IIHIIINHIIHIHHIHIIHIflIWIWWIllllllHlHlHllWl “”3246 We LIBRARY f‘ a n State .1 Universlty M iCh f. *- This is to certify that the dissertation entitled Gonadal hormone mediation of neural plasticity in the adult rodent amygdala presented by John A. Morris has been accepted towards fulfillment of the requirements for the Doctoral degree in Neuroscience MajorPTofessor’s Signature 5 a $1949 5:. J Date PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5108 K;IProj/Acc&Pres/CIRCIDateDue.indd GONADAL HORMONE MEDIATION OF NEURAL PLASTICITY IN THE ADULT RODENT AMYGDALA By John A. Morris A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Neuroscience Program 2008 ABSTRACT GONADAL HORMONE MEDIATION OF NEURAL PLASTICITY IN THE ADULT RODENT AMYGDALA By John A. Morris That behaviors differ by sex seems obvious, but how structures differ in the brain areas underlying these behaviors is not easily perceived. One factor driving these differences however is clear--testosterone, the gonadal steroid hormone, appears to be involved in most known sex differences in vertebrates. Testosterone may shape the nervous system quickly or over long periods of time, from the level of the synapse and the membrane up to the circuit level, often by influencing cell death or preservation during development, but also by continuing such influences, to some extent, into adulthood in the form of cellular remodeling, or plasticity. This must be true because androgens and their metabolites can act to change behavior across the lifespan. As changes in structure underlie function, effects that appear consistent by some measures, yet variable by others, indicate that closer inspection and comparison of hormonal effects on brain structures is needed to infer and predict how hormones relate to the nervous system. This dissertation found several cellular aspects of the brain changed following the manipulation of adult circulating gonadal hormones, including soma size, regional volume, number of glia, and rostrocaudal extent in a brain nucleus, the posterodorsal medial amygdala (MePD), that receives olfactory and pheromonal information and is known to play a role in reproductive behaviors. Our findings showed first that somal size and regional volume are reduced in males with dysfunctional androgen receptors (ARs), but that the specific effects depend on the nucleus and the nature of cellular morphology being considered. Based on these findings, it seems reasonable to conclude that although estrogen receptors affect these characteristics, AR effects cannot be ignored. Secondly, the number of neurons and glia in the MePD was greater in males than in females. While neuron number was unaffected by androgen manipulations, glial number was affected, mostly by increasing in the androgen treated females. Our experiments followed the time course of morphological change in the MePD, showing again that androgen effects vary by sex, and the structural features are being analyzed, as somal size changing in females faster than in males. Lastly, we found that the mouse MePD, like the rat, is sexually dimorphic in regard to volume and mean somal area. Also, the mouse MePD could be maintained by E or T, even when deprived of a primary input following bulbectomy. Taken together, these experiments show differences in hormone plasticity in adulthood are influenced by many variables including sex, hormone receptor type, brain nuclei, cell type, time, and even hemisphere; yet effects may still be generalized to some extent across species (male biased dimorphisms, equivalent somal size response in the two hemispheres). I interpret these findings as a clear demonstration that hormones precisely regulate many aspects of neural structure. Given the widespread administration of hormones to adult humans, this dissertation research may contribute to the understanding of behaviors correlated with neuro-structural changes, hormone mediated disease ontology, and hormone based therapies and treatments. DEDICATION AND ACKNOWLEDGMENTS Dedicated in memory of: Hazel Tigert, Connie Osborn, Jose de Olmos, Leonard Heimer, and Wally Welker. Acknowledgements I like to thank these instructors for imbuing me with these concepts Jayne Morris and Bob Switzer for Inspiration Andrew Morris for Encouragement Cheryl Sisk for The Bench Keith Lookingland for Persistence John I. Johnson for Connectivity Cynthia Jordan for Precision Marc Breedlove for Accuracy Lynwood Clemens for Healthy Skepticism Rebecca Watson for Love and Patience Special Thanks Candace Flynn et al., for animal care Everyone in the lab(s), good times... TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................... vii LIST OF TABLES ........................................................................................................ xii KEY TO ABBREVIATIONS ...................................................................................... xiii CHAPTER ONE: INTRODUCTION ......................................................................... 1 CHAPTER TWO: PARTIAL DEMASCULINIZATION OF SEVERAL BRAIN REGIONS IN THE ADULT MALE (XY) RATS WITH A DYSFU NCTION AL ANDROGEN RECEPTOR GENE Rationale ............................................................................................................. 9 Methods ............................................................................................................... 12 Results ................................................................................................................. 16 Discussion ........................................................................................................... 21 Appendix H ......................................................................................................... 28 CHAPTER THREE: SEXUAL DIMORPHISM IN N EURONAL NUMBER OF THE POSTERODORSAL MEDIAL AMYGDALA IS INDEPENDENT OF CIRCULATING ANDROGENS AND REGIONAL VOLUME IN ADULT RATS Rationale ............................................................................................................. 37 Methods ............................................................................................................... 40 Results ................................................................................................................. 46 Discussion ........................................................................................................... 50 Appendix III ........................................................................................................ 56 CHAPTER FOUR: TIME COURSE OF GONADAL HORMONE MEDIATED PLASTICITY IN THE ADULT RAT MEDIAL AMYGDALA Rationale ............................................................................................................. 62 Methods ............................................................................................................... 64 Results ................................................................................................................. 68 Discussion ........................................................................................................... 72 Appendix IV ........................................................................................................ 76 CHAPTER FIVE: SEXUAL DIMORPHISM AND STEROID RESPONSIVENESS OF THE POSTERODORSAL MEDIAL AMYGDALA IN ADULT MICE Rationale ............................................................................................................. 81 Methods ............................................................................................................... 83 Results ................................................................................................................. 88 Discussion ........................................................................................................... 92 Appendix V ......................................................................................................... 95 CHAPTER SIX: GENERAL SUMMARY AND CONCLUSIONS .......................................................... 100 BIBLIOGRAPHY ......................................................................................................... 109 vi. LIST OF FIGURES Figure 11-1. The posterodorsal medial amygdala (MePD) as revealed by Nissl stain of coronal sections from a wildtype male (top), a tfm male with a dysfunctional androgen receptor (middle), and a female rat (b_ott0m). The panels on the left are from the caudal- most appearance of the MePD, which served as an anchor point to assess changes in the nucleus across the rostrocaudal dimension. The appearance of the MePD, as well as the optic tract (0t), stria terminalis (st), the anterolateral part of the amygdalohippocampal transition area (AHiAL), and the lateral ventricle (v) are equivalent in wildtype males (a), tfm males (b) and females (c), indicating that the caudal termination of the MePD occurs in the homologous region of the brain across groups. The panels on the right are from the approximate middle of the rostrocaudal extent of the MePD where the nucleus is larger in wildtype males ((1) than in females (0, and is intermediate in size in genetic males with a dysfunctional androgen receptor (e). The MePD extends farther rostrally in wildtype males and tfm males than in females (see text). Scale bars = 250 um. ......................................................................................................................................... 30 Figure 11-2. The volume of the sexually dimorphic nucleus of the preoptic area (SDN- POA) as revealed by Nissl stained coronal sections is larger in a wildtype male (a) than in a wildtype female rat (c). SDN-POA volume in a tfm rat, a genetic male with a dysfunctional androgen receptor (b), is masculine: larger than in females and equivalent to that of males. The sexual dimorphism in the volume of the suprachiasmatic nucleus (SCN), while more subtle than in the SDN—POA, again reflects a larger nucleus in wildtype males (top) than in females (bottom), but the SCN volume in tfm males (middle) is feminine: less than that of wildtype males and equivalent to that of females. Scale bar = 250 um. ......................................................................................................................................... 31 Figure 11-3. MePD regional volume Maud soma size (b_ottom) in adult tfm males, and wildtype male and female litterrnate controls. MePD volume is significantly greater in the right than in the left hemisphere in wildtype males (p<0.03) and androgen—insensitive tfm males (p< 0.03), but not in females (p=0.55). On each side of the brain, all three groups were significantly different from one another, with values for {firm intermediate between those of males and females (ps <0.001). There is also a robust sex difference in MePD soma size (p<0.0001) with wildtype males having larger somata than do females. Tfm males have significantly smaller somata than wildtype male littermates (p<0.03), but significantly larger somata than female littermates (p<0.005). There was no laterality of MePD soma size in any of the groups. All error bars represent the standard errors of the means (SEM). ......................................................................................................................................... 32 Figure 11-4. Mean (:SEM)1'ostrocaudal extent of the MePD was estimated by counting the number of sections sampled for each animal that contain this region. MePD extent is significantly greater on the right than on the left hemisphere in wildtype males (p< 0.005) and androgen-insensitive tfin males (p< 0.007), but not in females (p: 0.55). Wildtype vii males have a significantly longer extent than did females on each side (L, p < 0.02; R, p< 0.0003). The tfm males are as masculine as wildtype males in this measure. ......................................................................................................................................... 33 Figure 11-5. Rostrocaudal extent of MePD in the right _(t_ob) and left hemispheres (bbttom) of androgen-insensitive rfm males and wildtype male and female littermates binned by average area per section (:SEM). The sex differences in MePD are found primarily in the middle and rostral end of the nucleus. Tfin males resemble females in some regions, are indistinguishable from males in caudal-most and rostral-most regions, and have volumes intermediate between males and females in others. . ........................................................................................................................................ 34 Figure 11-6. Total SDN-POA volume (t_op_)_and mean cross-sectional area of neuronal somata (bottom) in adult tfm males and wildtype male and female littermates. SDN-POA volume is not significantly different between wildtype and tfm males, but both groups displayed a greater volume than did females (p < 0.0001). In contrast, tfin males have smaller somata than wildtype males, indicating a role for AR in maintaining SDN-POA soma size in male rats. However, there is no significant sex difference in the size of SDN-POA somata (p: 0.16), nor is soma size in tfm males different from that in females (p: 0.31). ......................................................................................................................................... 35 Figure 11-7. Total volume (t_ob)_and mean cross sectional area of neuronal somata (bottom) in SCN of androgen-insensitive tfm affected males, wildtype males, and female littermates. SCN volume was significantly larger in wildtype males than in control females (p<0.01) with tfm males having the same size SCN as female controls (p=0.55). SCN soma area is greater in wildtype males compared to tfm males, but there is no significant sex difference in this measure (p: 0.23). SCN soma size did not differ between tfm males and females (p: 0.16). ......................................................................................................................................... 36 Figure III-l. Boundaries of the rat posterodorsal medial amygdala (MePD). Photomicrographs of coronal sections of the MePD in an adult, gonadally intact male rat (left column) and an adult, ovariectomized female rat without androgen treatment (right column). The caudalmost portion of the MePD appears as a capsule ventral to the optic tract (0t) surrounded by the relatively soma-sparse area of the stria terminalis (st), at a rostrocaudal level intersecting the lateral ventricle (v) and the anterolateral part of the amygdalohippocarnpal transition area (AHiAL). At this extreme end, the MePD is of a similar size in males (a) and females ((1). More rostrally, the MePD in males (b) has a triangular or wedge-shaped pro.le, with a slight reduction in cell density in the center, which gives the appearance of an inverted letter “v.” In females (e), the nucleus is considerably smaller and has a less cuneate appearance. The rostral end of the MePD is viii still adjacent to the ventral-lateral aspect of the ot and is more prominent in males (c) than in females (f). In both sexes, the ovoid intercalated nucleus of the amygdala is visible to the right of the MePD at this level. Scale bar = 250 um. ......................................................................................................................................... 58 Figure III-2. Classification of cell types. Typical cells viewed with a _100 objective in a sham male adult rat MePD as revealed by thionin stain for Nissl. Arrow indicates a neuron; black arrowheads indicate glia (oligodendrocyte or astrocyte); white arrowhead indicates a microglia. Scale bar = 10 um. ......................................................................................................................................... 59 Figure III-3. Androgen regulates medial amygdala volume and soma size in adult rats. A) As seen in previous studies, manipulations of androgen in adult rats alter the regional volume of the posterodorsal medial amygdala (MePD). Males were either castrated (low androgen) or subjected to sham surgery (high androgen), while females were ovariectomized and given capsules containing either testosterone (high androgen) or nothing (low androgen). Three way AN OVA revealed a sex difference (males > females; main effect of sex p < 0.0001), asymmetry (right > left; main effect of hemisphere; p < 0.005) and a response to androgen manipulation (main effect of hormone; p < 0.0003), with no significant interactions. B) Neuronal soma size in the rat MePD also responded to androgen manipulations (main effect of androgen; p = 0.0005), but there was no evidence of asymmetry (p > 0.2). While there was not a significant sex difference overall, there was an interaction of sex and androgen status (p = 0.02) because soma size was more responsive in females than in males. The previously reported sex difference in MePD soma size in gonadally intact animals is represented here by the larger somata in sham males sham males compared with females receiving no androgen. ......................................................................................................................................... 60 Figure III-4. Androgen has no effect on neuronal number in the adult rat MePD. A) Males have more MePD neurons than females (main effect of sex; p < 0.002), and there are more neurons in the left MePD than the right (main effect of hemisphere; p < 0.0001), but there was no effect of androgen manipulations in either sex or in either hemisphere (no significant interactions; ps > 0.14). B) Males also have more glial cells in the MePD than do females (main effect of sex; p < 0.003), and there are more glia in the right MePD than the left (main effect of hemisphere; p < 0.001). However, there was an interaction of androgen status and hemisphere (p < 0.03). Subsequent separate analyses of the left and right MePD revealed that androgen is associated with more glia in both sexes, but only in the right MePD. ......................................................................................................................................... 61 Figure IV -1. Mean regional volume (i SEM) of the posterodorsal medial amygdala (MePD) in response to androgen manipulations in adult rats. Top) Male rats that are castrated as adults show a significant shrinkage in MePD volume only in the right hemisphere and only 28 days following surgery, not 2 or 14 days after surgery. Bottom) Female rats that are ovariectomized (OvX) as adults and given either testosterone (T) or blank capsules also display an MePD volume response to androgen treatment only in those animals sacrificed 28 days later. However, in female rats MePD regional volume responsiveness to androgen appears equivalent in the two hemispheres. ......................................................................................................................................... 79 Figure IV-2. Figure IV -2. Mean neuronal somata size (i SEM) in the posterodorsal medial amygdala (MePD) in response to androgen manipulations in adult rats. As in past studies, we found no laterality in the soma size of MePD neurons in any group, so mean bilateral size is displayed. Top) MePD somata size showed no response to castration 2 days following surgery. Fourteen days after surgery, MePD somata were not significantly smaller in castrated males than males subjected to sham surgery. However, variance in this measure was significantly greater in castrates than sham males at this time point (note error bars), suggesting that a subset of males may have begun showing a response to surgery, increasing variability at this time. As in past studies, MePD somata are significantly smaller in castrate males than in sham males 28 days after surgery. Bottom) MePD soma size was significantly larger in ovariectomized females given testosterone than those given blank capsules both 14 and 28 days after treatment commenced. There was no discernible difference between groups examined 2 days after treatment began. .. .................................................... . ................................................................................... 80 Figure V-l. Mean size of neuronal somata in the MePD of BALB/c mice. ANOVA revealed no significant laterality of neuronal soma size in any group of animals, so soma sizes averaged across the two hemispheres are depicted here. Comparison of mice subjected to sham surgery revealed MePD somata to be larger in males than in females (p < 0.0001). Castration reduced MePD somata in males (p < 0.0001), while estrogen (E) treatment of ovariectomized (OvX) females enlarged somata size (p < 0.002). Olfactory bulbectomy (OBX) had no effect on MePD soma size in OvX females. In males, ANOVA revealed no significant main effect of surgery or hemisphere, but a marginally significant interaction (p < 0.05). Post-hoe tests indicated MePD soma size was slightly smaller in OBX males than sham males (p < 0.04) in the right hemisphere only ......................................................................................................................................... 97 Figure V-2. The volume of the posterodorsal medial amygdala (MePD) in BALB/c mice. ANOVA of sham-gonadectornized mice revealed that MePD volume is greater in males than in females (p < 0.0001) and larger in the left hemisphere than the right (p < 0.01). Post-hoc repeated-measures t-tests revealed the asymmetry was statistically significant in females (p < 0.01), but not in males (p > 0.4). Castration of males reduced MePD volume (p < 0.0003) equally in both hemispheres (interaction p > 0.3). Estrogen (E) treatment of ovariectomized (OvX) females marginally increased MePD volume (p = 0.06, two-tailed). Olfactory bulbectomy (OBX) had no discernible effect on MePD volume in either sex. ......................................................................................................................................... 98 Figure V-3. The posterodorsal medial amygdala (MePD) in BALB/c mice. In these Nissl-stained coronal sections taken approximately in the middle of the rostrocaudal extent of the MePD, the wedge-shaped MePD abuts the ventrolateral margin of the cell- body sparse optic tract (running from lower left corner of photograph to upper right) and is more slender in female (upper) than male mice (lower), resulting in sexual dimorphism in overall volume of the nucleus. ......................................................................................................................................... 99 xi LIST OF TABLES Table 11-] Body weight and anogenital distance of subjects. ......................................................................................................................................... 29 Table III-1 Average weights of the body, preputial glands, seminal vesicles and bulbocavernosus/levator ani muscles (BC/LA) for each group i standard errors of the means. ......................................................................................................................................... 57 Table IV-l Responses to androgen manipulations. ......................................................................................................................................... 77 Table IV-2 Rostrocaudal extent, as measured by the average number of sections i standard errors of the means (N = number of animals) containing the MePD, in each group. ......................................................................................................................................... 78 Table V-l. Rostrocaudal extent of the posterodorsal medial amygdala (MePD) in mice. ......................................................................................................................................... 96 xii AHiAL AR BC/LA DHT ER E or E2 MEA MePD OVX Ot SDN-POA SCN St- T Tfm KEY TO ABBREVIATIONS Amygdalohippocampal transition area Androgen receptor Bulbocavernosus/levator ani muscles Dihydrotestosterone Estrogen receptor Estrogen Medial amygdala Posterodorsal medial amygdala Ovariectomy or Ovariectomized Optic tract Sexually dimorphic nucleus of the preoptic area Suprachiasmatic nucleus Stria terminalis Testosterone Testicular feminization mutation xiii CHAPTER ONE INTRODUCTION That behaviors differ by sex seems obvious, but how structures differ in the brain areas underlying these behaviors is not easily perceived. One factor driving these differences however is clear--testosterone, the gonadal steroid hormone, appears to be involved in most known sex differences in vertebrates. Testosterone may shape the nervous system quickly or over long periods of time, from the level of the synapse and the membrane up to the circuit level, often by influencing cell death or preservation during development, but also by continuing such influences, to some extent, into adulthood in the form of cellular remodeling, or plasticity (Morris et al., 2004). This must be true because androgens and their metabolites can act to change behavior across the lifespan. As changes in structure underlie function, effects that appear consistent by some measures, yet variable by others, indicate that closer inspection and comparison of hormonal effects on brain structures is needed to infer and predict how hormones relate to the nervous system. Following a brief summary on the background of hormonal influence on neural structure and function, I will introduce the series of experiments I conducted that focused on hormone-mediated structural changes in the adult rodent brain. One reason that sex differences came to be a model for exploring structure/function relationships is that the same variables, gonadal hormones, that cause changes in behaviors will create concomitant changes in brain structure. These variations occur naturally or may be easily manipulated with straightforward surgeries of orchidectomy and/or hormone implantation. Sexual behavior itself, probably the most robustly dimorphic behavior between the sexes, is studied because it serves as such a convenient model, with a hormone sensitive suite of stereotypic behavioral patterns that are easily manipulated and measured (Meisel & Sachs, 1994). So as lesion studies began to implicate the medial basal hypothalamus and preoptic area as a critical structural locus for sexual behavior in males and females (Larsson & Heimer, 1964), other studies showed that developmental hormone exposure during critical periods could organize the systems responsible for behavioral differences (Phoenix et al., 1959). The degree and extent of hormonal effects that tied neural structure to function would depend in some part on the tools available, but also on knowing when, and where, to look. One seminal paper established the theoretical framework for understanding when hormones affect the brain. The “organizational/activational hypothesis” was derived from studies looking at the influence of steroid hormones on guinea pig behaviors, which suggested there are two ways in which steroid hormones might act on the nervous system to affect behavior (Young et al., 1964). The paper formulated the hypothesis that in adulthood, hormones can activate the nervous system to induce specific behaviors, such as the effect estrogen (E) and progesterone (P) may have on female guinea pigs that cause them to engage in feminine copulatory behaviors. These hormones would act on previously ordered brain tissue, and without such hormone exposure, the behaviors don’t occur or are much diminished. These adult effects are in contrast to the hormone effects during a perinatal critical period that may induce a different organization of the nervous tissue, so that administration of testosterone (T) to neonatal female guinea pigs will cause them to become in adulthood behaviorally defeminized (show fewer sexual behaviors than non-treated females) and masculinized (show more male—like sexual behaviors). That hormones could have an organizational effect on the nervous system in utero was suggested by Pheonix et al., 1959. The predictions then are that both neural and behavioral measures would resemble males if exposed to perinatal androgens, or resemble females without androgen exposure during development; and that once organized, the nervous system may only be activated according the direction organized. This idea is supported by a great deal of evidence in the literature. I will henceforth refer to this temporal theory as the organization/activational hypothesis. As for where in the nervous system hormone-sensitive structures reside; at first, structure-based reports seemed to indicate that sex differences in the brain occurred at the synaptic level, where interactions between terminals and spines that were only observable with electron microscopy deep within the preoptic area held the subtle differences responsible for sex differentiated behaviors (Raisman & Field, 1971). But as researchers began to further investigate sex differences in the brain, at least one brain nucleus in the same preoptic region displayed such a prominent difference in size and density that it could be seen with the naked eye, the sexually dimorphic nucleus of the pre-optic area (SDN-POA) was found to be several fold larger in males compared to females (Gorski et al., 1978). Once cellular level sex differences were found, several reports of dimorphisms in other interconnected nuclei, showing a range of differences, began to be published (See review by Guillamon & Segovia, 1993). Later, it became clear that some areas of the brain could differ at the gross level (Gur et al., 2004; Gur et al., 1999). Thus, the level of resolution matters in regards to establishing that a sex difference in structure exists and also in order to study such a difference; one must be able to observe changes in the structure. The SDN-POA offered an accessible model that could test the temporal predictions of the organization/activational hypothesis on structure. It was shown that perinatal hormone exposure during the critical period could drastically alter the phenotype of the nucleus (Gorski et al., 1981). Furthermore, studies that manipulated hormones in adulthood did not seem to affect cellular measures (Bloch & Gorski, 1988; Rhees et al., 1990), so the idea that the SDN—POA cytoarchitecture was organized during the critical period and then later acted on in adulthood, took root. Follow-up studies in other dimorphic areas tended to produce the same results. The medial amygdala receives major afferent input from the accessory olfactory bulb along with additional input from the main olfactory bulbs and then projects axons to the medial preoptic area (Coolen & Wood, 1998). As such, it is a major node in a steroid- sensitive vomeronasal network and several subsequent studies appeared to fit the standard model of a structure organized by gonadal hormones during the perinatal critical period (Staudt & Dorner, 1976; Mizukami, 1983). While some studies began to suggest a role for the medial amygdala in dimorphic behaviors (Kendrick, 1981), other studies began to tease apart the contributions of sub-regions within the nucleus to the overall dimorphism (Hines et al., 1992). Of the four subdivisions found in the rat, the posterodorsal aspect of the medial amygdala (MePD) exhibited the largest male—biased volumetric difference (85%) between the sexes. Soon thereafter, a study designed to examine dimorphic peptidergic systems in the medial amygdala found that castration of male rats caused drastic changes in the fiber expression of substance-P inununoreactivity in the MePD 8 weeks later (Malsbury & McKay, 1994). A second part of that study was meant to ensure that this finding wasn’t due to changes in the cytoarchitecture, but found instead that the volume of the nucleus shrank by up to 27% 8 weeks after castration. This relatively large scale difference in cytoarchitecture was both an extension, and a challenge, to the idea of steroid mediated brain organization. At the time of these experiments, studies had found evidence that an extension of the perinatal window for hormonal organization in the SDN-POA was in order, but the dichotomy of organization/activational hormone effects still dominated the field (Davis et al., 1995). The Malsbury & McKay experiment established the MePD as a possible exception however, and that idea was tested again soon thereafter in an experiment designed to vary stress in pregnant rat dams, which would presumably alter perinatal testosterone exposure and hence the hormonal organization of the MePD structure (Kerchner et al., 1995). The study found no effect of stressing the dams on the MePD, and concluded that perhaps the stress was not properly timed to suppress the testosterone levels sufficiently. It couldn’t be ruled out that the MePD maintained or retained a hormone sensitive window that extended into adulthood. This adumbration was first fully tested by Cooke et al., 1999, where the circulating hormonal milieu was reversed in both males and females. Surprisingly, the effect was to fully obviate, if not completely reverse, the sex differences in the volume of the MePD and cell soma size as well. This finding offered the first example by which circulating hormones in adulthood alone could maintain the dimorphism of a mammalian brain area necessary for sexual behavior. To determine the cellular targets of testosterone in maintaining dimorphisms of the MePD, it is necessary to first to explain in more detail how testosterone may act on cells. In some cases, e.g. the SDN-POA, brain masculinization occurs via a metabolite of testosterone. The enzyme aromatase, which is abundant in the hypothalamus and the medial amygdala (Roselli et al., 1987), converts androgens (such as testosterone) into estrogens (such as estradiol). Estrogen then interacts with estrogen receptors (ER), not androgen receptors (AR), to induce a masculine SDN-POA. Cell death seems to regulate sexual differentiation of the SDN-POA. There are more dying cells in the SDN-POA of neonatal females than males, and treating newborn females with testosterone reduces the number of dying cells, which presumably leads to a larger SDN-POA (Davis et al., 1996). In contrast, hormone manipulations in adulthood, after the period of naturally occurring neuronal death, have no effect on the volume of this nucleus (Gorski et al., 1978). Such work in the SDN-POA, and work in the medial amygdala (Mizukami, 1983), had indicated that estrogen, and thus aromatized testosterone, could explain the entire effect of testosterone on masculinization of brain nuclei. A study by Cooke et al., 2003 was designed to confirm the receptors though which testosterone could act on the MePD. They showed that ER activation, via treatment with estradiol, was indeed sufficient to maintain both volume and somal size in adult males, but dissociated the contribution of somal size to volume by showing that AR activation, via treatment with the non-aromatizable androgen dihydrotestosterone (DHT) could only maintain somal size. This result was interesting because it implicated AR in the MePD could have a role in brain sexual differentiation, which hadn’t previously been reported, as well as suggesting that elements in the neuropil might also contribute to the volumetric dimorphism. Other cellular elements have been reported to vary with adult hormone exposure. In another rodent model, mean somal area, mean highest branch order, and dendrite length were shown to decrease following long term castration (3 months) in male hamsters, or with DHT treatment which acts predominantly on AR, (Gomez & Newman, 1991). This result suggested that differences in the neurites that comprise the neuropil could change in adulthood following hormone manipulations, and a possible species difference in that somal areas were not maintained by AR stimulation. Other studies have since shown that dendrites may change in the MePD after the perinatal critical period (Zehr et al., 2006). Again in another rodent model, the meadow vole, neurogenesis has been shown to increase in the medial amygdala of adult females following estrogen treatment (Fowler et al., 2005). But that experiment did not find any such effect in prairie voles, which again hints at a species difference in MePD cellular regulation by hormones. Lastly, in a study by Rasia-Filho et al., 2002, glial fibrillary acidic protein (GFAP) irnmunoreactivity differed between males and females in the rat MePD, suggesting that glial number or extent could vary by sex, and possibly by hormone exposure. Taken together, it is not yet understood how AR, sex, circulating androgens, time, or species interact to affect MePD volume, cell somal size, or cell number in the MePD. The goal of this research was to test some of these relationships in order to better understand the structural responsiveness of the adult rodent MePD. I first hoped to challenge the idea that AR had little to no role in the sex difference in regional volume, somal size, and rostrocaudal extent. This question has implications for understanding sex differences in the human brain which may be AR mediated (Femandez-Guasti et al., 2000). Secondly, quantifying the sex difference in cell number in the MePD could better explain which cells are affected by hormones in adulthood, which led me to count neurons and glia in hormone-manipulated rats of both sexes. In addition to testing sex specific responses in an anatomical substrate, I thought this could inform interpretations of adult hormone regimens which may be neuroprotective in disease (Gillies et al., 2004). The absence in the literature of tests of short term temporal effects on cellular level changes in the MePD led me to test shorter periods of hormone manipulation in adult rats of both sexes. By dissecting sex- or time-specific contributions of hormone mediated effects, I wanted to look for another sex difference in the MePD that could be exploited to probe implications of short term hormone therapeutics. Lastly, by measuring adult mediated effects of gonadal hormones on the mouse medial amygdala, I wanted see if sex differences in the rat MePD would generalize to other rodent brains that may offer unique opportunities, such as the capacity to manipulate the genome in mice. I now report the findings of these experiments. CHAPTER TWO PARTIAL DEMASCULINIZATION OF SEVERAL BRAIN REGIONS IN THE ADULT MALE (XY) RATS WI I H A DYSFUNCTIONAL ANDROGEN RECEPTOR GENE RATIONALE Mammals exhibit sex differences in behaviors and brain morphology that are influenced by gonadal hormones during development and adulthood. For example, the medial amygdala is a sexually dimorphic brain area implicated in the display of male sexual behaviors (Meisel and Sachs, 1994). Both the morphology of this region and the behaviors associated with it are influenced by gonadal hormones. In adult rats, the posterodorsal aspect of the medial amygdala (MePD) is sexually dimorphic and depends on sex differences in circulating adult androgens (Cooke et al., 1999). As in other mammalian brain regions such as the sexually dimorphic nucleus of the preoptic area (SDN-POA), the encapsulated region of the bed nucleus of the stria terminalis, and the bed nucleus of the accessory olfactory tract (Segovia and Guillamon, 1993), the volume of the adult MePD is larger in males than in females. However, only in the MePD can the sex difference be eliminated by adult hormone manipulations. Current evidence suggests gonadal hormones affect the adult morphology of the MePD by acting via androgen receptors (ARs) and/or estrogen receptors (ERs). The presence of AR and ER mRNA (McAbee and DonCarlos, 1998); (Shughrue et al., 1997) and protein (Shughrue and Merchenthaler, 2001); (Roselli, 1991); (Li et al., 1997); (Greco et al., 1 998a) in neurons of the MePD suggests that these neurons may be directly influenced by these hormones. Estrogen levels in the MePD are also likely to be higher than in other areas, since aromatase activity in the medial amygdala as a whole is among the highest in the rat brain (Roselli et al., 1985). These findings are consistent with the idea that testosterone can either act as an androgen and/or be aromatized to an estrogen to influence MePD morphology. A recent study treating castrated rats with estradiol (E2) or the non-aromatizable androgen dihydrotestosterone (DHT) indicates that both androgenic and estrogenic metabolites of T influence the adult morphology of the MePD. Systemic treatment with DHT in adult castrated males maintains MePD soma size but not volume, while E2 treatment maintains both measures, suggesting that gonadal hormones may act via ER and AR to influence MePD morphology. These results also suggest that other factors besides soma size, such as neuron number or dendritic branching, contribute to volume differences, since increases in MePD soma size after DHT treatment did not lead to increases in volume (Cooke et al., 2003). To address further the role of endogenous steroids in the sexually dimorphic MePD, we studied male rats with the testicular feminization mutation (tfm) of the AR gene, which renders them insensitive to androgens. Tfin male rats have above normal levels of circulating testosterone (Roselli et al., 1987), but because they have a mutation in the AR gene involving a single nucleotide substitution (Yarbrough et al., 1990), only 10-15% of the AR protein binds androgen; (Naess et al., 1976). As {fin rodents have decreased AR binding but apparently normal ER binding (Attardi et al., 1976), these animals offer a way to examine the contribution of the AR to adult MePD morphology in 10 gonadally intact genetic males. Distribution of ER subtypes have not been studied in tfm rodents. so effects mediated by ER alpha or beta cannot be distinguished by this model. We compared regional volume and soma size in the MePD of adult tfm males to that of wildtype male and female littermates. We also examined the SDN-POA of these animals, which is thought to be masculinized through activation of ER during a perinatal sensitive period (Dohler et al., 1984). If masculinization of the SDN-POA requires ER but not AR stimulation, then tfm males would be expected to display a masculine SDN- POA, as suggested by Gorski and J acobsen (Gorski et al., 1981). Finally, we examined the suprachiasmatic nucleus (SCN) in these animals as a control nucleus in which morphology was not expected to depend upon either AR or ER activation. We found evidence that a defective AR gene affects the morphology of all three brain regions. 11 METHODS Animals Litterrnates (N=15/group) of wildtype male, female (tfm carriers and non- carriers), and affected male tfm rats from our colony at Michigan State University, aged 85-95 days, were used. These animals approximate a Long Evans strain, as female carriers of the mutation have been crossed with commercially supplied Long-Evans males for over 10 generations. After weaning, animals were housed 3 to a cage (males, (fins, and females separately) in standard rat cages with food and water freely available. Lights turned off at 1900 h and on at 0700 h. Animal care followed standards set by the National Institutes of Health and were approved by the institutional animal care and use committee at Michigan State University. On the day of sacrifice, animals were injected IP with an overdose of sodium pentobarbital (120 mg/kg). Deep anesthesia was noted by lack of reflexes to tail and foot pinch as well as lack of a corneal reflex. Animals were then perfused transcardially with 0.9% saline, followed by 10% neutral buffered formalin (~300mL/animal). Phenotype was confirmed at sacrifice by examining external genitalia and the gonads (adult tfm males have small, undescended testes and a blind vagina). Brains were removed and placed into the same fixative solution. Body weights and anogenital distance were measured before sacrifice. 12 Histology After at least one month post-fixation in formalin, the brains were placed overnight in 20% phosphate-buffered sucrose (pH 7.4) at 4°C prior to slicing. Each brain was scored along the left cortex to mark laterality, blocked at the cerebellum and olfactory tubercle, and coronally sliced on a freezing sliding rrricrotome set to 40pm. Sections were collected into a phosphate buffer (0.1M P04, 0.1% gelatin, 0.3% Triton; pH 7.4), every third section mounted onto gel-subbed glass slides, with a random start from the first series of each brain to ensure every section had an equal probability of being chosen for sampling. Missing sections due to damage were replaced by one of the two remaining sections within that interval, as determined by a coin flip, or a space was left on the slide. Mounted tissue was allowed to air-dry. stained with thionin for Nissl substance, and coverslipped with Perrnount. Analysis An investigator, blind to group status, measured the regional volume of the MePD, SDN-POA, and SCN on both sides of the brain. MePD boundaries were determined following nomenclature from a standard rat atlas (Paxinos and Watson, 1998) using criteria from Hines et al. (1992), and Canteras et a1. (1995). The MePD is located in the medial-most aspect of amygdala, and abuts the ventro-lateral margin of the Optic tract (Figure 11-1). The MePD contains larger and more darkly stained cells with higher packing density than the surrounding relatively cell-sparse area. The following landmarks indicated that the caudal most aspect of the MePD occupied the same rostrocaudal level of the brain in all three groups of animals: size and shape of the lateral l3 ventricle, position and shape of the optic tract with respect to the MePD, and size and shape of the stria terminalis (Figure 11-1 ). Moreover, the caudal-most end of the MePD shows a distinct encapsulation by the stria terminalis, and therefore was chosen as the anchor point in all measurements. Boundaries of the intensely staining cells of the SDN- POA were separate from lighter surrounding areas as delineated by Bloch and Gorski (1988a) and similarly the SCN highly contrasted with the surrounding areas (Figure 11-2) as delineated in Paxinos and Watson (1998). Stereolnvestigator (Microbrightfield, VT) software was used to estimate the volume of the MePD, SDN-POA, and SCN and the average neuronal soma size within each brain region of both sides of the brain. A digital camera captured at 50X an image containing the area of interest from a Zeiss (Axioplan) compound microscope and a computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections throughout the rostrocaudal axis. Total volume for each nucleus in each hemisphere was calculated taking into account sampling ratio (1 out of every 3 sections) and section thickness (40pm). Data from animals with damaged or missing tissue in the region of interest were excluded from the analysis. To estimate rostrocaudal extent, the number of sections used for volume estimates were counted. For soma size estimates, neurons were randomly selected by the Stereolnvestigator software, which positioned points within the traced sections without bias for location or appearance so that an investigator could trace the soma of the nearest neuron. An average of 5 neuronal somata from each section were measured throughout the MePD (sampling 25- 55 neurons/side), SDN—POA (15-30 neurons/side), and SCN (20 neurons/side) from each hemisphere, traced at 630x. Neuronal somata measures were averaged within each 14 hemisphere yielding a mean soma size per hemisphere for each animal. Neurons were identified by the presence of a distinct Nissl-stained cytoplasm and nucleolus. Statistical analysis Body weight and anogenital distance were each analyzed by 1-way ANOVA with Fisher’s PLSD post-hoe tests to determine which groups of animals differed significantly from one another. For each brain measure, groups were compared using a mixed-design 2 x 2 ANOVA with genotype (male, female, or tfm) as an independent variable, and hemisphere (left, right) as a repeated measure. When there was no significant effect of hemisphere nor a significant interaction between genotype and hemisphere, the means for each hemisphere were collapsed and l-way ANOVAs were conducted to determine which genotypes differed from one another. If there was a significant main effect of laterality, or an interaction of laterality and genotype, we conducted matched-pairs t-tests within each genotype to ask which groups displayed a significant hemispheric asymmetry. If there were significant main effects of both genotype and hemisphere, but no interaction between the two, we conducted separate l-way ANOVAs on each henrisphere to determine which genotypes were significantly different from one another in each hemisphere. For each analysis, Fisher’s PLSD post-hoc tests were used to determine which groups of animals differed significantly from one another. A two-tailed probability value of 0.05 was used as the significance criterion, with N representing the number of animals in all analyses. Adobe Photoshop (version 7.0) was used to store and manipulate photographs. No processing of images occun'ed except for resizing. 15 RESULTS The body weights of tfm males were significantly heavier than females and significantly lighter than wildtype males (1 way ANOVA, all ps < 0.0001; Table 1). Similarly, anogenital distance was also intermediate in tfm males (all ps < 0.0001), but much closer to that of females than that of wildtype males (Table 1). Several landmarks confirmed that the caudal-most aspect of the MePD arises in the same region of the brain in all three groups. These landmarks include the caudal-most occurrence of a prominent stria terrrrinalis, distinctive shape and size of the lateral ventricle, and the abutment of the optic tract on the dorsomedial comer of the MePD (see Figure 11-1). Furthermore, the size of the MePD was equivalent in this caudal-most section across the three groups. There was more variability across the groups in the rostral extent of the MePD, with the nucleus ending sooner in females than in the other two groups. MePD Regional Volume MePD volume in tfm males was significantly greater than in females, yet significantly less than in wildtype males, whether considering the left hemisphere, right hemisphere or the two hemispheres combined (l-way ANOVAs, all ps < 0.001). This meant that there was also a significant sex difference in MePD volume among the wildtype animals. The mean total volume of the MePD in females was 59% of that in wildtype males, comparable to previous reports (65 %; Cooke et a1, 1999). In wildtype males and tfm males, MePD volume was asymmetrical, with the right hemisphere volume 16 greater than the left (ps < 0.03, matched-pairs t-tests), but there was no asymmetry in MePD volume in females (p = 0.55; Figure II-3). MePD Soma Size There was no significant laterality of MePD soma size in any of the groups of animals (two-way ANOVA ps > 0.10), so we used soma size averaged across the two hemispheres to compare the different groups of animals. As with MePD regional volume, soma size of MePD neurons in tfm males was significantly larger than in females (p < 0.005), yet significantly smaller than in wildtype males (0 < 0.03; see Figure II-3). Among the wildtype males and females there was also a large sex difference in MePD soma size (p < 0.0001). Thus, there is a robust sex difference such that wildtype males have larger MePD volumes and soma size than females, with tfm males displaying intermediate values that are significantly different from both females and wildtype males. MePD Rostrocaudal Extent In both hemispheres, wildtype males had a significantly longer MePD rostrocaudal extent than did females (L: p < 0.02; R: p < 0.0003; l-way ANOVAs followed by Fisher’s PLSD, all ps < 0.02). The tfm males were similar to wildtype males on this measure in both hemispheres (L: p = 0.84; R: p = 0.57) with greater rostrocaudal extents than in females (L: p < 0.02; R: p < 0.0008; Figure 11-4). As with MePD volume, there was an asymmetry of MePD extent, with the nucleus being longer in the right hemisphere than the left, in wildtype males (p < 0.005; matched-pairs t-test) and tfm males (p < 0.007), but not in females (p = 0.55, Figure Il-4). l7 By examining the volume of the MePD at various rostrocaudal levels, we found that MePD volume seemed equivalent across the three groups of animals at the caudal- most region of the nucleus, but that differences emerged more rostrally (Figure II-5). Interestingly, wildtype and {fin males did not appear to differ in MePD volume at the rostral—most extent of the nucleus, where females display no MePD at all. SDN-POA Regional Volume There was no significant laterality of SDN-POA volume (2-way ANOVA ps > 0.60), so we examined group differences in total regional volume (Figure II-6). Wildtype males had a significantly larger SDN-POA volume than did females (p < 0.0001; 1-way ANOVA and Fisher’s PLSD). The tfin males were not significantly different from wildtype males (p < 0.20) and, like wildtype males, had a significantly greater volume than did females (p < 0.0001; see Figure 11-2). SDN-POA Rostrocaudal Extent There was also no laterality of SDN-POA rostrocaudal extent (2-way ANOVA ps > 0.20), so we compared mean rostrocaudal extent across the groups of animals. Wildtype males had a significantly longer extent than did females (p < 0.0001; l-way ANOVA and Fisher’s PLSD). The mean rostrocaudal extent of the SDN-POA of tfm males (3.6 i 0.20) was less than that of wildtype males (4.2 i- 0.28), but this difference was not statistically significant (p = 0.07). Rostrocaudal extent of the SDN-POA was greater in tfm males than in females (2.3 i 0.16; p < 0.003). In short, as with the MePD, 1.8 it appears that the longer rostrocaudal extent of the SDN-POA that is typical of males does not depend upon a functional AR. SDN-POA Soma Size There was no laterality of SDN-POA soma size (ps > 0.20) and therefore we used soma size averaged across the two sides to examine group differences. Mean soma size in 0"": males was smaller than in wildtype males (p < 0.02), but not significantly different from females (p = 0.31). Comparing wildtype males to females, there was not a significant sex difference in this measure (p = 0.16, 1-way ANOVA and Fisher’s PLSD; Figure II-6), although the means of males was greater than that of females. Therefore it is possible that a larger sample size might have detected a sex difference in SDN-POA soma size. SCN Volume There was no significant laterality of SCN volume (p > 0.05), so we combined the data across sides. Wildtype males had a significantly larger SCN volume than did females (p < 0.01, Figure II-7). The tfm. males were similar to female littermates on this measure (p = 0.55) and like female littermates, had a smaller SCN volume than did males (p < 0.03). SCN Rostrocaudal Extent and Soma Size SCN rostrocaudal extent did not differ across the three groups of animals, nor was there any significant laterality in this measure (data not shown). While there was no 19 significant sex difference in SCN soma size among wildtype males and females (p > 0.20), this measure was smaller in tfm males than in wildtype males (p < 0.02). SCN soma size in tfm males did not differ from that found in females (p = 0.16). There was no significant laterality of SCN soma size in any group (ps > 0.05). 20 DISCUSSION Although masculinization of the rodent brain is widely assumed to be due to estrogenic metabolites of testosterone interacting with ERs, we found that genetic male rats with a dysfunctional AR were demasculinized to some extent in every nucleus we examined. These results suggest that full masculinization of brain morphology in rats requires activation of ARs. On the other hand, the brains of genetic males with dysfunctional ARs were not entirely feminine in any region examined, which is further evidence that ER activation also plays a role in masculinization of the brain. Taken together, these results indicate that testosterone and its metabolites must activate both ERs and ARs to fully masculinize the rat brain. The tfm male rats have the same or higher circulating T as wildtype males (Roselli et al., 1987), as might be expected due to lack of negative feedback. Aromatase activity levels remain high in the medial amygdala of tfm males, equivalent to that seen in wildtype males. Having higher available T would likely increase the amount of E available for activating ERs in the MePD. If the volume and soma size in the MePD were solely ER dependent, one would expect fully masculine measures, yet the MePD of tfm rats was only partially masculinized. The body weights of tfm rats were less than that of males, yet the volume of the SDN was not demasculinized, suggesting that these effects were not due to a generalized decrease in brain volume. We found MePD volume and somata to be greater in males than in females, similar to the results of others (Cooke et al., 1999, 2003); (Kerchner et al., 1995 ); (Malsbury and McKay, 1994); (Hines et al., 1992). An effect of laterality (R>L 21 hemisphere) in volume was shown by Cooke et al. (2003) in male Long Evans rats, which we also see in both mutant and wildtype males of the Long—Evans strain, but not in females. In contrast to Cooke et al. (2003), we did not find an effect of laterality on MePD somata in any group. However, we measured somata from throughout the entire MePD, whereas Cooke et al. measured somata from a representative section in the more caudal-medial MePD corresponding to a dense concentration of AR. It may be that somata size in that subregion of the MePD is lateralized in its responsiveness to androgens. The rostrocaudal extent of the MePD was similar in wildtype and tfm males, and both groups of males displayed a greater extent that did females, which indicates that the sex difference in MePD volume reflects differences in both the length and width of the nucleus. The greatest differences between wildtype and tfm males in cross-sectional area occurred around the middle third of the nucleus (Figure H—5). However, the region displaying the greatest sex difference in MePD volume was at the rostral-most extent where tfm and wildtype males are equivalent, which suggests that ARs may not contribute to masculinization of this portion of the MePD. The MePD was the only brain region examined that displayed an asymmetry; specifically, total volume and rostrocaudal extent was greater in the right hemisphere than the left in tfm and wildtype males, but not in females. These results indicate that laterality of MePD morphology is a masculine trait that does not depend upon the presence of functional AR. Presumably the masculine lateralization of MePD structure is mediated solely by aromatized metabolites of androgen acting upon ER. 22 Circulating gonadal steroids can influence many aspects of cellular morphology, including neuron size and number, dendritic branching, and synaptic connectivity. Since Cooke et al., (2003) showed that changes in MePD volume were not fully explained by changes in soma size following castration, other elements of the neuropil and neuronal number might also contribute to the volume differences. Gomez and Newman (1991) reported diminished dendritic arborization in the medial amygdala of hamsters following castration. Greater number and/or size of glia could also contribute to volume differences. However, recent evidence indicates that female rats have a denser staining of glial fibrillary acid protein in the posterior medial amygdala than do males, suggesting that glia may not account for the increased volume seen in male MePD and implicates neuronal elements instead (Rasia-Filho et al., 2002). Adult neurogenesis cannot be excluded as a contributor to the increased volume of MePD in male rats. For example, female prairie voles given bromodeoxyuridine in adulthood show both labeled neurons and glia in the MePD following exposure to males (Fowler et al., 2002), suggesting that both neurogenesis and gliogenesis can occur in the adult MePD in response to sexually relevant stimuli. Sex differences in amygdala morphology and function have also been found in primates. Neurons in the medial amygdala of squirrel monkeys are smaller in females than in males (Bubenik and Brown, 1973). The human amygdala, measured by MRI, is sexually dimorphic beginning at puberty (Brierley et al., 2002). While a study of the Yakovlev collection of human brains did not find a sex difference in the volume of the medial amygdala (Murphy, 1986), the sample size was small (n=17) and included data from across the lifespan, which might easily mask a sex difference if it arises during 23 puberty. Allen and Gorski (1990) found a sex difference in the human bed nucleus of the stria terminalis (BNST), part of the so called extended amygdala that shares connectivity with the medial amygdala. As a node in the circuit responsible for male sexual behavior, the medial amygdala and the effects of gonadal hormones on it have been well documented. Malsbury and McKay (1994) showed in castrated adult males that systemic testosterone maintains both sexual behavior and the density of substance-P staining in the MePD for at least 8 weeks. Lesions of the medial amygdala in inexperienced males leave erections intact during copulation, but abolish non-contact erections (NCEs), a measure of sexual arousal (Kondo et al., 1997). Intracranial implants of either DHT or E2 into the medial amygdala delay the loss of NCEs following castration of males (Bialy and Sachs, 2002). Discrete neuronal populations in the MePD show irnmunostaining for both Fos and AR following ejaculation (Greco et al., 1998b). Taken together, these results suggest that AR activation in the MePD is important for male sexual behavior. Behavioral studies of tfm rats implicate both ER and AR in the activation of male copulatory behaviors. Castrated tfm males show mounting and intromissive patterns following treatment with T or E, but notDHT, whereas male littermates responded to treatments with DHT as well (Olsen, 1979). These same sexual behaviors were also deficient in gonadally intact tfm males in an earlier study (Beach and Buehler, 1977). The present findings suggest two explanations for this deficit. Perhaps tfm males experience only a decreased activational effect caused by the loss of functional AR in adulthood, leading to an underrnasculinized MePD and SDN-POA. Alternatively, tfm males may not receive a fully masculinizing organizational effect if perinatal AR 24 stimulation augments hypothalamic estrogen levels by increasing aromatase activity and therefore increasing local ER activation. As expected, the volume of the SDN-POA was fully masculine in tfm rats, confirming the report of Jacobson and Gorski (Gorski et al., 1981). However, we found the somata of neurons in the SDN-POA were smaller in tfm males than in wildtype males. While Gorski et al. (1981) report a sex difference in neuronal soma size in the SDN- POA, they do not provide mean size or details about the strain or age of the rats examined, so we cannot readily compare the present study with their results. In our material the mean soma size of SDN-POA neurons in females was less than that of males, albeit not statistically significant (p = 0.16, two-tailed). It is possible that with a larger sample size we might have replicated the sex difference in SDN-POA soma size reported by Gorski et al. (1981) and Madiera et al. (1999). In any case, finding that SDN-POA somata are smaller in tfm than wildtype males suggests that a functional AR is required for full masculinization of this characteristic. Dohler et al. (1986) found that SDN—POA volume can be demasculinized by blocking perinatal exposure to estrogen, while blocking perinatal androgen binding had no effect. However, Roselli et al. (1987) showed that aromatase was diminished in the medial preoptic area of tfm rats compared to normal males, supporting the idea that AR may normally have a role in regulating SDN-POA morphology by augmenting aromatase activity in this region, and in turn providing more ligand to activate ER to increase SDN-POA volume. If AR activation acts in this manner, it would explain why Lund et al. (2000) found that postnatal androgen receptor blockade with flutamide reduced the SDN-POA volume in adult males comparable to that of control females: AR-mediated conversion of T to E in the 25 hypothalamus may be compromised by lack of AR activation. In tfm rats, the decreased hypothalamic aromatase activity caused by lack of AR may be partially compensated by the elevated circulating T compared to wildtype males. Roselli et al. (1987) found aromatase activity to be normal in the medial-cortical amygdala of tfm male rats, so we have no reason to think that AR functions by modulating aromatase activity in the MePD. We found a sex difference in total SCN volume (males > females). Our absolute estimate of SCN volume in males (0.060 mm3 per side) is similar to that of Guldner (1983), who reported a unilateral SCN volume of 0.064mm3. Robinson et al. (1986) and Gorski et al. (1978) also reported sex differences in volume of SCN, consistent with the present results. However, there are reports of a lack of a sex difference in SCN volume (Madeira et al., 1995), including a subsequent study from Gorski’s laboratory GSloch and Gorski, 1988b), so the sexual dimorphism may be subtle and/or strain dependent. The SCN in mice has a smaller volume in males that are missing the gene for steroid-receptor coactivator—l (Monks et al., 2003), so perhaps AR activation requires this cofactor to masculinize the SCN. Castrating male gerbils at birth reduces the size of the SCN in adulthood G-lolman and Hutchison, 1991), and the human SCN contains ARs (Femandez- Guasti et al., 2000), all of which is consistent with AR having an influence on this nucleus. Swaab et al. (1992) also found that the human SCN was larger in homosexual men than in heterosexual men. If androgens play a role in human sexual orientation, the morphology of the SCN may serve as a marker for this influence. The present analysis cannot determine whether the morphological effects of a defective AR are due to the protein dysfunction during the perinatal, pubertal, or adult period since tfm rats have defective AR throughout development. For the MePD, stress 26 of the dam on embryonic days 17-21, which drastically reduces testosterone available during the prenatal critical period, does not change MePD volume in adulthood (Kerchner et al., 1995), suggesting that prenatal androgens may not influence adult volume of the MePD. Because adult MePD dimorphism is fully dependent on circulating androgens (Cooke et al., 1999), it may be that AR contributes to MePD morphology primarily in adulthood. Whether AR stimulation acts on the developing and/or the adult SDN-POA or SCN must await further study. 27 CHAPTER 11 APPENDIX 28 Table H-l Body weight and anogenital distance of subjects. Body Weight (g) AGD (mm) Males (n=18) 489.4 (x 11.7) 42.6 ($0.6) TFMs (n=16) 368.8 (1- 12.9) 17.8 (1- 0.7) Females (n=l9) 287.6 (i 5.9) 13.1 ($0.4) Average body weights and anogenital distance (AGD) for each group, with standard errors of the means in parentheses. For each measure, all groups are significantly different from each other (1 -way ANOVA for each measure, post—hoe tests indicate all ps < 0.0001 ). 29 orsal medial amygdala (MePD) as revealed by Nissl stain of coronal sections from a wild-type male (top), a rfin male with a dysfunctional androgen receptor (middle), and a female rat (bottom). The panels on the left are from the caudalmost appearance of the MePD, which served as an anchor point to assess changes in the nucleus across the rostrocaudal dimension. The appearance of the MePD, as well as the optic tract (ot), the stria terminalis (st), the anterolateral part of the amygdalohippocampal transition area (AHiAL), and the lateral ventricle (v) are equivalent in wild-type males (a), yin males (b), and females (c), indicating that the caudal termination of the MePD occurs in the homologous region of the brain across groups. The panels on the right are from the approximate middle of the rostrocaudal extent of the MePD where the nucleus is larger in wild-type males (d) than in females (0 and is intermediate in size in genetic males with a dysfunctional androgen receptor (e). The MePD extends farther rostrally in wild-type males and {fin males than in females (see text). Scale bar = 250 pm in a (applies to a—c), (1 (applies to d-f). 30 Figure II-2. The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) as revealed by Nissl stained coronal sections is larger in a wildtype male (a) than in a wildtype female rat (c). SDN-POA volume in a {fin rat, a genetic male with a dysfunctional androgen receptor (b), is masculine: larger than in females and equivalent to that of males. The sexual dimorphism in the volume of the suprachiasmatic nucleus (SCN), while more subtle than in the SDN-POA, again reflects a larger nucleus in wildtype males (top) than in females (bottom), but the SCN volume in {fin males (middle) is feminine: less than that of wildtype males and equivalent to that of females. Scale bar = 250 um. 31 ,. _,_P<9-0°01__ 0.001 0.001 0.6« ——-»..P..‘_..-_-_H...__ t?_f__________1 <0.03 «6‘ 05 l lp__l E .5, 0.4 . 2 NA 3 03 7““1 2 g 0.2. l " l é’ 0'1 L l R 0.04 , A i . . Male TFM Female (n=1 1) (n=15) (n=14) p<0-009.1_ 200 . l A . p<0.03 p< 0.005 0* 18° F ii 1 3 160 140 g 120~ 'J—I E 100 . / g 30‘ / I in E 60 / s ‘°‘ / 20 l ,__ / Males TFMs Females (n=10) (n=13) (n=11) Figure II-3. MePD regional volume (t_qp_)_and soma size (bottom) in adult tfm males, and wildtype male and female littermate controls. MePD volume is significantly greater in the right than in the left hemisphere in wildtype males (p<0.03) and androgen-insensitive {fin males (p< 0.03), but not in females (p=0.55). On each side of the brain, all three groups were significantly different from one another, with values for {fins intermediate between those of males and females (ps <0.001). There is also a robust sex difference in MePD soma size (p<0.0001) with wildtype males having larger somata than do females. Tfm males have significantly smaller somata than wildtype male littermates (p<0.03), but significantly larger somata than female littermates (p<0.005). There was no laterality of MePD soma size in any of the groups. All error bars represent the standard errors of the means (SEM). 32 all ps < 0.025 12 ‘ p<0.00 0| MePD extent (it of sectlons) OJ O I Melee (n=11) NA l——1 4‘ —L L R TFM: Females (n=14) (n=15) Figure 114. Mean (:SEM) rostrocaudal extent of the MePD was estimated by counting the number of sections sampled for each animal that contain this region. MePD extent is significantly greater on the right than on the left hemisphere in wildtype males (p< 0.005) and androgen-insensitive {fin males (p< 0.007), but not in females (p: 0.55). Wildtype males have a significantly longer extent than did females on each side (L, p < 0.02; R, p< 0.0003). The {fin males are as masculine as wildtype males in this measure. 33 MePD Right Hemi (7) 4 Ch 4 h N A Section Area (mm2 x 10") cu. L MePD Left Hemi 0') Mr 0" LL & I“. ITFM DFemde N 4 Section Area (mm’ x 10") d L CD \\\\\\\\\\\\\\\\ to .\\\\\\\\\\ 1 2 3 4 5 6 7 10 11 12 Candi Secflon mar-her Rostrd Figure II-5. Rostrocaudal extent of MePD in the right (top) and left hemispheres (bottom) of androgen-insensitive tfm males and wildtype male and female littermates binned by average area per section (:SEM). The sex differences in MePD are found primarily in the middle and rostral end of the nucleus. Tfin males resemble females in some regions, are indistinguishable from males in caudal-most and rostral-most regions, and have volumes intermediate between males and females in others. 34 0.10 - "A 0.08 . p<0.0001 . 5 Hm I v 0.06 « 0 s 7r g 0.044 // z 9, 0.02m / m. a F? Males TFMs Females (n=1 1) (n=7) (n=8) 140 « P < 0-02 f E E 1 z 130 . g l 7.: 120 « g / / a) / 110 « é / z. 100 l/ / / o 10/ w , l/////.1 l l Males TFMs Females (n=12) (n=8) (n=8) Figure 116 Total SDN-POA volume (top_) and mean cross-sectional area of neuronal somata (b_ottom) in adult {fin males and wildtype male and female littermates. SDN-POA volume is not significantly different between wildtype and {fin males, but both groups displayed a greater volume than did females (p < 0.0001). In contrast, {fin males have smaller somata than wildtype males, indicating a role for AR in maintaining SDN-POA soma size in male rats. However, there is no significant sex difference in the size of SDN-POA somata (p: 0.16), nor is soma size in {fin males different from that in females (p: 0.31). 35 0.16 - ,. 125.993,,“ _. 0.14 . [fl—l E 7 fit e44 7; 0.10 « %/ ; g 0.08 . V / a /’ I > 0 06 r . z % 8 004- , 0.02 - % 0.00— Males TFMs Females (n=10) (n=9) (n=8) < 0.02 i'£"““i SCN somal area (pmz) Males TFMs Females (n=9) (n=8) (n=9) Figure II-7. Total volume (top) and mean cross sectional area of neuronal somata (bottom) in SCN of androgen-insensitive {fm affected males, wildtype males, and female littermates. SCN volume was significantly larger in wildtype males than in control females (p<0.01) with tfm males having the same size SCN as female controls (p=0.55). SCN soma area is greater in wildtype males compared to {fin males, but there is no significant sex difference in this measure (p: 0.23). SCN soma size did not differ between {fm males and females (p: 0.16). 36 CHAPTER THREE SEXUAL DIMORPHISM IN NEURONAL NUMBER OF THE POSTERODORSAL MEDIAL AMYGDALA IS INDEPENDENT OF CIRCULATING ANDROGENS AND REGIONAL VOLUME IN ADULT RATS RATIONAL In mammals, sex differences in brain morphology typically result from differential exposure to gonadal steroid hormones during critical periods in development, although steroids also exert effects on the brain during puberty and in adulthood. As an example, the medial amygdala is influenced by both perinatal and adult androgen manipulations (Mizukami et al., 1983, Malsbury & McKay, 1994, Cooke et al., 1999, Stefanova & Ovcharov, 2000). One quadrant of the medial amygdala, the posterodorsal aspect (MePD), is particularly sensitive to androgen manipulations in adult rodents. Adult levels of circulating androgen maintain the larger volume of the MePD in male rats compared to females. Castration of adult males, or testosterone (T) treatment of females, abolishes the sex difference by decreasing or increasing MePD volume in males or females respectively (Cooke et al., 1999). Although many brain regions are sexually dimorphic in volume, the MePD appears unusual in that it is sensitive to hormones in adulthood. Gonadal steroids may activate andr0gen receptors (AR) and/or estrogen receptors (ER) to cause the sexual dimorphism in MePD volume. Systemic treatment of adult gonadectorrrized males with estradiol, but not dihydrotestosterone (DHT), maintains MePD volume in rats (Cooke et al., 2003), yet male rats with a dysfunctional AR exhibit 37 MePD volume intermediate to that of control males and females (Morris etal., 2005), indicating that AR also contributes to the masculinization of the MePD. In adulthood, this region contains abundant neurons expressing AR and/or ER protein (Roselli, 1991; Li etal., 1997; Yokosuka et al., 1997) and mRN A (McAbee and DonCarlos, 1998; Shughrue et al., 1997), suggesting that gonadal hormones may act directly in the MePD to influence its morphology. Sex differences in MePD volume may be caused by a number of integral anatomical components: cell size (soma and processes), cell number, vasculature, neuropil, and extracellular space. Although the size of neurons in the MePD is dimorphic (Bubenik & Brown, 1973; Cooke et al., 1999; Morris et al., 2005; Hermel et al., 2006) and therefore might account for the difference in MePD volume, previous work has shown that DHT treatment maintains neuronal size in the male MePD, without maintaining volume (Cooke et al., 2003). This dissociation of neuronal size and regional volume indicates that other components of the MePD are probably affected by circulating androgen to alter MePD volume. For example, no one has determined whether the sex difference in adult medial amygdala volume is accompanied by a sex difference in neuronal number, nor whether androgen manipulations in adulthood affect the number of neurons. This possibility is raised because T increases neurogenesis in the medial amygdala of castrated adult male meadow voles (Fowler et al., 2003) and prevents apoptosis in the preoptic area of deve10ping male rats, resulting in a larger adult volume (Davis et al., 1996). We report here that male rats have more neurons and glia in the MePD than do females. While neuronal number is unaffected by hormone manipulations in adults of 38 either sex, glial number is affected in the MePD of the right hemisphere only. These results further delineate the cellular mechanisms involved in the hormone-regulated plasticity of the rat MePD, indicating that a permanent sex difference in neuronal number is organized earlier in life, and does not contribute to fluctuations in regional volume in adulthood. 39 METHODS Animals 60 day old male and female Long Evans rats (Charles River, Wilmington, MA) were housed three to a cage by gender in standard rat cages with food and water freely available. Males and females were housed in separate rooms. Lights were turned off at 1900 hours and on at 0700 hours. Animal care followed standards set by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at Michigan State University. After one week of acclimation, surgeries and hormone capsule implantations were performed during isoflurane inhalant anesthesia using aseptic procedures. Animals were randomly assigned to one of 4 groups of N=10 animals each. Females were ovariectomized (OVX) and implanted 5.0. with two Silastic capsules (each having 20mm effective release length, 30mm total length, i.d. 0.062 inch; o.d. 0.125 inch) containing either crystalline testosterone (T) or nothing (“blank”) and sacrificed 28 days later. Capsules were incubated for 48hrs in phosphate buffered saline (pH 7.4) before implantation. Males were either castrated or subjected to sham surgery and then sacrificed 28 days later. On the day of sacrifice, animals were injected IP with an overdose of sodium pentobarbital (120 mg/kg). Deep anesthesia was noted by lack of reflexes to tail and foot pinch as well as a lack of a corneal reflex. Blood was taken via cardiac puncture for radioimmunoassay. Animals were then perfused intracardially with 0.9% saline, followed by 10% neutral buffered formalin (~300mIJanimal). Gonadectomies and 40 hormone implants were confirmed at sacrifice. Brains, spinal cords, preputial glands, seminal vesicles, and perineal muscles were removed, placed into the same fixative solution and, after at least a month of fixation, trimmed and weighed. Hormone assay Plasma testosterone concentrations were measured in duplicate using the Coat-a- Count Total Testosterone Kit (Diagnostic Products Corp., Los Angles, CA) and sample volumes of 50uL of plasma. The lower limit of detectability was 0.1 ng/mL and the intra—assay coefficient of variation was 9.0 %. Histology After at least 1 month post-fixation in buffered formalin, the brains were placed overnight in 20% phosphate-buffered sucrose (pH 7.4) at 4°C prior to coronal sectioning on a freezing sliding microtome set to 40pm using Multi-Brain Technology (Neuroscience Associates, TN). An embedding matrix ensured no lost sections, and preserved hemispheric orientation. Brains were sectioned throughout the entire region of interest and alternate sections mounted onto gelatin subbed slides with a random start to ensure that every section had an equal probability of being chosen for sampling. Mounted tissue was allowed to air-dry, stained with thionin for Nissl substance, and coverslipped with Permount. Stereological Analysis 41 All analyses were conducted by an investigator blind to group status. The investigator first measured the regional volume of the MePD on both sides of the brain. MePD boundaries were determined as previously described (Morris et al., 2005) following nomenclature from a standard rat atlas (Paxinos and Watson, 2005) and using criteria from Hines et al. (1992) and Canteras et al. (1995). Furthermore, in defining the MePD borders, at least two cytoarchitectonic features (size, shape, distribution, orientation, staining intensity, packing density, and heterogeneity of cells) were used to distinguish it from the surrounding areas, which allowed the entire reference space to be accurately and consistently traced (Figure Hl-l). Stereolnvestigator (Microbrightfield, Colchester, VT) software was used to estimate the volume of the MePD and the average perikaryal size (n: 8 animals per group). A digital camera on a Zeiss Axioplan 2 compound microscope captured an image containing the area of interest from which the perimeter of the area was traced in successive sections throughout the rostrocaudal axis. Total volume of the MePD in each hemisphere was calculated by multiplying the sampling ratio (2) times section thickness (40pm) times the total two-dimensional area traced. We estimated the size of neuronal cell bodies only and not apparent glia. For perikaryal estimates, sample regions were randomly selected by the Stereolnvestigator software, which positioned points within the MePD without bias for location or appearance. On average, the two-dimensional profile area of 10 perikarya from each section were measured throughout the rostrocaudal extent of the MePD (sampling 55-95 neurons per hemisphere), and were traced using a 100X Plan-NeoFluar, 1.3 NA oil- 42 immersion objective. Neuronal perikarya measures were averaged within each hemisphere yielding a mean soma size (area in umz) per hemisphere for each animal. Neurons were identified by the presence of a distinct Nissl-stained cytoplasm and nucleolus (Fig. 2). Glia were identified by the presence of many dark stained heterochromatic bodies in the nucleus that were interspersed with strands of thionin- stained material. Such presumptive glial nuclei were generally smaller and were not surrounded by a distinct cytoplasmic shell. The smallest cells that also had a very dark stained nucleus with several large stained bodies within the nucleus were categorized as microglia. Cells that could not be classified with confidence into these three categories were designated as “unknown”. The optical fractionator method was used for estimating number of cells (West et al., 1991). This method estimates total number of objects (Nobj) from the sum of objects sampled (Z N) in a known fraction of the reference space. The calculation then is: Nobj = (Z N)(l/ssf)( 1/asf)(1/ttf) where section sampling fraction (ssf) is the number of sections sampled divided by the entire number of sections through the reference space, area sampling fraction (asf) is the total area sampled by the array of counting frames divided by the total area of all sampled sections, and thickness sampling fraction (tsf) is the height of the disector divided by the average total section thickness. Cells in the MePD were counted by the optical fractionator method (West et al., 1991) using a Plan-NeoFluar 100X oil-immersion (1.3 NA.) objective which allowed us to distinguish in most cases neurons from glia and to ensure discrimination of discrete objects. Sampling parameters were set to allow a coefficient of error (m=1, Gundersen, 1999) of no more than 0.10 for each animal, and percentage of total observed variance 43 due to inter-animal variance to be at least double that of variance due to sampling error (n=5 per group). A pilot study indicated that sampling boxes with the following measures were appropriate: sampling depth, Sum; guard height, 1.5 pm minimum; sampling frame area, 625um2; x-y spacing 125nm. An average of 272 cells was counted in each side of each brain. Average thickness of stained sections for each brain was estimated by measuring section thickness in every third counting frame and was used to adjust cell counts for that brain. Overall average section thickness was 8 pm. DIC optics used in a pilot study indicated that potential slicing artifact was limited to about 1pm. Neuronal nucleoli and glial nuclei were used as unique counting points. Adobe Photoshop (v7.0) was used to generate figures containing photomicrographs. No processing of images occurred except for resizing and brightness. Statistical Analysis Results are expressed as means _+_ SEM. A three way mixed design analysis of variance (ANOVA), with left and right sides as a repeated measure, and two independent factors [sex and androgen status (high in gonadally intact males and T-treated females, low in castrate males and blank-treated females)], was conducted for each dependent variable (MePD volume, average perikaryal area, neuronal number, glial number). One statistically significant interaction was detected for each of two measures (soma size and glial number), prompting follow up 2-way AN OVAs to confirm trends detected by the 3- way ANOVA. T-tests comparing sham operated males and blank-treated females were used to assess previously reported sex differences in MePD regional volume and soma size (Cooke et al., 1999; Morris et al., 2005). T-tests were also used to assess the well- documented effects of T on the periphery to confirm androgen manipulations. For all analyses, a value of 0.05 was used as the significance criterion, with N representing the number of animals in each group. 45 RESULTS Bioassays of testosterone treatment Testosterone manipulations for 28 days had gross effects in both males and females (Table 1). Castration of males significantly decreased the average weight of the preputial glands, seminal vesicles, bulbocavernosus/levator ani muscles, and overall body weight (all ps < 0.05). Testosterone implants in females significantly increased the average weight of preputial glands (p < 0.05), but not overall body weight (p = 0.17). As expected, circulating T was beneath the detection limit in castrated males and blank- treated females. As T levels did not differ significantly between sham castrated males and T treated females (p = 0.10), the treatment appeared to provide physiologically relevant androgen stimulation to females. Regional Volume As in previous studies, the volume of the MePD was greater in males than in females and, in both males and females, was greater in the right hemisphere than the left and was affected by adult androgen levels (Figure III-3A). The 3-way AN OVA revealed these differences as significant main effects of sex (male greater than female; p< 0.0001), of androgen (greater in the presence of androgen than absence of androgen; p< 0.003) and of hemisphere (right greater than left; p< 0.005), with no significant interaction terms. Hence no posthoc comparisons for regional volume were called for, although examination of the data suggests that the left MePD of males may be less responsive to androgen than the right (Figure III-3A). We have twice reported a sex difference in 46 MePD volume in gonadally intact rats. A posthoc comparison of the closest comparison groups available from this study (sham males and untreated ovariectorrrized females) also revealed volumetric differences in both the left and right MePD (t—tests; ps < 0.05), presumably a result of higher circulating androgen in the males than in the females (Table 1). Neuronal Soma Size Also as in previous studies, there was no laterality of MePD soma size, but this measure was affected by adult hormone levels in both sexes (Figure III-3B). The ANOVA revealed these effects as a significant main effect of androgen (p < 0.001), with no significant main effect of laterality, nor any significant interaction of any factors with laterality. There was no significant main effect of sex, indicating that adult androgen status is more important than sex in determining soma size. There was a significant sex by androgen interaction (p = 0.02) because androgen status had a greater effect in females than in males. We previously reported sex differences in MePD soma size in gonadally intact rats, and in the present study found that the somata are larger in sham males than untreated females in both the left and right MePD (ps < 0.05; t-tests), presumably a result of higher circulating androgen in the males than in the females. The absence of laterality in MePD soma size, despite the robust asymmetry in regional volume, indicates that the asymmetry in regional volume cannot be attributed to asymmetry in soma size. Sham males have also been reported to have equal sized neurons across hemispheres, despite asymmetry in regional volume. 47 Neuron Number Males have more MePD neurons than do females (main effect of sex p< 0.002), and there are more neurons in the left hemisphere than the right in both sexes (main effect of side; p< 0.0001; Figure III—4A). However, these sex differences and laterality in neuronal number are unaffected by adult androgen status, as there was no main effect of androgen status (p > 0.5) nor any statistically significant interaction terms (ps > 0.13). Thus, the left MePD is smaller in volume than the right MePD, yet the left contains more neurons than the right, and this dissociation is seen in both sexes. Furthermore, the absence of androgenic effects on neuronal number indicates that other factors must underlie the effects of androgen on regional volume in the MePD of adult rats, and is another dissociation of neuronal number and regional volume. Glial Number Males have more MePD glial cells than do females, in both hemispheres (main effect of sex p < 0.003 and no significant interaction of sex with any other factor). The number of glial cells is greater on the right than on the left (main effect of hemisphere p< 0.001) and there was no significant main effect of androgen status (p > 0.4). However, there was a significant interaction of androgen status and hemisphere (p < 0.03), which prompted a follow up analysis of the left and right hemispheres separately (Figure 111- 4B). Two way ANOVA (sex and androgen status as independent factors) of glial numbers in the left hemisphere revealed a marginally significant sex difference (p = 0.06) but no effect of androgen. In contrast, in the right hemisphere there was both a sex difference (main effect p < 0.003) and an effect of androgen (p < 0.01), with no 48 interaction. Thus high androgen is associated with a greater number of glial cells in the MePD in the right hemisphere only, and this effect is seen in both sexes. We considered the density of glia by treatment group, using the cell counts and regional volume measures reported above. These indicated that the right MePD contains a higher density of glia, and the left MePD contains a higher density of neurons, in all treatment groups. Overall, there does not appear to be a significant sex difference in neuronal density, but OVX females that receive no T have a higher density of glia than sham males. This result seems to confirm the report that female rats display a greater density of GFAP staining than males in the MePD (Rasia-Filho et al., 2002). Other cell types Our sampling parameters were not designed to obtain reliable counts of rarely encountered microglia or “unknown” cells, but the mean estimates for the number of microglia suggest that if there is a difference, it also favors males (sham males = 3428 i 427; castrated males = 5118 i 576; T-treated females = 2720 t 323; blank-treated females = 2605 i 260). Likewise, the estimated number of unknown cells was only slightly greater in males than in females (sham males = 8148 ;l-_ 1491; castrated males = 11833 i 1296; T-treated females = 8134 i 1184; blank—treated females = 6678 i 1424). Because the mean estimates of microglia and unidentified cells is greater in males than in females, the sex differences in numbers of neurons and glia favoring males cannot be accounted for by mis-classification of cells. 49 DISCUSSION The adult rat MePD shows a remarkable level of plasticity that is regulated by gonadal hormones. We found that the adult female rat MePD responds to T treatment with increases in volume and neuronal size in both hemispheres, and number of glia in the right hemisphere. The male MePD also responds to gonadal hormones—adult castration leads to reductions in regional volume and neuronal size in both hemispheres and the number of glia in the right hemisphere. Unlike the other measures, however, the number of neurons in males and females was not affected by changes in adult androgen. These results limit hypotheses about cellular mechanisms underlying androgen’s effects on MePD regional volume in adult rats, and how hormonally-induced plasticity of this nucleus may affect reproductive behavior. In turn, adult behaviors that induce hormonal variations, such as exposure to an estrous female and social conflict, may influence the regional volume and neuronal size in this nucleus, but are unlikely to affect neuronal number. Because adult hormone manipulations can abolish sex differences in MePD volume (Cooke et al., 1999), one might have expected that the number of neurons was equivalent in the two sexes and that adult fluctuations in volume might reflect changes in neuropil that simply alters the density of neurons within the nucleus. But we found that males in fact have more MePD neurons than do females in adulthood, no matter what the androgen status of the animals. Thus castration of adult males causes the MePD to shrink in volume without any loss of neurons, while androgen treatment of females enlarges MePD volume without adding to neuronal number. As change in regional volume cannot, be explained by changes in neuronal number, other factors must contribute to volumetric 50 changes. We found evidence that androgen affects glial number, but only in the right hemisphere. In the present study, changes in MePD soma size generally reflected changes in volume, so soma size may contribute to some of the volume changes we detected. However, the persistent asymmetry in MePD volume stands in contrast to a lack of any asymmetry in MePD neuronal somata size. Furthermore, in previous studies, treatment with the various metabolites of testosterone can dissociate soma size from volume (Cooke et al., 2003). By elimination, these results together suggest that changes in synaptic neuropil, including the dendrites of neurons and processes of glia, probably underlie the bulk of change in MePD volume in adult rats. Reports of hormone-induced changes in MePD dendritic structure lend credence to this idea (Gomez & Newman, 1991; Rasia-Filho et al., 2005, Hermel et al., 2006). Cooke et al., (2007) report a sex difference in the proportion of MePD volume occupied by dendritic processes in prepubertal rats, consistent with this idea. They also found more neurons in males than females in the right MePD of prepubertal animals, but no sex difference in neuronal number on the left. Thus the sex difference in the left MePD may arise during or after puberty, which would suggest a dramatic reorganization during this period of ontogeny. There is also an interesting and pervasive dissociation of regional volume and neuronal number in these data. The MePD has a larger volume on the right than the left, yet there are more neurons on the left than the right. We found this pattern of results in both sexes, and androgen manipulations had no effect on neuronal number. Again, one might have expected that the hemisphere containing a larger MePD would also contain more MePD neurons. It seems clear that the several components that contribute to the 51 volume of a brain nucleus (neuronal number, glial number, neuronal soma size, etc.) are independent, at least in the rat MePD. The sex difference in neuronal number within the MePD is unaffected by adult androgen manipulations, so presumably T or its metabolites affect neuronal number earlier in life. Cooke & Woolley (2005), showed that in pre-pubertal rats at 25-29 days of age (when circulating androgens are equivalent in males and females), neither soma size, cell density, nor neuronal number in the left MePD were sexually differentiated, but volume was, being significantly larger in males. If the male amygdala contains more neurons only after puberty, it may be that rising levels of androgens promote increases in neuronal size and number in pubertal males. While speculative, this would explain why in humans, amygdalar volume increases faster in boys than girls between ages 4-18 (Giedd et al., 1997; Merke etal., 2003). Furthermore, neurogenesis has been reported in the amygdala of young adult male monkeys (Bedard etal., 2002). However, male and female children exposed to elevated circulating androgens prepubertally have smaller amygdalar volumes (Merke et al., 2003). Conversely, if females have fewer neurons after puberty than before, then androgens may prevent apoptosis in males to create the adult sex difference in neuronal number. In any case, future studies could ask when the sex difference in neuronal number arises in the developing MePD and whether androgen organizes this characteristic. There is also the question of how such differences in neuronal number emerge. What is the cellular mechanism involved? Studies of cell number in the MePD during ontogeny might indicate whether the sex difference in neuronal number arises due to sex differences in neurogenesis, neuronal migration, neuronal differentiation and/or 52 apoptosis. Although we saw no change in neuronal number in the adult MePD, this does not address whether neurogenesis or apoptosis occurs in adults. If neurogenesis and apoptosis are ongoing and in balance in adults, our counts of the net number of neurons would not detect those processes. Although castration can affect neuronal proliferation in the MePD of adult male meadow voles (Fowler et al., 2003), there may be species differences in neurogenesis, neurodegeneration, or both. Overall, the present findings add to the literature indicating that morphologic lateral asymmetry is common in the amygdala (Cooke et al., 2003, Morris et al., 2005). While androgen receptor distribution has been found to be asymmetrical in adult hippocampus (Xiao et al., 2002), no such laterality has been reported in the medial amygdala (Lu et al., 1998). Interestingly, the human amygdala has also been reported to be asymmetric in terms of both structure (Murphy et al., 1986) and function (Phillips et al., 2001; McClure et al., 2004; Cahill et al., 2004; Canli et al., 2002; Harnann et al., 2004). For glial number, only the right side of the MePD is sensitive to hormones. T- treated females had more glia than blank-treated females only in the right MePD. Conversely, castrated males have fewer glia than gonadally intact males only in the right MePD. These results suggest that the right MePD is more sensitive than the left to androgen’s effects on glial number. Whether this increased sensitivity in the right hemisphere is due to asymmetry in steroid receptors, metabolic enzymes (such as aromatase) or some other factor has yet to be addressed. Our results indicate that male rats have more glial cells in the MePD than do female rats. However, visualizing astroctyes in the rat MePD by glial fibrillary acidic 53 protein (GFAP) immunoreactivity, which is expressed by most (but not all) astrocytes (Eng et al., 2000), reveals a sex difference in the opposite direction, with females showing a greater density of GFAP staining than do males (Rasia-Filho et al., 2002). While GFAP marks only astrocytes, and our counts undoubtedly include both astrocytes and oligodendrocytes, our findings of a higher glial density in the female MePD could account for the sex difference in GFAP staining seen by Rasia-Filho et al. (2002). On the other hand, it is possible that despite the fewer glia found in females compared to males, the glia in females may have longer or more branched processes than those in males. In fetal rat hypothalamic cultures, astrocytic process elaboration, but not number, is increased by estradiol (Garcia-Segura et al., 1989). Other studies have shown that GFAP immunoreactivity in the dentate gyrus of the hippocampus and the MePD correlates with estrogen levels across the female’s estrous cycle (Luquin et al., 1993; Martinez et al., 2006). It will be important in future studies to count the number of GFAP-expressing cells in the MePD both to validate the identification of glia in this study, and to probe the relative contributions of astrocytes versus other glia to our counts, as well as any responses to steroid manipulations in adulthood. Although astrocytes have been shown to express steroid receptors in some parts of the rat brain (Lorenz etal., 2005; Tabori et al., 2005), we do not know based on the current data which type of glia is increased in response to androgen treatment of adult females, nor do we know whether androgen is acting directly on glial cells, glial precursors, or acting on some other cell type, including neurons, to increase glial numbers indirectly in the right MePD. Segovia et al. (2006) report a sex difference in the volume and number of cells in the medial amygdala of rabbits, but no difference in density, similar to the present report. 54 Although we counted more neurons than glia in the MePD of both males and females, it seems likely that neurons, with their Nissl-darkened cytoplasm, would be more readily detected than glia in our preparation. Similarly, it is possible that the T treatment did not increase the number of glia in the right MePD, but somehow altered glia morphology to make them more detectable. Thus any conclusions about the mechanisms of change in the number of glia found in the MePD must remain tentative. Our androgen manipulations did not affect MePD volume quite as dramatically as in a previous study (Cooke et al., 1999), where mean volumes in castrated males were smaller than in females given androgen. In the present study, sex difference in volume, seen as a main effect of sex in ANOVA, persisted even when androgen was controlled for (Figure III-3A). Although the androgen treatment was intended to duplicate that of Cooke et al., it is possible that providing more androgen and/or extending the androgen treatment might have more closely duplicated the results in the previous study. In any case, posthoc tests of MePD volume in the right hemisphere (the more androgen responsive side of the brain) show no significant difference between castrated males and females given androgen. Both studies demonstrate that the sex difference in MePD volume can be eliminated by manipulations of androgen in adulthood. Thus the plasticity of the adult MePD in rats continues to offer a model system to study structure/function relationships in hormone sensitive brain areas. Future studies should address directly whether adult hormone manipulations affect dendritic and/or synaptic structures in the MePD, the identity of glial cells affected, and the contributions each of these changes make to alterations in behavior of both sexes. 55 CHAPTER III APPENDIX 56 Table III-1 Average weights of the body, preputial glands, seminal vesicles and bulbocavernosus/levator ani muscles (BC/LA) for each group i standard errors of the means. Subject Wight 3:113?) vesseiICIIlelsuIlg) BC/LA (g) (ng/TmL) Male Sham 462(114) 0.21(:l:0.01) l.99(:t:0.l4) l.65(:0.05) 2.68(:I:0.43) exit]; 38209.1) 01200.02) 030(1003) 175(1005) N1) F e333+ T 319(t8.8) 0.24(:l:0.01) l.79(:l:0.28) F63: B 29mm) 04230.01) ND Plasma testosterone (T) levels were detectable only in sham males and OVX females given T, which were not significantly different from one another. For each measure, hormone manipulation (castration in males, T treatment in females) had a significant effect (t-test, p < 0.05), with the exception of body weight in females. 57 Figure III-1. Boundaries of the rat posterodorsal medial amygdala (MePD). Photomicrographs of coronal sections of the MePD in an adult, gonadally intact male rat (left column) and an adult, ovariectorrrized female rat without androgen treatment (right column). The caudalmost portion of the MePD appears as a capsule ventral to the optic tract (or) surrounded by the relatively soma-sparse area of the stria ternrinalis (st), at a rostrocaudal level intersecting the lateral ventricle (v) and the anterolateral part of the amygdalohippocampal transition area (AHiAL). At this extreme end, the MePD is of a similar size in males (a) and females (d). More rostrally, the MePD in males (b) has a triangular or wedge-shaped pro.le, with a slight reduction in cell density in the center, which gives the appearance of an inverted letter “v.” In females (e), the nucleus is considerably smaller and has a less cuneate appearance. The rostral end of the MePD is still adjacent to the ventral-lateral aspect of the 0t and is more prominent in males (c) than in females (f). In both sexes, the ovoid intercalated nucleus of the amygdala is visible to the right of the MePD at this level. Scale bar = 250 um. 58 Figure III-2. Classification of cell types. Typical cells viewed with a _100 objective in a sham male adult rat MePD as revealed by thionin stain for Nissl. Arrow indicates a neuron; black arrowheads indicate glia (oligodendrocyte or astrocyte); white arrowhead indicates a microglia. Scale bar = 10 pm. 59 > -Ht¢tAndrogen 4‘ [:lLoaAndrogq-n Volume (mm3x10'1) N 0 1 Male Female Male Female B Left MePD Right MePD 160 ' - High Anaogm [:3 LowAndrogen 140 120 Soma Slze (umz) 8 Male Female Male Female Left MePD Right MePD Figure 111-3. Androgen regulates medial amygdala volume and soma size in adult rats. A) As seen in previous studies, manipulations of androgen in adult rats alter the regional volume of the posterodorsal medial amygdala (MePD). Males were either castrated (low androgen) or subjected to sham surgery (high androgen), while females were ovariectomized and given capsules containing either testosterone (high androgen) or nothing (low androgen). Three way ANOVA revealed a sex difference (males > females; main effect of sex p < 0.0001), asymmetry (right > left; main effect of hemisphere; p < 0.005) and a response to androgen manipulation (main effect of hormone; p < 0.0003), with no significant interactions. B) Neuronal soma size in the rat MePD also responded to androgen manipulations (main effect of androgen; p = 0.0005), but there was no evidence of asymmetry (p > 0.2). While there was not a significant sex difference overall, there was an interaction of sex and androgen status (p = 0.02) because soma size was more responsive in females than in males. The previously reported sex difference in MePD soma size in gonadally intact animals is represented here by the larger somata in sham males sham males compared with females receiving no androgen. 6O > 40-4 - nghAnGogm A ‘ E LawAndrogen '3? : 5 304 I- 3 . S i 2 2° , é . 5 10. g . Z : o . Male Female Male Female Left MePD Right MePD B 30 _ - High mrooen I III] Lama-own 2‘5 1 r i o . i 20 1 — "" 4 B 1 g 15 ‘. :1 . z 10 i 5 1 o , 5J I : 1 I 0 Male Female Male Female Left MePD Right MePD Figure [II—4. Androgen has no effect on neuronal number in the adult rat MePD. A) Males have more MePD neurons than females (main effect of sex; p < 0.002), and there are more neurons in the left MePD than the right (main effect of hemisphere; p < 0.0001), but there was no effect of androgen manipulations in either sex or in either hemisphere (no significant interactions; ps > 0.14). B) Males also have more glial cells in the MePD than do females (main effect of sex; p < 0.003), and there are more glia in the right MePD than the left (main effect of hemisphere; p < 0.001). However, there was an interaction of androgen status and hemisphere (p < 0.03). Subsequent separate analyses of the left and right MePD revealed that androgen is associated with more glia in both sexes, but only in the right MePD. 61 CHAPTER FOUR TIME COURSE OF GONADAL HORMONE MEDIATED PLASTICITY IN THE ADULT RAT MEDIAL AMYGDALA RATIONALE Reports of hormone mediated structural changes in the rodent brain have emphasized the perinatal “organizational” period (Arnold & Breedlove, 1985). Recent findings suggest however that certain brain areas retain a capacity for morphological changes in adulthood (Malsbury & McKay,1994; Cooke et al., 2003; Morris et al., 2008a). Nuclei of the sexually dimorphic vomeronasal circuit have functional demands that vary over days or seasons in response to the environmental cues required for reproductive activity. One of the primary recipients of olfactory and pheromonal signals arising from the main and accessory olfactory bulbs is the medial amygdala, which contains subdivisions that exhibit morphological sex differences in several rodent species (Hines and Gorski, 1992; Morris et al., 2008b; Gomez & Newman, 1991). In particular, the posterodorsal medial amygdala (MePD) is sexually dimorphic in volume and soma size and appears sensitive to circulating gonadal hormones in adulthood. MePD volume, somal size, and glial number may increase or decrease following manipulation of circulating hormones in both males and females (Cooke et al., 1999; Cooke et al., 2003; Morris et al., 2008a). These changes in MePD morphology have been measured after a month or more of steroid treatment. However, because the MePD has been implicated in several behaviors that respond to hormones after different delays (Choi et al., 2005; Rasia—Filho et al., 2004; Bialy & Sachs, 2002; Rowe & Erskine, 1993; Frye and Walf, 62 2004), it is possible that the morphological changes within the MePD may occur in less than four weeks. This study examines the time course of change of three different hormone-sensitive morphological parameters of the MePD: regional volume, rostrocaudal extent and neuronal soma size. The MePD receives chemosensory derived bulbar input that conveys cues for attractive (Portillo & Paredes, 2004) or repellant (Muller and Fendt., 2005) social interactions. Vaginocervical (Polston & Erskine, 1995) or penile stimulation (Veening & Coolen, 1998) occurring over minutes show caused somatosensory induced FOS-ir activation in the MePD. Other studies have implicated the medial amygdala in slower responses, shrinking or expanding across the simulation of seasons along with variations in circulating gonadal androgens (Cooke, et al., 2001, Turek and Van Canter, 1994), or interactions of developmental and maternal experience(0xley & Fleming, 2000) including possible changes in cell number (Akbari et al., 2007). To extend previous research describing hormone mediated effects on the adult MePD a month after hormone manipulations in rats (Malsbury & McKay, 1994; Gomez & Newman, 1991; Cooke et al., 1999; Morris et al., 2008a), we examined the time course of response of various cellular attributes of the MePD (regional volume, neuronal soma size, rostrocaudal extent) at 2, 14, or 28 days following hormone manipulation. We found that neuronal soma size appears to respond more rapidly to androgen manipulations than does regional volume in the MePD. 63 METHODS Animals Male and female Long Evans rats (Charles River, Wilmington, MA) were housed 24 to a cage by gender in standard rat cages with food and water freely available. Lights were turned off at 1900 hours and on at 0700 hours. Animal care followed standards set by the national Institutes of Health and were approved by the Institutional Animal Care and Use Committee at Michigan State University. After one week of acclimation, when animals were 60- 70 days of age, ovariectomies/castrations and hormone capsule implantations were performed during isoflourane anesthesia using aseptic surgical procedures. Animals were divided into 12 groups of N =10 animals each. Females were ovariectomized and implanted so with two capsules of scaled Silastic tubing (i.d. 0.062 inch; o.d. 0.125 inch; 20mm effective length; 30mm total length) filled with either crystalline testosterone (T) or nothing (“blank”) and sacrificed 2, 14, or 28 days later. Capsules were incubated in phosphate buffer (pH 7.4) for 48 hrs before implantation. Males were either castrated or subjected to sham surgery and then sacrificed 2, 14, or 28 days later. On the day of sacrifice, animals were injected 1? with an overdose of sodium pentobarbital (120 mg/kg ip). Deep anesthesia was noted by lack of reflexes to tail and foot pinch as well as a lack of a corneal reflex. Blood was taken by transcardial puncture for radioimmunoassay for T. Animals were then perfused transcardially with 0.9% saline, followed by 10% neutral buffered formalin (~300mL/animal). Gonadectomies 64 were confirmed at sacrifice. Brains, preputial glands, seminal vesicles, and perineal muscles were removed and placed into the same fixative solution. Histology After at least 1 month fixation in formalin, the brains were placed overnight in 20% phosphate-buffered sucrose (pH 7.4) at 4°C prior to coronal sectioning on a freezing sliding microtome set to 40uM using Multi-Brain Technology (Neuroscience Associates, TN). An embedding matrix ensured no lost sections and preserved hemispheric orientation. Brains were sectioned throughout the region of interest and alternate sections mounted onto gelatin subbed slides with a random start to ensure that every section had an equal probability of being chosen for sampling. Mounted tissue was allowed to air- dry, stained with thionin for N issl substance, and Coverslipped with Permount. Analysis An investigator, blind to group status, measured the regional volume of the MePD on both sides of the brain as previously described Morris et al., 2005, Morris et al., 2008a). Briefly, the MePD was traced following cytoarchitectonic qualities (Hines et al., 1992; Paxinos & Watson, 2005) using Stereolnvestigator (Microbrightfield, Colchester, VT) software to estimate the volume of the MePD and the average perikarya size. Measures of MePD volume and peikaryal size at day 28 were previously reported (Morris eta1., 2008a). Total volume was calculated taking into account sampling ratio (one of every four sections) and section thickness (40uM). To estimate rostrocaudal extent, the number of sections used for volume estimates were counted. For perikarya estimates, neurons 65 were randomly selected by the Stereolnvestigator software, which positioned points within the traced sections without bias for location or appearance so that an investigator could trace (at 630x using a Plan-NeoF-luar objective, 0.95 NA) the perikarya of the nearest neuron. This resulted in sampling of 25-55 neurons per hemisphere throughout the MePD. Neuronal perikarya measures were averaged within each hemisphere yielding a mean soma size per hemisphere for each animal. Neurons were identified by the presence of a distinct Nissl-stained cytoplasm and nucleolus. Hormone assay Plasma T concentrations were measured in duplicate using the Coat-a—Count Total Testosterone Kit (Diagnostic Products Corp, Los Angles, CA) radioimmunoassay (RIA) using sample volumes of 50uL of plasma. The lower limit of detection was 0.1 ng/mL and the intra-assay coefficient of variation was 9.0 %. Statistical Analysis For each survival period (2, 14 or 28 days after surgery) we conducted separate three-way ANOVAs with sex and androgen status as independent variables and hemisphere as a repeated measure. At each time point, if there was a significant main effect of androgen status, or interaction of androgen status with any other factor, we conducted two-way ANOVAs for each sex (laterality as a repeated measure, androgen status as an independent measure). For soma size, the initial three way ANOVA indicated no laterality at any time, confirming our earlier findings (Morris et al., 2005). Consequently, we collapsed the data across hemispheres, averaging the left and right 66 estimates of soma size for each animal. When the ANOVA indicated an effect of androgen status, we used Fisher’s LSD post-hoc tests to isolate the effect of androgen on MePD morphology. Although we began with 10 animals in each group, due to attrition during the histological process, not all measures could be obtained from all subjects. For MePD morphology, final N = number of animals listed for mean body weight in Table 1. 67 RESULTS Hormone Manipulation Several indices confirmed androgen manipulations in the subjects. The mean (1; SEM) concentration of plasma T in male rats sacrificed 2, 14 and 28 days after sham surgery was 3.71 i 1.12, 4.52 i 1.04, and 2.68 i 0.43 ng/ml, respectively. Similarly, mean (i SEM) plasma T levels in females sacrificed 2, 14 and 28 days after ovariectomy and implantation of T capsules were 3.11 i 0.39, 1.85 i 0.14, and 1.79 :t 0.28 ng/ml, confirming that our treatment provided physiologically relevant levels of T slightly below that found in normal male rats. T concentrations were below the limit of detection in castrated males and ovariectomized females given blank capsules at all time three points. Body weight, seminal vesicle weight, and weight of the androgen-sensitive preputial glands also confirmed this pattern of androgen status (Table l). MePD Regional Volume Three-way ANOVAs for each time period after the hormone manipulation, with sex and androgen status as independent factors and hemisphere as a repeated measure, confirmed our previous reports of sex differences in MePD regional volume, soma size and rostrocaudal extent, as well as the laterality of MePD volume and absence of laterality in somata size (Morris et al., 2005; Morris et al., 2008). Specifically, two days after hormone manipulation, there was a significant effect of sex (male> female; p = 0.0001) and of hemisphere (right>left; p: 0.01), however there was no effect of androgen status at this early time point (p> 0.20), nor any significant interactions (ps > 0.18). 68 Fourteen days after the manipulation, statistical analysis of MePD volume yielded similar results: a sex difference (p: 0.0001) and laterality (p = 0.002), but no effect of androgen and no interactions. However, 28 days after manipulations there was, in addition to the effects of sex (p< 0.0001) and laterality (p< 0.004), a significant effect of androgen status (p < 0.0003). To confmn this effect, we performed a separate two-ANOVA for each sex, with androgen status as an independent factor and hemisphere as a repeated measure in animals after 28 days. In males four weeks after castration, this analysis suggested a side by treatment interaction (p < 0.1), as androgen status had a significant effect on MePD volume as revealed by post-hoc test in the right hemisphere (p = 0.04) but not the left (p > 0.20; Figure IV-l A). Cooke et al., (2003), reported a similar unilateral effect in males. In females, there was a significant main effect of androgen treatment (p < 0.0005) but not hemisphere (p > 0.06), and no interaction (p > 0.34), indicating that androgen has a significant effect on MePD regional volume after 28 days of treatment, and that the effect is equivalent in the two hemispheres (Figure IV-lB). It is interesting to note an asymmetry in MePD volume (R>L) that occurs only in the presence of androgen, in both males and females at the 28 day time point (ps < 0.03, T-test). Furthermore, in females there was an apparent increase in MePD volume between 2 and 14 days, whether the females are given T or blank capsules. This result suggests that some growth of MePD volume occurs in females between 62 and 74 days of age and that this is independent of gonadal steroids. Because both groups of females were ovariectomized, it is also possible that the growth between 62 and 74 days of age in this study was in response to ovariectomy itself. In either case, volume of the MePD was 69 maintained until 88 days of age in females given T, but shrank in females given blank capsules (see Figure lV-lB). Soma Size First we conducted 3-way ANOVAs of soma size for each time period after the hormone manipulation, with sex and androgen status as independent factors and hemisphere as a repeated measure. Two days after hormone manipulation, there was a significant main effect of sex (p < 0.0002), but not of hemisphere (p> 0.15) or androgen status (p> 0.26) on MePD soma size. No interactions were significant (ps > 0.10). Here, sex accounts for most of the somal size difference. Fourteen days after surgery, there was a significant effect of androgen status on soma size (p< 0.02), but no significant effect of sex (p> 0.80), indicating that androgen status accounted for most of the variance in soma size. This outcome conforms to earlier reports that either castration of males or T treatment of females can abolish the sex difference in MePD soma size (Cooke et al., 1999; Morris et al., 2008). There was no laterality of MePD soma size after 14 days of treatment. Twenty-eight days after androgen manipulations, MePD soma size again showed no effect of laterality, as in previous analyses (Morris et al., 2005), but there was a main effect of androgen status (p< 0.0001). There was no main effect of sex, but there was an interaction of sex and androgen status (p< 0.02), because T treatment of females caused a greater effect than did castration of males. These results led us to collapse across hemispheres and analyze MePD soma size averaged across the two hemispheres. Two way AN OVAs confirmed that androgen status had no significant effect on soma size at 2 days after manipulations (Figure IV-2). 70 Likewise, MePD soma size was not yet significantly different in castrated versus sham operated males at day 14 (p > 0.10). However, the variance in somata size increased dramatically in castrated males 14 days following surgery (note error bars in Figure IV- 2A), suggesting inter-individual variation in the degree to which neurons had begun to shrink. Soma size in the MePD was significantly larger at this time point in females given T versus those given blank capsules (p < 0.04). Twenty-eight days after hormone manipulations, mean soma size in the MePD was significantly affected by androgen status in both sexes (ps < 0.05). Rostrocaudal extent Three-way ANOVA of rostrocaudal extent, as measured by the number of sections containing the MePD, at each of the three time points revealed a significant main effect of sex at all time points (M>F, ps < 0.005) as had previously been shown (Malsbury & McKay, 1994; Morris et al., 2005), but no effect of hemisphere and no significant interactions of any factors (Table 2). There was a significant main effect of androgen status on rostrocaudal extent only in the animals examined 28 days after treatment, which post-hoe tests revealed to be due to effects in females only (p< 0.01). These results indicate that rostrocaudal extent of the MePD is longer in males that in females, and responds to androgen in females sometime between 14 and 28 days after treatment begins, approaching but not fully achieving the normal male length. 71 DISCUSSION We previously reported that MePD volume and soma size are affected 28 days after castration of adult males or commencement of androgen treatment in adult females (Cooke et al., 2003, Morris et al., 2005, Morris et al., 2008a). The new findings here are that neither regional volume nor soma size are significantly different in the MePD two days after a change in androgen status. This was true both in male rats responding to castration and in female rats given male-typical levels of T. Likewise, 14 days later, MePD volume still showed no significant response to androgen manipulations in either sex. However, MePD soma size was altered two weeks after androgen manipulations, being larger in females given T than those given blank capsules. The effect of castration in males was not significant at this time point, although there was much greater variability in this measure in castrated males 14 days after surgery. Thus individual differences in the time course of response to castration may have caused greater variability and made it more difficult to detect a statistically significant difference in males at this time point. Indeed, the mean soma size in castrated males 14 days after surgery is smaller than that seen in castrated males 28 days later (see Figure IV -2A). Overall, these findings indicate that MePD soma size responds more rapidly than MePD volume to androgen manipulations. These findings are also consistent with the idea that androgen acts similarly in males and females, since both sexes show evidence of a change in soma size sometime between 2 and 14 days after androgen manipulations, and . both display a change in MePD volume sometime between 14 and 28 days after androgen manipulations. 72 Because these responses to adult androgen manipulation require more than two days to become evident, androgen is presumably acting through so-called classical pathways (Jordan & DonCarlos, 2008), altering gene expression to slowly alter the size of MePD somata. Because at 14 days somata show evidence of androgen responsiveness while MePD volume has not yet responded, change in somata size may make little contribution to change in overall MePD volume. We have seen such dissociations of somata size and regional volume in the MePD on several previous occasions. For example, treatment of adult rats with the non-aromatizable androgen 5-alpha- dihydrotesosterone (DHT) increases MePD somata size without affecting regional volume (Cooke et al., 2003). Also, while we consistently see laterality in rat MePD volume, with the MePD in males being larger in the right hemisphere than the left, we also consistently see no such lateralization of MePD somata size (Morris et al., 2005). So it seems more likely that androgen regulation of MePD somata is independent of the slower regulation of regional volume. By elimination, the additional contribution to MePD volume, which takes longer to accomplish than the change in somata size in the adult, may be a change in dendritic structure and/or synaptic neuropil within the neurons of the MePD, and glial or vasculature variations. This was recently confirmed in pre- pubertal rats that showed the sex difference in volume of the MePD to be due primarily to differences in dendrites (Cooke et al., 2007). It would be interesting to compare the time course of the loss of various medial amygdala-related behaviors following castration to the time course of morphological responses in the MePD. Such comparisons might suggest which plastic aspects of MePD morphology, if any, are responsible for plasticity in the various behaviors influenced by this brain region. 73 It is also interesting that while some morphological features of the MePD, such as soma size and regional volume, are very responsive to changes in androgen in adulthood, others, such as the rostro-caudal extent of the nucleus, show little or no response. It is not easy to speculate why regional volume would be more structurally malleable in two dimensions (dorsal-ventral and medial-lateral) than in the third (rostral—caudal). Since neither overall brain weight nor sectional area is affected by these manipulations (Malsbury & McKay, 1994), the expansion of MePD volume either comes at the expense of another brain region shrinking, or makes so small a contribution to overall brain weight that it is difficult to detect. The parameters for the present experiment were tailored to capture overall volume changes most efficiently and it could be that the increased precision gained from sampling more sections in the MePD would reveal changes in the rostrocaudal extent of the nucleus. Also, in their report showing an effect of adult hormone manipulations on rostrocaudal extent, Malsbury et al. (1994) may not have sampled as far rostral as in the present study, or there may exist a strain difference (Sprague Dawley vs. Long Evans) in responsiveness. In conclusion, we found that neuronal soma size in the MePD responded more rapidly than regional volume to hormonal variations. These results suggest that in the hormone sensitive MePD, soma size changes occur in approximately two weeks, while volumetric changes take place between two and four weeks following androgen manipulation. MePD morphology was somewhat more responsive to adult hormone treatments in females than in males. If in the future it is possible to differentially stimulate the MePD to increase only some of the many morphological characteristics of the nucleus that normally respond to androgen, it may be possible to discern which 74 behavioral characteristics are mediated by the various structural characteristics of the MePD. 75 CHAPTER IV APPENDIX 76 TABLE IV-l Responses to androgen manipulations. BW, Sham Males: BW, GdX Males: BW, GdX+T Females: BW, GdX+B Females: PPG, Sham Males: PPG, GdX Males: PPG, GdX+T Females: PPG, GdX+B Females: SV, Sham Males: SV, GdX Males: DAY 2 317:5.7 (9) 30919.5 ('11) 204 3; 6.2 (10) 208 i 5.7 (9) 0.20 i 0.02 (6) 0.23 i 0.02 (7) 0.15 i0.01(10) 0.12 i 0.01 (9) 1.11:0.08(6) 0.85 i 0.14 (7) DAY 14 377 :I_- 5.3 (8) 331 i 7.2 (9)* 265 i 6.3 (10) 259 i 6.6 (10) 0.23 i 0.03 (8) 0.13 i 0.02 (9)* 0.21 $0.01 (10) 0.09 $0.01 (10)** 1.64 1; 0.10 (9) 0.33 _-t_- 0.04 (9)** DAY 28 462 _-l_- 13.9 (9) 382 i 9.1 (9)** 319 i 8.8 (10) 297 _-_t-_ 13.3 (9) 0.21 i 0.01 (9) 0.12 i 0.02 (9)** 0.24 i 0.01 (10) 0.12 i 0.01 (9)** 2.00 i 0.14 (9) 0.30 i 0.03 (9)** Data represent means i standard errors of the means (N = number of animals). BW= body weight; SV= seminal vesicles; PPG= preputial glands; Sham: sham gonadectomy; GdX: gonadectomy; T: testosterone capsule; B=blank capsule. All measures in grams. * p < 0.05; ** p < 0.01 Different from same sex control group. 77 TABLE IV -2 Rostrocaudal extent, as measured by the average number of sections i standard errors of the means (N = number of animals) containing the MePD, in each group. DAY 2 Sham Males: 8.14 i 0.24 (7) GdX Males: 8.50 i 0.19 (7) GdX+T Females: 7.06 -_1-_ 0.06 (8) GdX+B Females: 6.92 i 0.15 (6) DAY14 813:013w) 7.88 i 0.23 (8) 7.35 i 0.20 (10) 7.28 1; 0.15 (9) DAY28 8.31 i 0.21 (9) 8.11 i 0.29 (9) 7.75 i 0.11 (10)** 6.94 i 0.10 (9) *Significantly different from same-sex control group at that time point (p< 0.001). 78 [:1 Castrate Males - Sham Surgery p < 0.05 'or {— :3‘ ‘1': —} % m r- e ,i i E. gr- 22- O 1- > D n. °L 2 / / 01’ 11111111111111] d2 d28 d14 d2 d14 d28 Left Hemisphere Right Hemisphere 1. ,9 r C: OVX + Blank 'c _ - OVX + T 1'- 3 .. X ”E _ p O 01 p < 0.01 - < . .8, - — i O .. > a . a, . e 2 7 l / 01{_l_ll_Llll|II1llJll d2 d14 d28 d2 d14 d28 Left Hemisphere Right Hemisphere Figure IV-l. Mean regional volume (i SEM) of the posterodorsal medial amygdala (MePD) in response to androgen manipulations in adult rats. Top) Male rats that are castrated as adults show a significant shrinkage in MePD volume only in the right hemisphere and only 28 days following surgery, not 2 or 14 days after surgery. Bottom) Female rats that are ovariectomized (OvX) as adults and given either testosterone (T) or blank capsules also display an MePD volume response to androgen treatment only in those animals sacrificed 28 days later. However, in female rats MePD regional volume responsiveness to androgen appears equivalent in the two hemispheres. 79 : l:| Castrate Males 140 .- - Sham Surgery "5 I p < 0.05 3 . a . 1 a 120 1 fl 1. E . 0°, . o 100 ‘ n. . fl 1- : . IV of 1 l l 1 d2 1: OVX + Blank r - OVX + T J“ p < 0.05 N... ‘40 . —‘— p < 0.01 E . a .. 3 120 _ 17) . a _ I I E i L .L 1 (D " l D 100 " Q r 0 r- E +- /7 or I l- | I! l l- d2 d14 d28 Figure IV-2. Mean neuronal somata size (i SEM) in the posterodorsal medial amygdala (MePD) in response to androgen manipulations in adult rats. As in past studies, we found no laterality in the soma size of MePD neurons in any group. so mean bilateral size is displayed. Top) MePD somata size showed no response to castration 2 days following surgery. Fourteen days after surgery, MePD somata were not significantly smaller in castrated males than males subjected to sham surgery. However, variance in this measure was significantly greater in castrates than sham males at this time point (note error bars), suggesting that a subset of males may have begun showing a response to surgery, increasing variability at this time. As in past studies, MePD somata are significantly smaller in castrate males than in sham males 28 days after surgery. Bottom) MePD soma size was significantly larger in ovariectomized females given testosterone than those given blank capsules both 14 and 28 days after treatment commenced. There was no discernible difference between groups examined 2 days after treatment began. 80 CHAPTER FIVE SEXUAL DIMORPHISM AND STEROID RESPONSIVENESS OF THE POSTERODORSAL MEDIAL AMYGDALA IN ADULT MICE RATIONALE Sexual dimorphism in the nervous system offers a valuable perspective to learn more about the relationships between neuroanatomy and behavior, as well as the ways in which steroid hormones affect neural function and behavior (Morris et al., 2004). In rats, the posterodorsal medial amygdala (MePD) is larger in males than in females in several characteristics, including regional volume and neuronal soma size (Morris et al., 2005). These sexual dimorphisms can be accounted for by circulating hormones in adults, as the measures are reduced in males four weeks after castration and are increased in females after four weeks of testosterone (T) treatment (Cooke et al., 1999). Treatment of adult gonadectomized rats with two metabolites of T, either dihydrotestosterone, which activates androgen receptors, or estradiol (E), which activates estrogen receptors, indicates that while both receptors contribute to the masculinization of the rat MePD, E is the more effective agent, especially for MePD regional volume (Cooke et al., 2003). Examination of male rats with a genetic defect in the androgen receptor gene, the testicular ferrrinization mutant (Tfm) allele, confirms that both metabolites of T act upon this nucleus (Morris et al., 2005). Because the MePD has been implicated in male sexual arousal (Meisel & Sachs, 1994; Kondo etal., 1997), which waxes and wanes with adult androgen levels, the hormone responsiveness of adult MePD morphology may reflect hormonal activation of sexual arousal. 81 Aside from Tfm animals, there are very few rat models offering genetic variations that might permit a greater understanding of sexual differentiation of the MePD or other brain regions. Therefore, we asked whether the sexual dimorphism and hormone responsiveness of the MePD also occurs in mice, where many genetic manipulations can be readily accomplished. Because the olfactory bulbs are a major afferent to the MePD (Scalia & Winans, 1975), and because removal of the olfactory bulbs causes a profound reduction in male copulatory behavior (Wood & Newman, 1995), we also examined whether removal of the olfactory bulbs affects MePD morphology in mice. We found that the MePD of mice is indeed sexually dimorphic and responsive to hormone manipulations in adulthood, but shows virtually no response to the loss of olfactory bulb afferents in adulthood. 82 METHODS Animals Commercially supplied BALB/c mice (Charles River), approximately 6 months of age, were used. Animals were housed three to a cage (males and females separately) in standard cages with food and water freely available with the exception of the bulbectornized (OBX) females, which were housed singly. Lights were turned off at 1900 hours and on at 0700 hours. The animal care followed standards set by the National Institutes of Health and was approved by the institutional animal care and use committee at Michigan State University. Bulbectomy Olfactory bulbectomized (OBX) males and females were purchased from Charles River. Brains were removed from all animals and assessed for complete OBX at sacrifice. Animals with partial bulbectomies were excluded from analysis. Sham bulbectomies were not performed. Because the aim was to determine whether absence of olfactory afferents interfered with steroidal effects on the MePD, bulbectorrrized males were subjected to sham castration, while bulbectomized females were ovariectomized and treated with estradiol (E), which has been shown to affect MePD volume and soma size in adult rats. Hormone manipulation 83 Ten OBX Females and ten randomly selected bulb-intact females were ovariectomized (OvX) and implanted so with a Silastic capsule (0.058” id and 0.077” o.d.; 5mm effective release length, 15mm total length) containing crystals of 25% free E: 75% cholesterol. Capsules were incubated for 48hrs in phosphate buffered saline (pH 7.4) before implantation and were left in place until sacrifice, approximately 2 months later. Bulb-intact control females (n=10) received sham ovariectomies and blank capsules. Bulb-intact males were either castrated (n=10) or subjected to sham surgery (n=10), while OBX males were subjected to sham surgery (n=10). All animals were sacrificed 28 days after hormonal manipulations. Histology On the day of sacrifice, animals were injected IP with an overdose of sodium pentobarbital (210 mg/kg). Deep anesthesia was noted by lack of reflexes to tail and foot pinch as well as lack of a corneal reflex. Animals were then perfused transcardially with 0.9% saline (pH 7.4), followed by 10% neutral buffered formalin (pH 7.4; ~150 mL/animal). Phenotype was confurned at sacrifice by examining external genitalia and noting the presence or absence of gonads. Brains were removed and placed into the same fixative solution. After at least 1 month fixation, the brains were placed overnight in 20% phosphate-buffered sucrose (pH 7.4) at 4°C prior to slicing. Each brain was scored along the left cortex to mark laterality, blocked at the cerebellum and olfactory tubercle, and coronally sliced on a freezing sliding microtome set to 30 um. Sections were collected into a phosphate buffer (0.02 M P04; pH 7.4); every other section was mounted onto gel- 84 subbed glass slides, with a random start from the first series of each brain to ensure that every section had an equal probability of being chosen for sampling. Missing sections due to damage were replaced by the remaining section within that interval, or a space was left on the slide. Mounted tissue was allowed to air-dry, stained with thionin for Nissl substance, and coverslipped with Permount. Analysis An investigator, blind to group status, measured the regional volume of the MePD on both sides of the brain. MePD boundaries were determined following nomenclature from a standard mouse atlas (Paxinos & Franklin, 2004) using criteria from Hines et al. (1992) and Canteras et al. ( 1995). The MePD is located in the medial-most aspect of amygdala, and abuts the ventrolateral margin of the optic tract (Figure V-l). The MePD contains larger and more darkly stained cells with higher packing density than a surrounding, comparatively cell sparse area. The following landmarks indicated that the caudalmost aspect of the MePD occupied the same rostrocaudal level of the brain in all three groups of animals: size and shape of the lateral ventricle, position and shape of the optic tract with respect to the MePD, and size and shape of the stria terrrrinalis. Moreover, the caudalmost end of the MePD shows a distinct encapsulation by the stria terrrrinalis and therefore was chosen as the anchor point in all measurements. Stereolnvestigator (Microbrightfield, Colchester, VT) software was used to estimate the volume and average neuronal soma size of the MePD of both sides of the brain. A digital camera captured at 50X an image containing the area of interest from a Zeiss (Axioplan) compound microscope, and a computer mouse was used to trace the 85 perimeter of the area of interest on a computer monitor in successive sections throughout the rostrocaudal axis. Total volume for each nucleus in each hemisphere was calculated taking into account sampling ratio (every other section) and section thickness (30 um). Data from animals with damaged or missing tissue in the region of interest were excluded from the analysis. To estimate rostrocaudal extent, the number of sections used for volume estimates were counted. For soma size estimates, neurons were randomly selected by the Stereolnvestigator software, which positioned points within the traced sections without bias for location or appearance so that an investigator could trace the soma of the nearest neuron. An average of 5 neuronal somata from each section was traced (630X) and measured throughout each hemisphere (sampling 25—55 neurons/side). Neuronal somata measures were averaged within each hemisphere yielding a mean soma size per hemisphere for each animal. Neurons were identified by the presence of a distinct nucleolus and Nissl-stained cytoplasm. Statistical analysis For each brain measure, groups were compared using separate mixed-design 2 x 2 ANOVAs with hemisphere (left, right) as a repeated measure in every case, and either sex (within sham-operated mice), or gonadal state within males (castrate versus sham), or hormone manipulation within females (OVX plus E versus sham), or olfactory bulb status (OBX or not) within each sex as the independent variable. These were conducted separately for each dependent variable (volume, average perikaryal size, rostrocaudal extent). When there was no significant effect of hemisphere nor a significant interaction between sex and hemisphere, the means for each hemisphere were collapsed, and one- 86 way ANOVAs were conducted to determine whether groups differed from one another. If there was a significant main effect of laterality, or an interaction of laterality and groups, we conducted matched-pairs t-tests within each group of animals to ask which groups displayed a significant hemispheric asymmetry. For each analysis, Fisher’s PLSD post hoc tests were used to determine which groups of animals differed significantly from one another. A probability value of 0.05 was used as the significance criterion, with N representing the number of animals in all analyses. Adobe Photoshop (version 7.0) was used to store and manipulate photographs. No processing of images occurred except for resizing. 87 RESULTS Sex differences in MePD morphology We compared male and female shams using a two-way mixed design ANOVA (sex as an independent variable and hemisphere as a repeated measure) followed by PLSD post-hoc test, and found an overall effect of sex (males larger than females; p < 0.0001) and hemisphere (left larger than right; p < 0.01) on MePD volume (Figure V-2). The interaction was close to significant (p = 0.15), so we further probed with paired T- tests, revealing a significant lateral asymmetry in MePD volume in females (p < 0.01, L>R) but not in males (p = 0.42). This sex difference in asymmetry is opposite to that seen in rats where males have asymmetrical MePD volumes, but females do not (Morris et al., 2005). The asymmetry is reversed as well with female mice having larger left MePD volumes and male rats having larger right MePD volumes. Rostrocaudal extent of the MePD was greater in males than females (main effect of sex; p < 0.03), but there was no lateral asymmetry (main effect of hemisphere; p = 0.49), nor any interaction (p = 0.49) of the two variables among sham-operated males and females (Table 1). Unlike the asymmetry in male rats, where the herrrisphere with a greater MePD volume is also longer in extent, in female mice, the larger MePD hemisphere does not have correspondingly longer rostrocaudal extent. MePD soma size is larger in male shams than in female shams (main effect of sex: p < 0.0001) but there was no effect of hemisphere (p = 0.36) on soma size, and no interaction (p = 0.87). Therefore, Figure V-3 presents soma size averaged across the two hemispheres. These data indicate that soma size is sexually dimorphic but displays no 88 lateral asymmetry in mice. The asymmetry in female MePD volume cannot be attributed to an asymmetry in neuronal size. Manipulating steroids affects MePD morphology Male castrates were compared to sham-operated males using a two-way ANOVA (hormone manipulation as independent measure and hemisphere as repeated measure) followed by PLSD post-hoc test. Castration reduced MePD volume (main effect of castration; p < 0.0003), and there was only a marginal effect of hemisphere (p = 0.08), with no interaction (p = 0.39; Figure V—2). The marginally significant p value for hemisphere led us to conduct repeated-measures t-tests comparing values from the two hemispheres within castrates and sham males, but neither reached statistical significance. In females, we found OvX + E treated females have larger MePD volumes than intact females, although the effect was only marginally significant (p = 0.06 two-tailed; p = 0.03 one-tailed if one predicts the direction of effect seen in rats). The overall effect of laterality in females was again present (p < 0.0004), but there was no interaction of laterality with E treatment (p = 0.92). Rostrocaudal extents of the MePD in male and female shams were not affected by either castration in males or hormone treatment in females (ps > 0.30; Table 1). There was no laterality in rostrocaudal extent, nor any statistical interaction between laterality in either sex (all ps > 0.18). In males, MePD soma size was reduced by castration (p < 0.0001) but there was no difference between hemispheres (p = 0.76), and no interaction (p = 0.41). In females, MePD soma size was greater in E treated females (p < 0.002), but there was no effect of 89 hemisphere (p = 0.70), and no interaction (p = 0.54). Thus, hormone manipulations affect MePD soma size in both sexes, and the two hemispheres respond equivalently. Bulbectomy has little or no effect on the MePD of adult mice Male and female bulbectomized mice were compared to their respective same sex controls using ANOVAs within each sex (bulbectomy as an independent variable and hemisphere as a repeated measure) followed by PLSD post-hoc test. In males, MePD volume displayed an overall effect of hemisphere (p < 0.03), but not of bulbectomy (p = 0.97). As the interaction was non—significant but low (p = 0.17), we used repeated measures t-tests to find a slight asymmetry in bulbectomized males (p < 0.05; L>R) that was not seen in bulb-intact males, indicating that bulbectomy may induce an asymmetry in MePD volume in males. In females, there was no effect of bulbectomy on MePD volume among OVX + E treated females (p = 0.83). The main effect of laterality was still present (p < 0.002; L>R), but there was no statistical interaction (p = 0.84), indicating bulbectomy has no effect on MePD volume or asymmetry in OvX + E treated females. Because there was no effect of bulbectomy on MePD volume in OvX +E females, we collapsed the two groups of females and compared them to gonadally intact females (shams) to determine if the larger sample size would confirm an effect of OvX plus B treatment on MePD volume. Two way ANOVA followed by PLSD post hoc analysis did reveal an overall effect of E treatment on MePD volume (p < 0.02), and the lateral asymmetry (main effect of hemisphere; p < 0.0001; L>R), that did not interact with 90 hormone status (p > 0.9), indicating that hormone manipulation of females does increase MePD volume equally in both herrrispheres. In males, rostrocaudal extent of the MePD displayed no effect of bulbectomy ( p = 0.49) or hemisphere (p = 0.52), nor interaction (p = 0.52). Likewise, in females, there was no effect of bulbectomy (p = 0.51), or hemisphere (p = 0.83), nor an interaction (p = 0.83) on rostrocaudal extent. Thus, bulbectomy has no effect. on the length of the MePD in mice of either sex. In males, MePD soma size displayed no overall effect of bulbectomy (p = 0.12), or hemisphere (p = 0.24), but a significant interaction (p < 0.05) was found. Post-hoe t- tests revealed neurons in bulbectomized males are reduced in size compared to bulb intact males in the right MePD (122.4 i 4.6 vs. 133.6 i 1.8 umz; p < 0.04), but not the left (p = 0.72). In females, there was no effect of bulbectomy (p = 0.31), or hemisphere (p < 0.39), and no interaction (p = 0.33), on MePD soma size in OVX + E treated females. Thus, of the various MePD measures examined, only soma size in the right hemisphere of male mice was affected by bulbectomy. 91 DISCUSSION We found the MePD of mice to be sexually dimorphic in many ways. Regional volume, soma size and rostrocaudal extent are all greater in male than in female mice, mirroring the dimorphism in rats (Morris et al., 2005). The mouse MePD is also affected by hormone manipulations in adult mice. Castration of males causes regional volume and soma size to shrink, while ovariectomy and E treatment of females causes these same measures to increase. None of our manipulations affected the rostrocaudal extent of the MePD in mice, suggesting that this sexually dimorphic aspect is established early in life and is not susceptible to change in adulthood. The lack of effect on rostrocaudal extent also indicates that hormonal expansion of MePD volume occurs within the dorsoventral and/or lateral planes rather than the rostrocaudal axis. The sexual dimorphism and hormonal responsiveness of the MePD in adult mice indicate that this may be a fertile model for examining neural sexual differentiation because many tools for manipulating the genome are available in this species. We examined outbred BALB/c mice, but the question remains whether the MePD is sexually dimorphic in other mouse strains. In the hypothalamus, some strains of mice show sexual dimorphisms that other strains lack (Mathieson et al., 2000). There is one sexual dimorphism of the MePD that appears to be reversed in rats versus mice. In rats, MePD regional volume is asymmetric in males, where the MePD is larger on the right than the left, and females display no asymmetry. Furthermore, asymmetrical effects of castration have been seen before in rats (Cooke et al., 2003; Morris et al., 2008). But in mice, we found MePD regional volume to be asymmetric in 92 females, with the left MePD larger than the right, while males display no asymmetry. It will be important to see whether the asymmetry in female mice is replicated in other laboratories and/or in other mouse strains. The olfactory bulbs provide important inputs to the MePD and so the loss of these afferents might be expected to affect regional volume or soma size in this nucleus. Yet olfactory bulbectomy of adult mice had little or no effect on the MePD in mice. The only statistically significant effect we found was that in gonadally intact males: those subjected to OBX had smaller MePD somata in the right hemisphere than bulb-intact males. Because we see no laterality of MePD soma size in bulb-intact mice or rats of either sex, it is difficult to explain why OBX would affect only one hemisphere. Thus, given the number of comparisons made and the modest size of this effect, even this result of bulbectomy should be regarded tentatively until other laboratories have replicated it. Given the neuroanatomical inputs from the olfactory and pheromonal systems to the MePD (Canteras et al., 1995), the importance of such signaling to sexual behavior in rodents, and the evidence that this nucleus plays a role in sexual behavior in rats (Meisel & Sachs 1994; Kondo et al., 1997) and mice (Sipos & Nyby, 1998; Kang et al., 2006), the sexual dimorphism in the MePD likely reflects sex differences in the contribution of this nucleus to sexually differentiated behaviors. The fluctuation in morphology of the male MePD in response to changes in hormone levels, either by castration as in the present study and/or castration and testosterone replacement in studies of rats, presumably contributes to the concomitant changes in male reproductive behavior that accompanies these manipulations. Why should the MePD of rats and mice retain this plasticity in response to adult androgen manipulations? One possibility is that the 93 ancestors of laboratory rats and mice were seasonal breeders and that the MePD, and possibly other brain regions involved in reproduction, waxed and waned with the seasons to support sexual behavior only when it was likely to produce offspring. We have examined the MePD in one seasonally breeding rodent, the Siberian hamster, and find that indeed manipulations of photoperiod that suppress gonadal systems are accompanied by a reduction in MePD regional volume and soma size (Cooke et al., 2002). The hormone-induced plasticity of the MePD in mice may be a remnant of such plasticity that was regulated by seasons in ancestral mice. Because a wide variety of transgenic mice and knockout mice are available, the sexual dimorphism and hormone responsiveness of the MePD in this species may be amenable to a fuller understanding of the molecular and cellular processes underlying sex differences in the brain and behavior. 94 CHAPTER V APPENDIX 95 TABLE V-l. Rostrocaudal extent of the posterodorsal medial amygdala (MePD) in mice. LEFT HEMISPHERE RIGHT HEMISPHERE Male Sham 1.3.0 i 0.37 (9) 13.0 i 0.33 (9) Male Castrate 13.0 :1; 0.26 (6) 12.2 i 0.41 (6) Male OBX 12.9 :t 0.20 (9) 12.6 i 0.38 (9) Female Sham 12.0 i 0.44 (9) 11.7 i 0.33 (9) Female OvX+E 12.3 i 0.62 (8) 12.2 i 0.67 (8) Female OvX+E + OBX 11.7 i 0.29 (7) 11.7 :1; 0.36 (8) Means and standard errors of the mean for number of coronal sections containing the MePD are depicted with N = number of animals in parentheses. MePD rostrocaudal extent was significantly greater in males than in females, but there was no significant effect of hormone manipulations or olfactory bulb removal (OBX) in either sex, nor any interaction of hemisphere with those factors. 96 - Male Shah C1: Fem Shun 1222 Male Castrate E Female ME 160 m Male W [11111]] Fenfle OlX-FE‘t-CBX Mun Neuronal Some 8le (11112) 8 8 Males Females Figure V-l. Mean size of neuronal somata in the MePD of BALB/c mice. ANOVA revealed no significant laterality of neuronal soma size in any group of animals, so soma sizes averaged across the two hemispheres are depicted here. Comparison of mice subjected to sham surgery revealed MePD somata to be larger in males than in females (p < 0.0001). Castration reduced MePD somata in males (p < 0.0001), while estrogen (E) treatment of ovariectorrrized (OvX) females enlarged somata size (p < 0.002). Olfactory bulbectomy (OBX) had no effect on MePD soma size in OVX females. In males, ANOVA revealed no significant main effect of surgery or hemisphere, but a marginally significant interaction (p < 0.05). Post—hoe tests indicated MePD soma size was slightly smaller in OBX males than sham males (p < 0.04) in the right hemisphere only. 97 MOW“ Ilse 20 Satflhrmcuwtlm Lamtmzdlm > - “Shun :1 Female Shem 3 MePD Var-m (nun, x 1041 O 9.. e‘e r 4 1 5 ‘ '9" E331 We Ceetrete ' 'o’o‘ 122: Male Shun-003x v e ‘02 O'O'O' 32020.. e'o efefe‘ womrmu’ne") tn LettHemlephere c 2.0- 1:] Male Shem .- 22 Female ovx+e 2 15_ m Fenrele ovx+e+oex _ . g 1.0- ; . g os- i on ' Figure V—2. The volume of the posterodorsal medial amygdala (MePD) in BALB/c mice. ANOVA of sham-gonadectomized mice revealed that MePD volume is greater in males than in females (p < 0.0001) and larger in the left hemisphere than the right (p < 0.01). Post-hoc repeated-measures t-tests revealed the asymmetry was statistically significant in females (p < 0.01), but not in males (p > 0.4). Castration of males reduced MePD volume (p < 0.0003) equally in both hemispheres (interaction p > 0.3). Estrogen (E) treatment of ovariectomized (OvX) females marginally increased MePD volume (p = 0.06, two-tailed). Olfactory bulbectomy (OBX) had no discernible effect on MePD volume in either sex. 98 Figure V-3. The posterodorsal medial amygdala (MePD) in BALB/c mice. In these Nissl—stained coronal sections taken approximately in the middle of the rostrocaudal extent of the MePD, the wedge-shaped MePD abuts the ventrolateral margin of the cell-body sparse optic tract (running from lower left comer of photograph to upper right) and is more slender in female (upper) than male mice (lower), resulting in sexual dimorphism in overall volume of the nucleus. 99 CHAPTER SIX GENERAL SUMMARY AND CONCLUSIONS I have characterized in this dissertation how, and to what extent, the adult posterodorsal aspect of the medial amygdala (MePD) differs at the cellular level between: males versus females; animals with a functional versus dysfunctional AR; in the presence versus absence of gonadal hormone; in response to short- versus long-term exposure; and in rats versus mice. In brief, the MePD is sexually dimorphic (male biased) in adult rats and mice and responds to adult manipulations of circulating gonadal androgens by exhibiting plasticity in several measures of cellular morphology. Our findings showed first that somal size and regional volume varied in males with dysfunctional ARs, but that the specific effects depend on the nucleus and the measure of cellular level changes being considered. Based on these findings, it seems reasonable to conclude that although ER affects these characteristics, AR effects cannot be ignored. Secondly, the number of neurons and glia in the MePD was greater in males than in females. While neuron number was unaffected by androgen manipulations, glial number was affected, mostly by increasing in the OVX+T females. The data support the idea that hormone induced effects on brain structure vary according to cell type and sex. Our experiments that followed the time course of morphological change in the MePD showed again that androgen effects vary by sex, and what structural features are being analyzed, as somal size changing in females faster than in males. 100 Lastly, we found that the mouse MePD, like the rat, is sexually dimorphic in regard to volume and mean somal area. Also, the mouse MePD could be maintained by E or T, even when deprived of a primary input following bulbectomy. Taken together, these experiments show differences in hormone plasticity in adulthood are influenced by many variables including sex, hormone receptor type, brain nuclei, cell type, time, and even hemisphere; yet effects may still be generalized to some extent across species (male biased dimorphisms, equivalent somal size response in the two hemispheres). I interpret these findings as a clear demonstration that hormones precisely regulate many aspects of neural structure. The first study replicated earlier findings of sex differences in MePD volume, somal size, and rostrocaudal extent (Cooke et al., 2003; Malsbury & McKay, 1994). Additionally, I studied male XY rats that have the tfm (testicular feminization mutation) allele (TFM males), a mutant allele of the AR that renders males largely unresponsive to androgens (Yarbough et al., 1990). TFM males do not display lordosis despite their feminine appearance (Olsen, 1979), so they are defeminized, presumably by aromatized metabolites of testosterone acting on estrogen receptors. They also show infrequent and incomplete male copulatory responses to receptive females (Beach and Buehler, 1977), but that could be due to either incomplete masculinization of brain sites such as the MePD or to the absence of normal male genitalia. We found regional volume in brain nuclei of TFM males to be fully masculinized in SDN-POA, intermediate in MePD, and fully demasculinized in the SCN. That cell somal size did not follow similar patterns as volume, shows again that these various models offer advantages for dissecting structure/function relationships. The TFM rat offers an advantage in testing the role of 101 AR compared to a pharmacological approach that utilizes DHT administration. DHT can be metabolized to 3u-androstanediol (3a-diol) which has a low affinity for ARs but a high affinity for GABA receptors, and 5a-androstan-3B,l7B-diol (3B-diol), an estrogenic compound which binds ERs (Kuiper et al., 1998). Additionally, DHT treatment might not reveal a contribution of AR in cases where T acts synergistically upon both ERs and ARs. This model may therefore offer a unique opportunity to help understand the role of AR in behaviors as well as the neural substrate of behavior. While we are limited with this model in interpreting the timing of AR activity in mediating these effects, additional studies could measure perinatally or pubertally mediated AR effects to further understand whether a window exists for these influences. We tested the dissociation of MePD volume and cell size again in the second study, also determining whether cell number in adulthood was affected by hormone manipulations. Sex differences in MePD volume may be caused by a number of integral anatomical components: cell size (soma and processes), cell number, vasculature, neuropil, and extracellular space. Although the size of neurons in the MePD is sexually dimorphic (Bubenik & Brown, 1973; Cooke et al., 1999; Morris et al., 2005; Hermel et al., 2006) we again found dissociations of neuronal size and regional volume indicating that other components of the MePD are probably affected by circulating androgen to alter MePD volume. We found no evidence to suggest that neuron numbers vary in adult rats following a month of androgen exposure or deprivation. Other reports show effects of T on cell number by showing that T increases neurogenesis in the medial amygdala of castrated adult male meadow voles (Fowler et al., 2003), and prevents apoptosis in the preoptic area of developing male rats, which resulted in a larger adult volume (Davis et 102 al., 1996). If similar effects occur in adult rats, they may be so slight as to escape detection by our estimates of neuronal number. We did find a hormone-mediated effect on glial number, derived from morphology based estimates, indicating this element is more plastic in adulthood. Recent studies implicate differences in synaptic number and the glial processes that may occur with them, contribute to MePD volumetric sexual dimorphism. (Cooke & Woolley, 2005; Cooke et al., 2007). Future studies may measure astrocytic vs oligodendritic contributions to the glial variances we found, as well as silver degeneration stains or Fluoro—Jade staining for to determine whether neuronal pruning or death are other indices of MePD plasticity. Time was shown to be another factor in MePD plasticity in our study that compared effects 2, 14, or 28 days after androgen manipulations in adulthood. Because at 14 days somata show evidence of androgen responsiveness while MePD volume has not yet responded, change in somata size may make little contribution to change in overall MePD volume, reinforcing again the dissociation of cell size and volume. Neither regional volume nor soma size is significantly different in the MePD two days after a change in androgen status. This was true both in male rats responding to castration and in female rats given male-typical levels of T. Likewise, 14 days later, MePD volume still showed no significant response to androgen manipulations in either sex. However, MePD soma size was altered two weeks after androgen manipulations, being larger in females given T than those given blank capsules. The effect of castration in males was not significant at this time point, although there was much greater variability in this measure in castrated males 14 days after surgery. Thus individual differences in the time 103 course of response to castration may have caused greater variability and made it more difficult to detect a statistically significant difference in males at this time point. Overall, these findings indicate that MePD soma size responds more rapidly than MePD volume to androgen manipulations. How responses differ across individuals may be an important factor given the wide range of effects of hormones in humans. I wanted to first consider whether rodent species differ in the same cellular measures of hormone responsiveness, because if they do not it may be because the effects may be generalized to even more evolutionarily separate species that would increase confidence in extrapolating findings from rodent models. On the other hand, if hormones vary between two rodent species in their influence on structural measures, then that would limit the extrapolation of hormonal effects to other species. At the same time, such species differences might yield an interesting means of understanding how differential effects of hormones on brain structures might correlate with species differences in behavior. My last experiment looked to mice for comparison in order to make some generalizations about steroid mediated effects in the MePD, but also to take advantage of genetic tools available in mouse models. Therefore, we set out to establish, with a similar paradigm, whether adult mice respond to circulating hormones. We found that the MePD is sexually dimorphic in volume, rostrocaudal extent, and somal area in BALB/c mice. Four weeks after castration of adult male mice, MePD regional volume and soma size are reduced, but rostrocaudal extent is not, compared to sham-castrated males. Estradiol treatment of adult ovariectonrized females for eight weeks increased MePD volume and soma size, but not rostrocaudal extent. To probe the possible role of afferents in the 1.04 steroid—induced plasticity of the MePD, we examined the effect of removing the olfactory bulbs in gonadally intact males and in estrogen-treated females. Bulbectomy had no effect on MePD morphology except among gonadally intact males where neuronal soma size was slightly smaller in the right MePD of bulbectomized males compared to males with intact bulbs. Thus the sexual dimorphism and hormone responsiveness of the MePD that has been extensively studied in rats is also present in mice. We detected little or no evidence that olfactory bulb afferents play a role in maintaining MePD morphology in adult mice. Although these studies provide evidence of gonadal hormone effects on brain nuclei, we still cannot be certain that the effects are direct. Studies have shown that the neurotrophic effects of hormones may act indirectly on other cells (Rand & Breedlove, 1995; Blurton-Jones et al., 2004), which then influence the neurons. This possibility complicates simple conclusions that hormone binding cells are changed by hormones directly. Future studies might target specific types of cells in order to determine what the target site of action for circulating hormones, such as been shown in the in the spinal cord, where cells showing functional AR were indeed the site of androgen action on motoneurons (Watson et al., 2001). To use tfm affected carrier (mosaic) females, the half of the AR cells affected by the mutation must be phenotyped by nuclear localization of AR using immunocytochemistry. However, a similar approach may prove difficult to conduct in the MePD due to the smaller size of MePD neurons, which makes phenotype based on relative nuclear AR—ir difficult. Unfortunately, it would also be more difficult to draw similar conclusions, as the populations of neurons in the brain are much more dense and heterogeneous than those in motor pools of the spinal cord, making it more 105 difficult to presume similar conditions across cells. Another strategy might include marking only astrocytes to determine if they respond differently than neurons. That glia change in number (as reported in Chapter 3) strengthens the idea that glial subtypes must be considered in future studies of the MePD. In fact, studies of the preoptic area (McCarthy et al. ,2003) have shown a clear hormone responsiveness of astrocytes to estradiol. Another concern regarding these studies would be that whenever a sexual dimorphism in the volume of a brain region is found, one question that normally arises is whether the dimorphism of the region simply reflects a sexual dimorphism of the entire brain. Indeed, we have found that in general adult male brains weigh more than female brains. However, evidence suggests that the effects detailed in this dissertation are specific to these particular brain regions. Malsbury & McKay, 1994, found no overall effect of androgen regimens on either brain weight or on the complete sectional area. Furthermore, in our study of tfm rodents, our effects varied by region in opposite directions (fully masculine in SDN-POA volume, yet lower than feminine in the SCN, showing that overall brain effects cannot explain the pattern of sexual dimorphisms we see in the MePD. Second, androgen effects in male rats are more prominent in the right MePD, whereas we would expect equivalent effects were the mechanism general. Additionally, while the MePD in TFM males are demasculinized, unpublished observations from our laboratory have noted overall brain weight in TFM rats to be fully masculine, again indicating that MePD dimorphism reflects a specific rather than a general effect. 106 These finding may have some implication or predictive power in humans. Brains of that species also contain a medial amygdala, but unlike the rodent, it does not appear to be parcellated (unpublished observations), which may be due to the reduced contribution of the primate olfactory bulbs to the medial amygdala. However, there are some interesting findings in the literature that may be considered in light of my results. The human amygdala is sexually dimorphic in structural growth (Giedd et al., 1997), possibly in hormone mediated asymmetries (Merke et al., 2003), and in response to sexual stimuli (Harnann et a1, 2004). The primate amygdala contains dense concentrations of hormone receptors, but the distribution varies in that the cortical amygdala appears to have a higher concentration than the nearby medial amygdala (Osterlund et al., 2000; Roselli et al., 2001). Finally, the amygdala of primates shows sexual dimorphisms of function and disease state. The human amygdala may be asymmetrical in size (Murphy 1987) and activity (Savic et al., 2005). Thus, there are enough similarities between species to predict that the olfactory driven human amygdala is sexually dimorphic and may respond to hormones in adulthood, both in function and structure. One study did compare the medial amygdala using the Yakovlev brain collection, but measures varied widely enough between individuals such that no sexual dimorphism was found (Murphy, 1986). However, because the subjects varied extensively in age, genetic background, and likely therefore in levels of circulating androgens, my results suggest a more controlled comparison might yield evidence of hormone mediated plasticity in the human amygdala. In surrunary, these experiments show the medial amygdala of rodents to be extremely responsive to gonadal steroids in adulthood. Several cellular aspects changed following the manipulation of adult circulating gonadal hormones including soma size, 107 regional volume, number of glia, and rostrocaudal extent. The influence of sex, cell type, hemisphere, and time were shown to be important considerations for the judging the effect of hormone exposure on brain structure. The time necessary for some of these changes to occur following hormone manipulation was shown to be less than reported before and may therefore refine understanding of contemporaneous changes in behavior. The role of the androgen receptor in sex differences was revealed to be greater than previously understood, which may have implications for neural and behavioral sex differences in humans. Characterization of hormone effects on plasticity in the adult mouse medial amygdala will allow for future research to utilize genomic tools to further reveal how hormones may affect the adult nervous system. 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