Anatomy, development, and plasticity of the neurosecretory hypothalamus in zebrafish.

The paraventricular nucleus (PVN) of the hypothalamus harbors diverse neurosecretory cells with critical physiological roles for the homeostasis. Decades of research in rodents have provided a large amount of information on the anatomy, development, and function of this important hypothalamic nucleus. However, since the hypothalamus lies deep within the brain in mammals and is difficult to access, many questions regarding development and plasticity of this nucleus still remain. In particular, how different environmental conditions, including stress exposure, shape the development of this important nucleus has been difficult to address in animals that develop in utero. To address these open questions, the transparent larval zebrafish with its rapid external development and excellent genetic toolbox offers exciting opportunities. In this review, we summarize recent information on the anatomy and development of the neurosecretory preoptic area (NPO), which represents a similar structure to the mammalian PVN in zebrafish. We will then review recent studies on the development of different cell types in the neurosecretory hypothalamus both in mouse and in fish. Lastly, we discuss stress-induced plasticity of the PVN mainly discussing the data obtained in rodents, but pointing out tools and approaches available in zebrafish for future studies. This review serves as a primer for the currently available information relevant for studying the development and plasticity of this important brain region using zebrafish.

Molecular neuroanatomy of the mouse medial preoptic area with reference to parental behavior.

The medial preoptic area (MPOA), an anterior part of the hypothalamus, is one of the most important areas for the regulation of instinctively motivated behaviors, such as parental behavior, mating behavior and aggression. Consistent with its role in reproductive behaviors, the MPOA abundantly expresses gonadal steroid hormone receptors and shows distinct sexual dimorphism in its morphology. Despite the functional importance of the MPOA, the anatomical demarcations of the mouse MPOA subregions have been confusing and remained undefined because of their heterogeneity and complexity. In this review, I first introduce our histological examination showing differential expression of various molecules among the MPOA subregions. I also provide useful molecular markers to delineate the mouse MPOA subregions showing sexual dimorphism. Based on this anatomical study at the subregion level, I also summarize the current understanding of the role of the mouse MPOA and adjacent bed nucleus of the stria terminalis in parental motivation: the central part of the MPOA is essential for parental motivation, and this area exerts an inhibitory effect on the neural activity in the BNST rhomboid nucleus resulting in suppressed infanticide.

Gonadal Hormone-Dependent Sexual Differentiation of a Female-Biased Sexually Dimorphic Cell Group in the Principal Nucleus of the Bed Nucleus of the Stria Terminalis in Mice.

We recently reported a female-biased sexually dimorphic area in the mouse brain in the boundary region between the preoptic area and the bed nucleus of the stria terminalis (BNST). We reexamined this area and found that it is a ventral part of the principal nucleus of the BNST (BNSTp). The BNSTp is a male-biased sexually dimorphic nucleus, but the ventral part of the BNSTp (BNSTpv) exhibits female-biased sex differences in volume and neuron number. The volume and neuron number of the BNSTpv were increased in males by neonatal orchiectomy and decreased in females by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. Sex differences in the volume and neuron number of the BNSTpv emerged before puberty. These sex differences became prominent in adulthood with increasing volume in females and loss of neurons in males during the pubertal/adolescent period. Prepubertal orchiectomy did not affect the BNSTpv, although prepubertal ovariectomy reduced the volume increase and induced loss of neurons in the female BNSTpv. In contrast, the volume and neuron number of male-biased sexually dimorphic nuclei that are composed of mainly calbindin neurons and are located in the preoptic area and BNST were decreased by prepubertal orchiectomy but not affected by prepubertal ovariectomy. Testicular testosterone during the postnatal period may defeminize the BNSTpv via binding directly to the androgen receptor and indirectly to the estrogen receptor after aromatization, although defeminization may proceed independently of testicular hormones in the pubertal/adolescent period. Ovarian hormones may act to feminize the BNSTpv during the pubertal/adolescent period.

Maternal care associates with differences in morphological complexity in the medial preoptic area.

The medial preoptic area (MPOA) is implicated in the expression of maternal behavior including the frequency of pup licking/grooming (LG) in the rat. Cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB) is a transcription factor that regulates the expression of many genes. We found that lactating rats that are more maternal towards their pups showing increased licking/grooming (i.e. high-LG mothers) had increased levels of phosphorylated CREB (pCREB) in the MPOA following a nursing bout and they displayed a reduced population of greater dendritic complexity index (DCI) neurons compared to less maternal rats showing decreased licking/grooming (i.e. low-LG mothers). CREB overexpression in MPOA neuronal cultures associated with a decrease in dendritic complexity and an increase in the expression of Rem2 and brain-derived neurotrophic factor (BDNF), genes implicated in dendritic pruning. While there were no differences in Rem2 expression in virgin high and low-LG female rats, Rem2 was significantly increased in the MPOA of high-LG compared to low-LG lactating rats. CREB activity in the MPOA associates with maternal behavior and reduced dendritic complexity possibly by increasing Rem2 expression.

Genetic sex and the volumes of the caudate-putamen, nucleus accumbens core and shell: original data and a review.

Sex differences are widespread across vertebrate nervous systems. Such differences are sometimes reflected in the neural substrate via neuroanatomical differences in brain region volume. One brain region that displays sex differences in its associated functions and pathologies is the striatum, including the caudate-putamen (dorsal striatum), nucleus accumbens core and shell (ventral striatum). The extent to which these differences can be attributed to alterations in volume is unclear. We thus tested whether the volumes of the caudate-putamen, nucleus accumbens core, and nucleus accumbens shell differed by region, sex, and hemisphere in adult Sprague-Dawley rats. As a positive control for detecting sex differences in brain region volume, we measured the sexually dimorphic nucleus of the medial preoptic area (SDN-POA). As expected, SDN-POA volume was larger in males than in females. No sex differences were detected in the volumes of the caudate-putamen, nucleus accumbens core or shell. Nucleus accumbens core volume was larger in the right than left hemisphere across males and females. These findings complement previous reports of lateralized nucleus accumbens volume in humans, and suggest that this may possibly be driven via hemispheric differences in nucleus accumbens core volume. In contrast, striatal sex differences seem to be mediated by factors other than striatal region volume. This conclusion is presented within the context of a detailed review of studies addressing sex differences and similarities in striatal neuroanatomy.

Sexually dimorphic distribution of calcium-binding protein, calretinin in the preoptic area of the freshwater catfish, Clarias batrachus (Linn.).

Preoptic area (POA) plays an important role in the hormonal regulation of the pituitary gland in vertebrates. In this study we report the sexually dimorphic distribution of calcium-binding proteins calretinin (CR) in the POA in the freshwater catfish, Clarias batrachus. Nissl staining highlighted the presence of the nucleus praeopticus periventricularis (NPP) and other subdivisions of the nucleus praeopticus (NPO), including supraoptic (NPOs), paraventricular (NPOp) and magnocellular (NPOm) divisions. In NPO, CR immunoreactivity was noted only in females but not in males. In both sexes, CR stained perikarya were found in the NPP. Sexually dimorphic localization of CR in the POA supports the notion that CR may play a gender-specific role and may be involved in hormonal regulation in fishes.

Social status and GnRH soma size in female convict cichlids (Amatitlania nigrofasciatus).

Gonadotropin-releasing hormone (GnRH) neurons in the preoptic area (POA) of the hypothalamus play a key role in regulating reproductive function. These neurons in turn are modulated by environmental influences, including the social environment. In both the Old World cichlid Astatotilapia burtoni and the New World cichlid Amatitlania nigrofasciatus, the size of the soma of GnRH expressing neurons in the POA varies with social status in males and breeding state in females. Dominant males have larger GnRH-releasing cells than subordinate males, and spawning females have larger GnRH-releasing cells than brooding females. A. nigrofasciatus is monogamous and both sexes engage in similar levels of aggression and territorial defense. Here we test whether female A. nigrofasciatus display GnRH-releasing cell plasticity as a function of dominant status. We find that GnRH-releasing neuron soma sizes are larger in dominant females and that this difference is independent of differences in gonado-somatic index in A. nigrofasciatus.

Molecular neuroanatomy and chemoarchitecture of the neurosecretory preoptic-hypothalamic area in zebrafish larvae.

The paraventricular nucleus (PVN) in mammals is the main hypothalamic nucleus controlling hormone release in the pituitary and plays pivotal roles in homeostasis. While the location of a PVN-homologous region has been described in adult fish as the neurosecretory preoptic area (NPO), this region has not been clearly defined in larval zebrafish due to the difficulty in defining cytoarchitectonic nuclear boundaries in the larval brain. Here we identify the precise location of the larval zebrafish NPO using conserved transcription factor and neuropeptide gene expressions. Our results identify the dorsal half of the preoptic-hypothalamic orthopedia a (otpa) domain as the larval NPO and the homologous region to the mammalian PVN. Further, by reconstructing the locations of cells producing zebrafish neuropeptides found in the mammalian PVN (CCK, CRH, ENK, NTS, SS, VIP, OXT, AVP), we provide the first 3D arrangement map of NPO neuropeptides in the larval zebrafish brain. Our results show striking conservation of transcription factor expression (otp, arx, dlx5a, isl1) in and around the NPO/PVN together with neuropeptide expression within it. Finally, we describe the exact anatomical location of cells producing Oxt and Avp in the adult zebrafish. Thus, our results identify the definitive borders and extent of the PVN homologous region in larval zebrafish and serve as an important basis for cross-species comparisons of PVN/NPO structure and function.

Neuroanatomy of the kisspeptin signaling system in mammals: comparative and developmental aspects.

Our understanding of kisspeptin and its actions depends, in part, on a detailed knowledge of the neuroanatomy of the kisspeptin signaling system in the brain. In this chapter, we will review our current knowledge of the distribution of kisspeptin cells, fibers, and receptors in the mammalian brain, including the development, phenotype, and projections of different kisspeptin subpopulations. A fairly consistent picture emerges from this analysis. There are two major groups of kisspeptin cell bodies: a large number in the arcuate nucleus (ARC) and a smaller collection in the rostral periventricular area of the third ventricle (RP3V) of rodents and preoptic area (POA) of non-rodents. Both sets of neurons project to GnRH cell bodies, which contain Kiss1r, and the ARC kisspeptin population also projects to GnRH axons in the median eminence. ARC kisspeptin neurons contain neurokinin B and dynorphin, while a variable percentage of those cells in the RP3V of rodents contain galanin and/or dopamine. Neurokinin B and dynorphin have been postulated to contribute to the control of GnRH pulses and sex steroid negative feedback, while the role of galanin and dopamine in rostral kisspeptin neurons is not entirely clear. Kisspeptin neurons, fibers, and Kiss1r are found in other areas, including widespread areas outside the hypothalamus, but their physiological role(s) in these regions remains to be determined.

Sex differences in the rapid control of aromatase activity in the quail preoptic area.

Adult male quail show high levels of aromatase activity in the preoptic area-hypothalamus (POA-HYP), which parallels the high number of aromatase-immunoreactive cells and elevated mRNA concentrations detected in this brain region by in situ hybridisation. Interestingly, females display considerably lower aromatase activity than males but have almost equal numbers of aromatase-immunoreactive cells and express similar levels of aromatase mRNA. Aromatase activity in the male POA-HYP can be rapidly regulated by calcium-dependent phosphorylations, in the absence of changes in enzyme concentration. In the present study, we investigated whether aromatase activity is differentially regulated by phosphorylations in males and females. A linear increase in accumulation of aromatisation products was observed in both sexes as a function of time but the rate of conversion was slower in females. Saturation analysis confirmed the lower maximum velocities (V(max) ) in females but indicated a similar affinity (K(m) ) in both sexes. Aromatase activity in females reacted differentially to manipulations of intracellular calcium. In particular, chelating calcium with ethylene glycol tetraacetic acid (EGTA) resulted in a larger increase of enzymatic activity in males than in females, especially in the presence of ATP. A differential reaction to kinase inhibitors was also observed between males and females (i.e. a larger increase in aromatase activity in females than in males after exposure to specific inhibitors). These findings suggest that the nature of aromatase is conserved between the sexes, although the control of its activity by calcium appears to be different. Additional characterizations of intracellular calcium in both sexes would therefore be appropriate to better understand aromatase regulation.

The development of male-oriented behavior in rams.

The sheep offers a unique mammalian model in which to study paradoxical same-sex sexual partner preferences. Variations in sexual partner preferences occur spontaneously with as many as 8% of rams in a population exhibiting a sexual preference for other rams (male-oriented). The current review presents an overview and update of the male-oriented ram model and discusses several theories that have been invoked to explain same-sex preferences in this species. Although our understanding of the biological determinants and underlying neural substrates of sexual attraction and mate selection are far from complete, compelling evidence is discussed that supports the idea that neural substrates regulating sexual partner preferences are organized during prenatal development. The challenge for future research will be to construct an integrated picture of how hormones, genes, and experience shape sexual partner preference.

Sex steroid-induced neuroplasticity and behavioral activation in birds.

The brain of adult homeothermic vertebrates exhibits a higher degree of morphological neuroplasticity than previously thought, and this plasticity is especially prominent in birds. In particular, incorporation of new neurons is widespread throughout the adult avian forebrain, and the volumes of specific nuclei vary seasonally in a prominent manner. We review here work on steroid-dependent plasticity in birds, based on two cases: the medial preoptic nucleus (POM) of Japanese quail in relation to male sexual behavior, and nucleus HVC in canaries, which regulates song behavior. In male quail, POM volume changes seasonally, and in castrated subjects testosterone almost doubles POM volume within 2 weeks. Significant volume increases are, however, already observable after 1 day. Steroid receptor coactivator-1 is part of the mechanism mediating these effects. Increases in POM volume reflect changes in cell size or spacing and dendritic branching, but are not associated with an increase in neuron number. In contrast, seasonal changes in HVC volume reflect incorporation of newborn neurons in addition to changes in cell size and spacing. These are induced by treatments with exogenous testosterone or its metabolites. Expression of doublecortin, a microtubule-associated protein, is increased by testosterone in the HVC but not in the adjacent nidopallium, suggesting that neuron production in the subventricular zone, the birthplace of newborn neurons, is not affected. Together, these data illustrate the high degree of plasticity that extends into adulthood and is characteristic of avian brain structures. Many questions still remain concerning the regulation and specific function of this plasticity.

How it's made: organisational effects of hormones on the developing brain.

Gonadal steroid hormones exert permanent organisational effects on the developing brain and thereby direct adult hormonal responsiveness to dictate sex-specific behaviour and physiology. Considerable progress has been made in elucidating the cellular mechanism of action of androgens and oestrogens during the perinatal sensitive period during which organisation occurs. This review highlights the findings obtained with respect to differential cell death and synaptogenesis with an emphasis on region-specific mechanisms that involve diverse signalling molecules including tumour necrosis factor-alpha, BAX, GABA, glutamate, prostaglandin E(2), FAK and paxillin.

Sexual experience changes sex hormones but not hypothalamic steroid hormone receptor expression in young and middle-aged male rats.

Testosterone is well known to regulate sexual behavior in males, but this is dependent upon prior sexual experience. Aging is associated with decreased libido and changes in testosterone, but the role of experience in these age-related processes has not been systematically studied. We examined effects of age and sexual experience on serum hormones (total testosterone, free testosterone, estradiol, LH) and on numbers of androgen receptor (AR) and estrogen receptor alpha (ERalpha) immunoreactive cells in the hypothalamus. Extensive sexual experience was given to male rats at 4 months of age. Rats were euthanized at either 4 months (young) or 12 months (middle-aged (MA)). Comparable sexually naïve male rats were handled and placed into the testing arena but did not receive any sexual experience. Thus, we had four groups: young-naïve, young-experienced, MA-naïve and MA-experienced. Serum hormone levels were assayed, and numbers of AR and ERalpha cells were quantified stereologically in the medial preoptic nucleus (MPN) and the anteroventral periventricular nucleus (AVPV). Sexually experienced males had significantly elevated serum testosterone and free testosterone in both age groups. Both total and free testosterone were higher, and estradiol lower, in middle-aged than young rats. Experience did not alter either AR or ERalpha expression in the preoptic brain regions studied. Aging was associated with increased expression of AR, but no change in ERalpha. These results show that sexual experience can induce short-term and long-term alterations in serum hormones but these effects are not manifested upon their receptors in the hypothalamus.

Dopaminergic projections to the medial preoptic area of postpartum rats.

Dopamine receptor activity in the rodent medial preoptic area (mPOA) is crucial for the display of maternal behaviors, as well as numerous other physiological and behavioral functions. However, the origin of dopaminergic input to the mPOA has not been identified through neuroanatomical tracing. To accomplish this, the retrograde tracer Fluorogold was iontophoretically applied to the mPOA of postpartum laboratory rats, and dual-label immunocytochemistry for Fluorogold and tyrosine hydroxylase later performed to identify dopaminergic cells of the forebrain and midbrain projecting to the mPOA. Results indicate that the number of dopaminergic cells projecting to the mPOA is moderate ( approximately 90 cells to one hemisphere), and that these cells have an unexpectedly wide distribution. Even so, more than half of the dual-labeled cells were found in either what has been considered extensions of the A10 dopamine group (particularly the ventrocaudal posterior hypothalamus and adjacent medial supramammillary nucleus), or in the A10 group of the ventral tegmental area. The rostral hypothalamus and surrounding region also contained numerous dual-labeled cells, with the greatest number found within the mPOA itself (including in the anteroventral preoptic area and preoptic periventricular nucleus). Notably, dual-labeled cells were rare in the zona incerta (A13), a site previously suggested to provide dopaminergic input to the mPOA. This study is the first to use anatomical tracing to detail the dopaminergic projections to the mPOA in the laboratory rat, and indicates that much of this projection originates more caudally than previously suggested.

Effects of estradiol, sex, and season on estrogen receptor alpha mRNA expression and forebrain morphology in adult green anole lizards.

Steroid hormones, especially estradiol, facilitate reproductive behaviors in male and female rodents and birds. In green anole lizards estradiol facilitates receptivity in females but, unlike in some other species, is not the activating hormone for courtship and copulatory behavior in males. Instead, testicular androgens directly facilitate male courtship and copulation. Yet, activity of the estradiol synthesizing enzyme aromatase is higher in the brain of male than female green anoles, and it is increased during the breeding compared to the non-breeding season. The functional relevance of these differences in local estradiol production is unknown. They might prime the male forebrain to facilitate production of appropriate sexual behaviors, perhaps by modifying morphology of relevant brain regions. In addition, we recently reported increased expression of estrogen receptor alpha (ERalpha) in selected brain regions in females compared to males [Beck LA, Wade J (2009b) Sexually dimorphic estrogen receptor alpha mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards. 55:398-403]. Thus, it is possible that the hormone serves to downregulate its receptor in males to inhibit the expression of estradiol-dependent receptive behaviors. To begin to address these ideas, the present study examines the effects of estradiol treatment, sex, and season on forebrain morphology and ERalpha mRNA abundance in three regions important for anole reproductive behavior-the preoptic area, ventromedial amygdala, and ventromedial hypothalamus. While a number of effects of sex and season on forebrain morphology were detected, direct effects of estradiol treatment on these measures were minimal. ERalpha expression was greatest in the ventromedial hypothalamus, and a large female-biased sex difference was detected in this area alone; it resulted from estradiol-treated animals. These results indicate a sex- and region-specific mechanism by which estradiol can modify ERalpha expression in the green anole and could impact the expression of female-typical receptivity.

Circuit projection from suprachiasmatic nucleus to ventral tegmental area: a novel circadian output pathway.

The suprachiasmatic nucleus (SCN) is a circadian pacemaker that synchronizes a number of vital processes. Although a great deal of research has focused on input pathways to SCN and on the central clock itself, relatively little is known about SCN output signaling pathways. The ventral tegmental area (VTA) has been extensively studied for its influence in motivated learning and, recently, for a potential role in arousal and sleep-wake regulation. Here we present data that SCN indirectly projects to VTA via the medial preoptic nucleus (MPON). Microinjection of the retrograde, transynaptic tracer pseudorabies virus (PRV) in rat VTA consistently labeled SCN neurons at time points indicative of an indirect circuit projection. To specify intermediate relay nuclei between SCN and VTA, putative relays were lesioned 1 week prior to PRV injections in VTA. Unilateral lesions of MPON reduced PRV labeling in SCN by 81.6% in the ipsilateral hemisphere and 75.8% in the contralateral hemisphere. Bilateral lesions of the caudal-dorsal lateral septum, another putative relay nucleus and dorsal injection control, did not significantly reduce PRV labeling in the SCN. Single-unit extracellular recordings under halothane anesthesia revealed a novel population of VTA neurons that selectively fired during the active circadian phase. These results show that SCN provides an indirect circuit pathway to VTA via MPON, and that VTA neurons exhibit a circadian rhythm in their impulse activity. This pathway may function in the circadian regulation of numerous behavioral processes including arousal and motivation.

Gonadal steroid action and brain sex differentiation in the rat.

Gonadal steroids that establish sexually dimorphic characteristics of brain morphology and physiology act at a particular stage of ontogeny. Testosterone secreted by the testes during late gestational and neonatal periods causes significant brain sexual dimorphism in the rat. This results in both sex-specific behaviour and endocrinology in adults. Sexual differentiation may be due to neurogenesis, migration or survival. Each mechanism appears to be uniquely regulated in a site-specific manner. Thus, the volume of an aggregate of neurones in the rat medial preoptic area (POA), termed the sexually dimorphic nucleus of the POA (SDN-POA), is larger in males than in females. The anteroventral periventricular nucleus (AVPV) is packed with neurones containing oestrogen receptor (ER)beta in female rats but, in males, ERbeta-positive neurones scatter into the more lateral portion of the POA. POA neurones are born up to embryonic days 16-17 and not after parturition. Therefore, neurogenesis is unlikely to contribute to the larger SDN-POA in males. DNA microarray analysis for oestrogen-responsive genes and western blotting demonstrated site-specific regulation of apoptosis- and migration-related genes in the SDN-POA and AVPV.

Age-related changes in hypothalamic androgen receptor and estrogen receptor alpha in male rats.

The control of reproductive function involves actions of sex steroids upon their nuclear receptors in the hypothalamus and preoptic area (POA). Whether hypothalamic hormone receptors change their expression in aging male mammals has not been extensively pursued, although such changes may underlie functional losses in reproductive physiology occurring with aging. We performed a stereologic analysis of immunoreactive androgen receptor (AR) and estrogen receptor alpha (ERalpha) cells in three POA nuclei of male Sprague-Dawley rats (anteroventral periventricular nucleus [AVPV], median preoptic area [MePO], and medial preoptic nucleus [MPN]), at young (3 months), middle-aged (12 months), and old (20 months) ages. Serum testosterone and estradiol levels were assayed. Testosterone concentrations decreased significantly and progressively with aging. Estradiol concentrations were significantly higher in middle-aged than either young or old rats. Stereologic analyses of the POA demonstrated that AR-immunoreactive cell numbers and density in the AVPV, MePO, and MPN were significantly higher in old compared with young or middle-aged rats. No change in the total number or density of ERalpha-immunoreactive cells was detected with age, although when cells were subdivided by intensity of immunolabeling, the most heavily labeled ERalpha cells increased in number with aging in the AVPV and MePO, and in density in the AVPV. There are several interpretations to our finding of substantially increased AR cell numbers during aging, including a potential compensatory upregulation of the AR under diminished testosterone concentrations. These results provide further information about how the neural targets of steroid hormones change with advancing age.

The volume of the ovine sexually dimorphic nucleus of the preoptic area is independent of adult testosterone concentrations.

The ovine sexually dimorphic nucleus (oSDN) is characterized by high levels of aromatase mRNA expression which can be used to delineate its boundaries. The volume of the oSDN is approximately 2 to 3-fold larger in rams that mate with ewes (female-oriented rams) than in rams that mate with other rams (male-oriented rams) and ewes. The sex difference in oSDN volume is present in late gestation fetuses and can be eliminated before birth by exposing genetic females to exogenous testosterone during midgestation, suggesting that early exposure to androgen masculinizes volume of the oSDN. The present study was performed to determine whether differences in oSDN volume are influenced by the adult hormonal environment. Adult rams, behaviorally characterized as female-oriented or male-oriented, and ewes were gonadectomized and treated with subcutaneous implants of testosterone to achieve physiologic concentrations of serum testosterone. Three weeks after implant placement brain tissue was prepared for histological assessment of oSDN volume using in situ hybridization for detection of aromatase mRNA expression. Quantitative analysis revealed that despite similar serum testosterone levels among the groups, the volume of the oSDN was greater in female-oriented rams than in male-oriented rams and ewes (P<0.05). Differences in oSDN volume were specific and not reflective of differences in preoptic area height or brain size. These results suggest that differences in the size of the oSDN in adult sheep were not influenced by adult exposure to testosterone.