factor?
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That stress might play a role in affecting cortical asymmetry was suggested by the study of Lin et al. (1987) who found that enriched and enriched/high population density environments altered cortical laterality pattems. The experimental conditions consisted of our usual enrichment design (12 rats per large cage, 70x70x46 cm plus "toys", changed twice weekly) and a high population density group (36 rats per large cage with the other conditions similar to the regular enrichment design). In area 17, primary visual cortex, the usual right dominance pattern was eliminated, and in area 3, a general sensory region, the usual right dominant pattern was reversed, the left cortex being significantly thicker than the right by 2% to 3%. Though these data do not speak to the mechanisms for these changes in laterality patterns, they illustrate the plasticity of such patterns under group living and crowded conditions. Other investigators attempting to elucidate the factors responsible for cortical laterality have turned to the adrenal gland. Adrenalectomized male and female rats show significant increases in overall brain dimensions and weight (Davenport, 1979; Meyer, 1983). Fisher (1989) showed that removal of the adrenals in male rats at 55 days of age caused an increase in cortical thickness and an alteration in brain laterality patterns in 85-day-old animals. The adrenalectomized animals (N= 9) showed a 3.5% to 7% (p <0.05) greater cortical thickness than that of the shamoperated animals (N = 12) in all nine areas of the cerebral cortex measured. There was an overall trend toward right hemispheric dominance for cortical thickness in both the adrenalectomized and sham-operated animals, with areas 17 and 18a showing a significant (/9<0.05) difference (Fig. 1). An exception to the right-greater-than-left pattern was found in the frontal cortex: The left side was greater than the right by 2% to 3% (Fig. 2). This reversal was of interest because of studies on the role of the cerebral cortex in goveming the immune system. Bardos et al. (1981) reported that the female mouse left cortex was more involved than the fight in certain immune functions and the right more in others. In earlier work, we found that both female mouse frontal lobes were smaller, as measured by cortical thickness, in the immunedeficient nude mouse, whereas many other cortical areas did not differ significantly from the control BALBc mouse (Diamond et al., 1986). In neonatally thymectomized male mice, the right cerebral cortex at 41 days of age shows a greater decrease in thickness than the left cortex (Gaufo, unpublished observations). All these findings are of interest as we begin to determine the interaction between the immune system and the cerebral cortex. Though this article is dealing primarily with sex hormones and cortical laterality patterns, it is intriguing to interweave other related factors as well, because all are interacting in one way or another. CEREBRAL LATERALIZATIONIN NORMAL FEMALE LONG-EVANS RATS In contrast to the male rats, in our studies the female Long-Evans rat had a thicker left cortex than the right in 45 of the 63 areas measured between 7 and 800 days of age (71%). But only three areas out of the 63 showed a statistically significant left-greater-than-right difference. Neuron and glial cell counts were taken from enlarged photographs of cortical samples of area 39 in both the right and left hemispheres from 90-day-old female rats (McShane et al., 1988). The cell counts per unit area supported the left-favored laterality demonstrated by the female cortical thickness measurements: The left area 39 cortical sample had more neurons by 13% (/9<0.05) and glial cells by 8% (NS) per unit area than did the right sample. Thus, both the male and female cortical cell counts supported the direction of the cortical thickness asymmetry differences in area 39. Different laterality pattems develop at different ages in the female, as well as they do in the male. In the female occipital cortical section, an intriguing finding appeared in our
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development and aging study. At 7 days of age, the right occipital cortical section was thicker than the left; this right-greater-than left pattern disappeared at 14, 21, and 90 days of age, only to return at 180, 390 and 800 days of age. In fact, area 39 became significantly different in favor of the right cortex at 390 days of age, and by 800 both areas 17 and 39 were significantly different. From these data, the female laterality pattern in the occipital cortex becomes more similar to that of the male as the animals aged. As far as cortical laterality is concerned, it appeared that males and females reverse patterns in old age. As with the males, the concentration of estrogen receptors was measured in the female left and right cerebral cortices for the first month after birth (Sandhu et al., 1986). As predicted, the estrogen receptors were greater in concentration in the right female cortex than in the left. The fact that both the male and female estrogen receptor concentrations are greater in the thinner cortex offers further support that estrogen plays some role in determining the initial different cerebral cortical laterality patterns between the sexes. Further evidence continued to support the role of estrogen in influencing asymmetry. If female rats are ovariectomized at birth and their right and left cerebral cortical thickness was measured at 90 days of age, the male laterality pattern in the occipital cortex was observed. In the intact female rats (N = 19-20), in seven areas out of nine, the left hemisphere was thicker than the right, though the differences were not significant (one comparison is equal). In the ovariectomized female rats (N= 18), in only two areas out of nine was the left hemisphere thicker than the right. But in the ovariectomized rats, areas 17, 18a and 39 all show the right cortex to be statistically significantly thicker than the left. Still further evidence has accumulated to indicate that ovarian hormones can affect cortical structure. If the ovaries are removed at birth and the cortical thickness is measured at 90 days of age and compared with that in sham-operated controls, the thickness is greater in the ovariectomized rat brains than in the controls (Pappas et al., 1978). Medosch and Diamond (1982) measured the number of discontinuous postsynaptic densities in these brains from ovariectomized rats and their sham-operated controls. Synapses in layer II of the right medial occipital cortex of the ovariectomized animals had significantly more split or discontinuous postsynaptic thickenings than the sham-operated controls. Greenough et al. (1978) showed that the relative frequency of perforations or discontinuities in the postsynaptic membrane increased markedly between 10 and 60 days of age. They also noted that rats reared in complex environments had postsynaptic thickenings with a significantly greater number of perforations than did rats raised in isolated conditions. Dyson and Jones (1988) also showed that the number of synapses with discontinuities in postsynaptic density increased with maturation. Since the rats without ovaries develop a thicker right occipital cortex with more synapses with a discontinuous postsynaptic thickening, it is possible that this cortex represents a more mature one than that in the sham-operated animal. With these results, it is important that the roles played by estrogen and progesterone in cortical development be clarified. Exogenous progesterone (2 mg/kg) given for 50 days (40 to 90 days of age) to rats ovariectomized on day 1 increased several areas of the cortex, whereas estrogen in the form of ethinylestradiol (1 p.g/kg), starting at day 40 and continuing until day 90, decreased cortical thickness (Pappas et al., 1979). Neither of these experiments covered the total time periods for the rats with the greatly reduced amount of ovarian hormones as experienced by ovariectomy at birth and studied 90 days later. But all these experiments indicate that sex steroid hormones play some role in developing and maintaining cortical morphology.
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SUMMARY To design experiments that would cover all the necessary variables to understand the precise role o f estrogen, progesterone, testosterone and other steroidal hormones on cortical asymmetry over an extended period of time will be a major undertaking. In the meantime, the status of the present results can be summarized. It is clear that the morphological and asymmetrical patterns in the c e r e b r a l cortex are different in the average m a l e and f e m a l e rat. N o t o n l y does the cortical thickness illustrate these differences, both both neuronal and glial cell counts do so as well. The cortical thickness patterns can be altered by changing the concentrations of the sex steroid hormones as well as other steroid hormones, for example those from the adrenal gland. Stress factors evidently play some role in cortical asymmetry. Not all areas o f the cortex have the same asymmetry pattems at a single age or over several ages. This is true for both males and females. It is both exciting and challenging to learn about the m a n y h o r m o n a l factors as well as others which alter cortical asymmetry. As I seem to conclude after every decade, look how far we have come, but in essence we have only just begun! However, I do find that in accumulating these data some greater tolerance, empathy, and appreciation for the behavior created by both halves of the brain has been provided. REFERENCES Bardos P, Degenne D, Lebranchu Y, Biziere K, Renoux G (1981) Neocortical lateralization of NK activity in mice. ScandJlmmuno113: 609-611. Davenport LD (1979) Adrenal modulation of brain size in adult rats. Behav Neural Bio127" 218-221. Denenberg VH (1984) Behavioral asymmetry. In: Geschwind N, Galaburda AM (Eds) Cerebral Dominance. Harvard, Cambridge MA, pp 114-133. Denenberg VH (1985) Hemispheric laterality, behavioral, asymmetry, and the effects of early experience in rats. In: Glick SD (Ed) Cerebral Lateralization in Nonhuman Species. Academic Press, New York, pp 110-131. Diamond MC (1984) Age, sex, and environmental influences. In: Geschwind N, Galaburda AM (Eds) Cerebral Dominance: The Biological Foundations. Harvard University Press, Cambridge MA, pp 134-146. Diamond MC (1988) Enriching Heredity: The Impact of the Environment on the Anatomy of the Brain. The Free Press, New York. Diamond MC, Johnson RE, Ingham CA (1975) Morphological changes in the young, adult and aging rat cerebral cortex, hippocampus and diencephalon. Behav Biol 14: 163-174. Diamond MC, Scheibel AB, Elson LM (1985) The Human Brain Coloring Book. Harper and Row, New York. Diamond MC, Rainbolt RD, Guzman R, Greet ER, Teitelbaum S (1986) Regional cerebral cortical deficits in the immune deficient nude mouse: a preliminary study. Exp Neuro192: 311-322. Dyson SE, Jones ~ (1980) Quantitation of terminal parameters and their interrelationships in maturing central synapses: a perspective for experimental studies. Brain Res 183: 43-59. Fisher E (1989) Effects of adrenalectomy on the rat forebrain: growth and asymmetry in cerebral cortex and hippocampus. University of California, Berkeley, Senior Thesis. Fleming BE, Anderson RH, Rhees RW, lOnghorn E, Bakaltis J (1986) Effects of prenatal stress on sexually dimorphic 21 asymmetries in the cerebral cortex of the rat. Brain Res Bull 16" 395-398. Galaburda AM, Kemper TL (1979) Cytoarchitectonic abnormalities in developmental dyslexia: a case study. Ann Neurol 6: 94-100. Geschwind N, Behan P (1982) Left-handedness: association with immune disease, migraine, and developmental learning disorder. Proc Natl Acad Sci USA 79: 5097-5100. Greenough WT, West RW, Devoogd JT (1978) Subsynaptic plate perforations: changes with age and experience in the rat. Science 202: 1096-1098. Krieg WJS (1946) Connections of the cerebral cortex. J Comp Neuro184: 221-275. Lewis D (1989) Sex differences in the cerebral cortex of the Long-Evans rat: the role of gonadal steroids during development. University of California, Berkeley, PhD Thesis.
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Lin JC, Greer ER, Diamond MC (1987) Altered patterns of cerebral cortical lateralization in male rats with increased levels of environmental complexity. Soc Neurosci Abs 13: 1594. MacLusky NJ, Lieberger I, McEwen BS (1979a) The development of estrogen receptor systems in the rat brain: perinatal development. Brain Res 178: 129-142. MacLusky NJ, Chaptal C, McEwen BS (1979b) The development of estrogen receptor systems in the rat brain: postnatal development. Brain Res 178: 143-160. McShane S, Glaser L, Greer ER, Houtz J, Tong MF, Diamond MC (1988) Cortical asymmetry - - neurons-glia, female-male: a preliminary study. Exp Neuro199: 353-361. Medosch CM, Diamond MC (1982) Rat occipital cortical synapses after ovariectomy. Exp Neurol 75: 120-133. Meyer JS (1983) Early adrenalectomy stimulates subsequent growth and development of the rat brain. Exp Neuro182: 432-446. Murphy G (1984) An evolutionary perspective on hemispheric asymmetry: the human striate cortex. University of California, Berkeley, PhD Thesis. Pappas CTE, Diamond MC, Johnson RE (1978) The effects of ovafiectomy and differential experience on rat cerebral cortical morphology. Brain Res 154: 53-60. Pappas CTE, Diamond MC, Johnson RE (1979) Morphological changes in the cerebral cortex of rats with altered levels of ovarian hormones. Behav Neurol Bio126: 298-310. Robinson TE, Becker JB, Camp DM, Mansour A (1985) Variation in the pattern of behavioral and brain asymmettles due to sex differences. In: Glick SD (Ed) Cerebral Lateralization in Nonhuman Species. Academic Press, New York, pp 185-226. Sandhu S, Cook P, Diamond MC (1986) Rat cerebral cortical estrogen receptors: male-female, fight-left. Exp Neuro192: 186-196.
0306-4530/91 $3.00 + 0.00 ©1991 Pergamon Press plc
H O R M O N A L EFFECTS O N THE DEVELOPMENT OF CEREBRAL LATERALIZATION MARIAN CLEEVES DIAMOND Department of Integrative Biology, University of California, Berkeley, California, U.S.A.
(Received 10 October 1989; in final form 13 April 1990)
SUMMARY The morphology of cerebral cortical laterality patterns differs between the sexes. In the male Long-Evans rat, the thickness of the cerebral cortex is, in general, greater on the fight side than on the left, with many areas showing statistically significant differences. In the female Long-Evans rat, the left side is thicker more often than the fight, but the differences, in general, are not statisticaily significant. These laterality patterns are maintained throughout the lifetime of the animal with few variations. Some of the male and female laterality patterns reverse with old age. The numbers of neurons and glial ceils in the area sampled, area 39, support the direction of cortical thickness measurements in male and female rats. Removal of the testes or ovaries at birth alters the usual cortical lateraiity patterns, illustrating that the sex steroid hormones play some role in determining laterality. In the neonates of both sexes, estrogen receptors are found in the cerebrai cortex, but the concentration is greater in the left male cortex than in the fight, the opposite being true for the female. Factors other than the sex steroid hormones, such as stress, can alter cortical laterality. Many studies indicate that plasticity of laterality is a factor to be considered when dealing with cortical morphology and, in turn, behavior.
INTRODUCTION "May knowledge of the brain provide people of all nations with greater tolerance, empathy, and appreciation of human behavior." IF THIS STATEMENT is s a t i s f a c t o r y f o r the p r e f a c e o f o u r " H u m a n B r a i n C o l o r i n g B o o k " (Diamond et al., 1985), then I think it might also serve as an appropriate introduction for an article on the role o f the sex steroid hormones on lateralization o f the cerebral hemispheres. One studies the similarities and differences in the structure o f male and female brains to gain a greater understanding o f the foundations of our complex behavior. By obtaining such knowledge we eventually will develop greater tolerance and more patience and respect, not only for those o f the opposite sex, but for those of the same sex. K n o w l e d g e o f b r a i n structure and function is one step toward peace, not only amongst nations in general, but also specifically between and within the sexes. Address correspondence and reprint requests to: Prof. Marian C. Diamond, 345 Mulford Hail, Department of Integrative Biology, University of California, Berkeley CA 94720, USA. 121
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Shakespeare has given us one lifetime sequence by defining the seven stages of man. Is there an equally suitable overall pattem which defines the various stages of woman? Perhaps her numerous life phases offer even greater complexity, because, with her menstrual cycle providing different ovarian hormonal levels daily throughout the month, her life stages might be considered monthly as well as during a complete lifetime. In other words, one discrete cycle of hormonal events continuously needs to fit into the larger developmental and aging circle of her total body, for neither process is independent of the other. This type of interaction typifies the complexity of problems involved in attempting to understand the role of hormones in establishing laterality in the cerebral cortex, that part of the brain which demonstrates such marked plasticity during a lifetime. Some of the many factors which need to be considered in studying laterality of the cortex are: age, gender, living conditions (including situations contributing to emotional status and stability), concentration of hormones (not just the sex hormones), and many others. Like most of life, the body is an ever-changing entity, responding constantly to its internal and extemal environment. A specific condition producing a discrete effect at one time may produce a quite different effect at another. Having thus hinted at the densely interconnected framework for the subject at hand, one now can begin to fill in some of the accumulated data which have become available as this field of brain laterality begins to unfold. CEREBRAL LATERALIZATION IN NORMAL MALE LONG-EVANS RATS In 1985 Robinson et al. presented a review table of examples of sex difference in neuroanatomical forebrain asymmetries in non-human mammals. They showed that sex differences in structural asymmetry were reported in three of the four studies existing at that time. In the same collection of review articles, Denenberg (1985) concluded that brain behavioral lateralization was a rather general property of all vertebrates, offering functional significance to the anatomical findings. However, observations in the literature on general cerebral cortical structural laterality were reported earlier. I am not aware of any reports on sex differences in rat cerebral cortical structural laterality before 1975. Prior to this time, for over a decade, we had been examining the effects of environmental conditions on the structure of the cerebral cortex, but we always pooled the data from the two hemispheres, assuming they were similar if not identical. In other investigations, we had been studying the normally developing and aging Long-Evans rat cortex, again combining the results from both hemispheres. However, with these latter data, we decided to separate the cortical thickness results from the fight and left hemispheres. Unexpectedly, we found that, in the male, Long-Evans rats, the right cerebral cortex was thicker than the left (Diamond et al., 1975). The areas measured included 10, 4, 3, 2, 18, 17, 18a, 39, according to Krieg's designations (Krieg, 1946). Out of 49 cortical areas measured, in male rats from 6 to 900 days of age, the differences, ranging on the average from 2% to 7%, between the thickness of the fight and left cortices were significant in 31 comparisons, or 63% (Diamond, 1988). Seventeen of the other cortical areas also showed right dominant asymmetry, but these differences were nonsignificant. Thus, 48 out of 49 areas (98%) measured in the male cortex of the Long-Evans strain were thicker on the fight side than on the left. These differences were present in the newborn and continued in varying degrees throughout the lifetime of the male rats, at least until 900 days of age, the last age we measured. However, by 900 days of age, none of the differences was significant. Is this lack of significant laterality in very old age responsible for some of the more docile, less aggressive, characteristics noted in aged males?
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The pattern of asymmetry was not uniform throughout the cortex. Some areas, such as 3 and 17, displayed strongly significant differences at every age (Diamond, 1988). This rat study was useful in that it inspired an investigation in humans which showed that the volume of area 17 was 5% greater in the right human cortex than in the left (Murphy, 1984). Though, in rats, areas 10, 3, 17 and 18 retained consistent, highly significant right-dominant patterns throughout most of life, area 2 showed no significant laterality differences during early life, i.e., 6, 14, 20, and 90 days of age. But, by 185 and 400 days of age, the right-left differences in area 2 became significant. Apparently the input to area 2 is symmetrical in each hemisphere until about 185 days of age, and then either the left hemisphere input decreases or the right increases as the right dominant asymmetrical pattern develops. The concentration of hormonal receptors in area 2 over the life span needs to be established and compared with other cortical areas. It also is necessary to consider some other factors that affect the development of the cortex, such as direct thalamic input or input from cortical areas on the same or opposite side. At present, it is not clear what factors might be causing these inputs to change at different ages in the separate hemispheres. Not only was the male right cortex thicker than the left in the Long-Evans strain of rats, but a similar pattern was found in the S1 strain, as well. This strain was developed in the University of California at Berkeley Psychology Department, where it was designated the "maze-bright" strain, in contrast to the S3s, the "maze-dull" strain. It is a pity that the $3 strain no longer exists (it was too expensive to maintain), for it would be intriguing to learn whether the asymmetry pattern was the same in the "maze-dull" as in the "maze-bright" animals. The greater thickness in the male right cortex compared to the left was supported by cell counts (McShane et al., 1988). Both neuron and glia counts indicated that more cells were present in the right cortex than in the left. Equal-sized samples of area 39 from right and left, male, cerebral cortical, thionine-stained, histological preparations were photographed to allow for neuron and glial counts on the same samples by two investigators. Final enlargement of the prints was 648×. The right sample had 10% (p<0.05) more neurons and 14% (p<0.05) more glial cells than the left. Thus, neurons and glial cell numbers account for some of the structural asymmetrical differences
Hormonal Factors Responsible for Male Cortical Asymmetry Several factors may account for cortical laterality. One is the role of the sex steroid hormones, previously mentioned by several investigators (e.g., Galaburda & Kemper, 1979; Denenberg, 1984; Geschwind & Behan, 1982). It is apparent that testosterone and stressrelated hormones are capable of altering cortical asymmetry in male rats. In an attempt to understand the role of testosterone in developing cortical asymmetry, we removed the testes on day 1 and measured cortical thickness at 90 days of age (Diamond, 1984). On histological sections of frontal, somatosensory and occipital cortices, in five out of seven areas measured (10, 4, 3, 2, 18, 17, 18a), the usual right-greater-than-left pattern of cortical dominance was reversed. Only areas 17 and 18a retained a significant right-greater-than-left pattern. With large samples of rats (N = 15 for intact males and N= 21 for gonadectomized males), these reversals in laterality were clearly evident, supporting the hypothesis that testosterone plays a role in determining cortical laterality in some cortical regions. It is known that, in order for testosterone to act on brain tissue, it is converted to estrogen by an aromatase enzyme. Though initially there was difficulty locating the aromatase enzyme in rat cortex, small amounts have been found in the cingulate cortex. Both male and female newborn rats possess estrogen receptors in their cortices for a least one month after birth
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(MacLusky et al., 1979a; 1979b). The concentration of these receptors is much less in later life. However, before we had this information, we administered estrogen (ethinylestradiol, 1 mg/kg) for 50 days, from 40 to 90 days of age, to day-1 ovariectomized rats and studied the effect on cortical thickness (Pappas et al., 1978). Areas 3, 4, 17, and 18 were significantly thinner ( 4 - 6 % ) in the rats receiving the estrogen, compared with sham-ovariectomized controls. These were adult rats, and estrogen receptors are found in greater concentration in the cerebral cortex of neonates. Nonetheless, these results helped form our hypothesis that the hemisphere with the greater concentration of estrogen receptors would be thinner than its counterpart. This prediction was supported by the study of Sandhu et al. (1968), with radioactive estradiol, showing that in the neonatal male rat (first postnatal month), the concentration of estrogen receptors was greater in the left cortex sample than in the fight. In a study of the role of estrogen in developing laterality, Lewis (1989) hypothesized that if testosterone is partially responsible for determining laterality in the male cerebral cortex, then blockade of the conversion of testosterone to estrogen might prevent the establishment of right dominance in the male cortex. He blocked aromatase action with ATD (1,4,6-androstatriene3,17-dione), which selectively inhibits aromatase during late prenatal and early postnatal life. ATD was given to pregnant females, and brain laterality patterns in the neonatal male pups were determined by examination of the size and structure of the cerebral cortex as it developed in the absence of active estrogen, as well as any effect ATD might have had on the female pups. In the third week of pregnancy, the females were given daily subcutaneous injections of ATD (5 mg in 0.1 ml sesame oil). At birth, the pups received 0.5 mg ATD in 0.1 ml of sesame oil. The intraperitoneal injections continued until postnatal day 14. The pups were weaned at 21 days of age and separated, three of like sex to each standard colony cage, until autopsy at postnatal day 41. The cerebral cortical laterality patterns were compared with age-matched, uninjected and sesame oil-injected controls. In the males, treatment with ATD caused a shift in laterality toward the left in seven of nine regions measured. This finding was particularly consistent and statistically significant in the somatosensory region (areas 4, 3, and 2) and in the hippocampus. The oil-treated males showed a smaller, statistically nonsignificant shift to the left. The ATD-treated females demonstrated a similar pattern favoring left laterality, generally not statistically significant and less robust than in the males. This study offers more consistent evidence in males of the role of testosterone in determining cerebral cortical laterality. Fleming et al. (1986) demonstrated that factors other than testosterone can alter cortical laterality. They showed that if the pregnant female was stressed during her last trimester (both temporarily confined and briefly exposed to increased temperature), her male pups no longer exhibited the dominant right cortical pattern. Behaviorally, these male pups later simulated female behavior during the sexual act by displaying lordosis and being submissive to other males. It also has been shown that prenatal stress reduces (feminizes) the size of the sexual dimorphic nucleus in the hypothalamus, an area directly involved in sexual behavior. Both these findings suggest that a biological basis for some forms of homosexuality may exist and underscore the importance of the relationship of brain structure with behavior. It is not clear what factors were involved in causing this reversal of cortical laterality in the male pups from stressed mothers. Fleming et al. (1986) reported that the left hemisphere increased in dimensions, and the right did not decrease. It has been reported that exposure of the fetus to an increase in temperature by 3°-4 ° C for 1 hr in the latter part of gestation will reduce fetal brain weight by 10%. These results suggest that it was not the exposure to the increased temperature that caused the laterality reversal but possibly the stress due to confinement. Could the maternal corticosterone released in response to stress be a determining factor?
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That stress might play a role in affecting cortical asymmetry was suggested by the study of Lin et al. (1987) who found that enriched and enriched/high population density environments altered cortical laterality pattems. The experimental conditions consisted of our usual enrichment design (12 rats per large cage, 70x70x46 cm plus "toys", changed twice weekly) and a high population density group (36 rats per large cage with the other conditions similar to the regular enrichment design). In area 17, primary visual cortex, the usual right dominance pattern was eliminated, and in area 3, a general sensory region, the usual right dominant pattern was reversed, the left cortex being significantly thicker than the right by 2% to 3%. Though these data do not speak to the mechanisms for these changes in laterality patterns, they illustrate the plasticity of such patterns under group living and crowded conditions. Other investigators attempting to elucidate the factors responsible for cortical laterality have turned to the adrenal gland. Adrenalectomized male and female rats show significant increases in overall brain dimensions and weight (Davenport, 1979; Meyer, 1983). Fisher (1989) showed that removal of the adrenals in male rats at 55 days of age caused an increase in cortical thickness and an alteration in brain laterality patterns in 85-day-old animals. The adrenalectomized animals (N= 9) showed a 3.5% to 7% (p <0.05) greater cortical thickness than that of the shamoperated animals (N = 12) in all nine areas of the cerebral cortex measured. There was an overall trend toward right hemispheric dominance for cortical thickness in both the adrenalectomized and sham-operated animals, with areas 17 and 18a showing a significant (/9<0.05) difference (Fig. 1). An exception to the right-greater-than-left pattern was found in the frontal cortex: The left side was greater than the right by 2% to 3% (Fig. 2). This reversal was of interest because of studies on the role of the cerebral cortex in goveming the immune system. Bardos et al. (1981) reported that the female mouse left cortex was more involved than the fight in certain immune functions and the right more in others. In earlier work, we found that both female mouse frontal lobes were smaller, as measured by cortical thickness, in the immunedeficient nude mouse, whereas many other cortical areas did not differ significantly from the control BALBc mouse (Diamond et al., 1986). In neonatally thymectomized male mice, the right cerebral cortex at 41 days of age shows a greater decrease in thickness than the left cortex (Gaufo, unpublished observations). All these findings are of interest as we begin to determine the interaction between the immune system and the cerebral cortex. Though this article is dealing primarily with sex hormones and cortical laterality patterns, it is intriguing to interweave other related factors as well, because all are interacting in one way or another. CEREBRAL LATERALIZATIONIN NORMAL FEMALE LONG-EVANS RATS In contrast to the male rats, in our studies the female Long-Evans rat had a thicker left cortex than the right in 45 of the 63 areas measured between 7 and 800 days of age (71%). But only three areas out of the 63 showed a statistically significant left-greater-than-right difference. Neuron and glial cell counts were taken from enlarged photographs of cortical samples of area 39 in both the right and left hemispheres from 90-day-old female rats (McShane et al., 1988). The cell counts per unit area supported the left-favored laterality demonstrated by the female cortical thickness measurements: The left area 39 cortical sample had more neurons by 13% (/9<0.05) and glial cells by 8% (NS) per unit area than did the right sample. Thus, both the male and female cortical cell counts supported the direction of the cortical thickness asymmetry differences in area 39. Different laterality pattems develop at different ages in the female, as well as they do in the male. In the female occipital cortical section, an intriguing finding appeared in our
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development and aging study. At 7 days of age, the right occipital cortical section was thicker than the left; this right-greater-than left pattern disappeared at 14, 21, and 90 days of age, only to return at 180, 390 and 800 days of age. In fact, area 39 became significantly different in favor of the right cortex at 390 days of age, and by 800 both areas 17 and 39 were significantly different. From these data, the female laterality pattern in the occipital cortex becomes more similar to that of the male as the animals aged. As far as cortical laterality is concerned, it appeared that males and females reverse patterns in old age. As with the males, the concentration of estrogen receptors was measured in the female left and right cerebral cortices for the first month after birth (Sandhu et al., 1986). As predicted, the estrogen receptors were greater in concentration in the right female cortex than in the left. The fact that both the male and female estrogen receptor concentrations are greater in the thinner cortex offers further support that estrogen plays some role in determining the initial different cerebral cortical laterality patterns between the sexes. Further evidence continued to support the role of estrogen in influencing asymmetry. If female rats are ovariectomized at birth and their right and left cerebral cortical thickness was measured at 90 days of age, the male laterality pattern in the occipital cortex was observed. In the intact female rats (N = 19-20), in seven areas out of nine, the left hemisphere was thicker than the right, though the differences were not significant (one comparison is equal). In the ovariectomized female rats (N= 18), in only two areas out of nine was the left hemisphere thicker than the right. But in the ovariectomized rats, areas 17, 18a and 39 all show the right cortex to be statistically significantly thicker than the left. Still further evidence has accumulated to indicate that ovarian hormones can affect cortical structure. If the ovaries are removed at birth and the cortical thickness is measured at 90 days of age and compared with that in sham-operated controls, the thickness is greater in the ovariectomized rat brains than in the controls (Pappas et al., 1978). Medosch and Diamond (1982) measured the number of discontinuous postsynaptic densities in these brains from ovariectomized rats and their sham-operated controls. Synapses in layer II of the right medial occipital cortex of the ovariectomized animals had significantly more split or discontinuous postsynaptic thickenings than the sham-operated controls. Greenough et al. (1978) showed that the relative frequency of perforations or discontinuities in the postsynaptic membrane increased markedly between 10 and 60 days of age. They also noted that rats reared in complex environments had postsynaptic thickenings with a significantly greater number of perforations than did rats raised in isolated conditions. Dyson and Jones (1988) also showed that the number of synapses with discontinuities in postsynaptic density increased with maturation. Since the rats without ovaries develop a thicker right occipital cortex with more synapses with a discontinuous postsynaptic thickening, it is possible that this cortex represents a more mature one than that in the sham-operated animal. With these results, it is important that the roles played by estrogen and progesterone in cortical development be clarified. Exogenous progesterone (2 mg/kg) given for 50 days (40 to 90 days of age) to rats ovariectomized on day 1 increased several areas of the cortex, whereas estrogen in the form of ethinylestradiol (1 p.g/kg), starting at day 40 and continuing until day 90, decreased cortical thickness (Pappas et al., 1979). Neither of these experiments covered the total time periods for the rats with the greatly reduced amount of ovarian hormones as experienced by ovariectomy at birth and studied 90 days later. But all these experiments indicate that sex steroid hormones play some role in developing and maintaining cortical morphology.
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SUMMARY To design experiments that would cover all the necessary variables to understand the precise role o f estrogen, progesterone, testosterone and other steroidal hormones on cortical asymmetry over an extended period of time will be a major undertaking. In the meantime, the status of the present results can be summarized. It is clear that the morphological and asymmetrical patterns in the c e r e b r a l cortex are different in the average m a l e and f e m a l e rat. N o t o n l y does the cortical thickness illustrate these differences, both both neuronal and glial cell counts do so as well. The cortical thickness patterns can be altered by changing the concentrations of the sex steroid hormones as well as other steroid hormones, for example those from the adrenal gland. Stress factors evidently play some role in cortical asymmetry. Not all areas o f the cortex have the same asymmetry pattems at a single age or over several ages. This is true for both males and females. It is both exciting and challenging to learn about the m a n y h o r m o n a l factors as well as others which alter cortical asymmetry. As I seem to conclude after every decade, look how far we have come, but in essence we have only just begun! However, I do find that in accumulating these data some greater tolerance, empathy, and appreciation for the behavior created by both halves of the brain has been provided. REFERENCES Bardos P, Degenne D, Lebranchu Y, Biziere K, Renoux G (1981) Neocortical lateralization of NK activity in mice. ScandJlmmuno113: 609-611. Davenport LD (1979) Adrenal modulation of brain size in adult rats. Behav Neural Bio127" 218-221. Denenberg VH (1984) Behavioral asymmetry. In: Geschwind N, Galaburda AM (Eds) Cerebral Dominance. Harvard, Cambridge MA, pp 114-133. Denenberg VH (1985) Hemispheric laterality, behavioral, asymmetry, and the effects of early experience in rats. In: Glick SD (Ed) Cerebral Lateralization in Nonhuman Species. Academic Press, New York, pp 110-131. Diamond MC (1984) Age, sex, and environmental influences. In: Geschwind N, Galaburda AM (Eds) Cerebral Dominance: The Biological Foundations. Harvard University Press, Cambridge MA, pp 134-146. Diamond MC (1988) Enriching Heredity: The Impact of the Environment on the Anatomy of the Brain. The Free Press, New York. Diamond MC, Johnson RE, Ingham CA (1975) Morphological changes in the young, adult and aging rat cerebral cortex, hippocampus and diencephalon. Behav Biol 14: 163-174. Diamond MC, Scheibel AB, Elson LM (1985) The Human Brain Coloring Book. Harper and Row, New York. Diamond MC, Rainbolt RD, Guzman R, Greet ER, Teitelbaum S (1986) Regional cerebral cortical deficits in the immune deficient nude mouse: a preliminary study. Exp Neuro192: 311-322. Dyson SE, Jones ~ (1980) Quantitation of terminal parameters and their interrelationships in maturing central synapses: a perspective for experimental studies. Brain Res 183: 43-59. Fisher E (1989) Effects of adrenalectomy on the rat forebrain: growth and asymmetry in cerebral cortex and hippocampus. University of California, Berkeley, Senior Thesis. Fleming BE, Anderson RH, Rhees RW, lOnghorn E, Bakaltis J (1986) Effects of prenatal stress on sexually dimorphic 21 asymmetries in the cerebral cortex of the rat. Brain Res Bull 16" 395-398. Galaburda AM, Kemper TL (1979) Cytoarchitectonic abnormalities in developmental dyslexia: a case study. Ann Neurol 6: 94-100. Geschwind N, Behan P (1982) Left-handedness: association with immune disease, migraine, and developmental learning disorder. Proc Natl Acad Sci USA 79: 5097-5100. Greenough WT, West RW, Devoogd JT (1978) Subsynaptic plate perforations: changes with age and experience in the rat. Science 202: 1096-1098. Krieg WJS (1946) Connections of the cerebral cortex. J Comp Neuro184: 221-275. Lewis D (1989) Sex differences in the cerebral cortex of the Long-Evans rat: the role of gonadal steroids during development. University of California, Berkeley, PhD Thesis.
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Lin JC, Greer ER, Diamond MC (1987) Altered patterns of cerebral cortical lateralization in male rats with increased levels of environmental complexity. Soc Neurosci Abs 13: 1594. MacLusky NJ, Lieberger I, McEwen BS (1979a) The development of estrogen receptor systems in the rat brain: perinatal development. Brain Res 178: 129-142. MacLusky NJ, Chaptal C, McEwen BS (1979b) The development of estrogen receptor systems in the rat brain: postnatal development. Brain Res 178: 143-160. McShane S, Glaser L, Greer ER, Houtz J, Tong MF, Diamond MC (1988) Cortical asymmetry - - neurons-glia, female-male: a preliminary study. Exp Neuro199: 353-361. Medosch CM, Diamond MC (1982) Rat occipital cortical synapses after ovariectomy. Exp Neurol 75: 120-133. Meyer JS (1983) Early adrenalectomy stimulates subsequent growth and development of the rat brain. Exp Neuro182: 432-446. Murphy G (1984) An evolutionary perspective on hemispheric asymmetry: the human striate cortex. University of California, Berkeley, PhD Thesis. Pappas CTE, Diamond MC, Johnson RE (1978) The effects of ovafiectomy and differential experience on rat cerebral cortical morphology. Brain Res 154: 53-60. Pappas CTE, Diamond MC, Johnson RE (1979) Morphological changes in the cerebral cortex of rats with altered levels of ovarian hormones. Behav Neurol Bio126: 298-310. Robinson TE, Becker JB, Camp DM, Mansour A (1985) Variation in the pattern of behavioral and brain asymmettles due to sex differences. In: Glick SD (Ed) Cerebral Lateralization in Nonhuman Species. Academic Press, New York, pp 185-226. Sandhu S, Cook P, Diamond MC (1986) Rat cerebral cortical estrogen receptors: male-female, fight-left. Exp Neuro192: 186-196.