Decoding neuronal composition and ontogeny of individual hypothalamic nuclei.

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.

Single cell transcriptome analysis of developing arcuate nucleus neurons uncovers their key developmental regulators.

Despite the crucial physiological processes governed by neurons in the hypothalamic arcuate nucleus (ARC), such as growth, reproduction and energy homeostasis, the developmental pathways and regulators for ARC neurons remain understudied. Our single cell RNA-seq analyses of mouse embryonic ARC revealed many cell type-specific markers for developing ARC neurons. These markers include transcription factors whose expression is enriched in specific neuronal types and often depleted in other closely-related neuronal types, raising the possibility that these transcription factors play important roles in the fate commitment or differentiation of specific ARC neuronal types. We validated this idea with the two transcription factors, Foxp2 enriched for Ghrh-neurons and Sox14 enriched for Kisspeptin-neurons, using Foxp2- and Sox14-deficient mouse models. Taken together, our single cell transcriptome analyses for the developing ARC uncovered a panel of transcription factors that are likely to form a gene regulatory network to orchestrate fate specification and differentiation of ARC neurons.

Endogenous amylin contributes to birth of microglial cells in arcuate nucleus of hypothalamus and area postrema during fetal development.

Amylin acts in the area postrema (AP) and arcuate nucleus (ARC) to control food intake. Amylin also increases axonal fiber outgrowth from the AP→nucleus tractus solitarius and from ARC→hypothalamic paraventricular nucleus. More recently, exogenous amylin infusion for 4 wk was shown to increase neurogenesis in adult rats in the AP. Furthermore, amylin has been shown to enhance leptin signaling in the ARC and ventromedial nucleus of the hypothalamus (VMN). Thus, we hypothesized that endogenous amylin could be a critical factor in regulating cell birth in the ARC and AP and that amylin could also be involved in the birth of leptin-sensitive neurons. Amylin+/- dams were injected with BrdU at embryonic day 12 and at postnatalday 2; BrdU+ cells were quantified in wild-type (WT) and amylin knockout (KO) mice. The number of BrdU+HuC/D+ neurons was similar in ARC and AP, but the number of BrdU+Iba1+ microglia was significantly decreased in both nuclei. Five-week-old WT and KO littermates were injected with leptin to test whether amylin is involved in the birth of leptin-sensitive neurons. Although there was no difference in the number of BrdU+c-Fos+ neurons in the ARC and dorsomedial nucleus, an increase in BrdU+c-Fos+ neurons was seen in VMN and lateral hypothalamus (LH) in amylin KO mice. In conclusion, these data suggest that during fetal development, endogenous amylin favors the birth of microglial cells in the ARC and AP and that it decreases the birth of leptin-sensitive neurons in the VMN and LH.

Distinct temporal requirements for Sonic hedgehog signaling in development of the tuberal hypothalamus.

Sonic hedgehog (Shh) plays well characterized roles in brain and spinal cord development, but its functions in the hypothalamus have been more difficult to elucidate owing to the complex neuroanatomy of this brain area. Here, we use fate mapping and conditional deletion models in mice to define requirements for dynamic Shh activity at distinct developmental stages in the tuberal hypothalamus, a brain region with important homeostatic functions. At early time points, Shh signaling regulates dorsoventral patterning, neurogenesis and the size of the ventral midline. Fate-mapping experiments demonstrate that Shh-expressing and -responsive progenitors contribute to distinct neuronal subtypes, accounting for some of the cellular heterogeneity in tuberal hypothalamic nuclei. Conditional deletion of the hedgehog transducer smoothened (Smo), after dorsoventral patterning has been established, reveals that Shh signaling is necessary to maintain proliferation and progenitor identity during peak periods of hypothalamic neurogenesis. We also find that mosaic disruption of Smo causes a non-cell autonomous gain in Shh signaling activity in neighboring wild-type cells, suggesting a mechanism for the pathogenesis of hypothalamic hamartomas, benign tumors that form during hypothalamic development.

Dlx1/2 and Otp coordinate the production of hypothalamic GHRH- and AgRP-neurons.

Despite critical roles of the hypothalamic arcuate neurons in controlling the growth and energy homeostasis, the gene regulatory network directing their development remains unclear. Here we report that the transcription factors Dlx1/2 and Otp coordinate the balanced generation of the two functionally related neurons in the hypothalamic arcuate nucleus, GHRH-neurons promoting the growth and AgRP-neurons controlling the feeding and energy expenditure. Dlx1/2-deficient mice show a loss-of-GHRH-neurons and an increase of AgRP-neurons, and consistently develop dwarfism and consume less energy. These results indicate that Dlx1/2 are crucial for specifying the GHRH-neuronal identity and, simultaneously, for suppressing AgRP-neuronal fate. We further show that Otp is required for the generation of AgRP-neurons and that Dlx1/2 repress the expression of Otp by directly binding the Otp gene. Together, our study demonstrates that the identity of GHRH- and AgRP-neurons is synchronously specified and segregated by the Dlx1/2-Otp gene regulatory axis.

Obesity during pregnancy in the mouse alters the Netrin-1 responsiveness of foetal arcuate nucleus neuropeptide Y neurones.

When individuals undergo gestation in an obese dam, they are at increased risk for impairments in the ability of the brain to regulate body weight. In rodents, gestation in an obese dam leads to a number of changes to the development of the hypothalamic neurones that regulate body weight, including reduced neuronal connectivity at birth. In the present study, we aimed to clarify how this neural circuitry develops normally, as well as to explore the mechanism underpinning the deficiency in connectivity seen in foetuses developing in obese dams. First, we developed an in vitro model for observing and manipulating the axonal growth of foetal arcuate nucleus (ARN) neuropeptide (NPY) neurones. We then used this model to test 2 hypotheses: (i) ARN NPY neurones respond to Netrin-1, one of a small number of axon growth and guidance factors that regulate neural circuit formation throughout the developing brain; and (ii) Netrin-1 responsiveness would be lost upon exposure to the inflammatory cytokine interleukin (IL)-6, which is elevated in foetuses developing in obese dams. We observed that ARN NPY neurones responded to Netrin-1 with a significant expansion of their growth cones, comprising the terminal apparatus that neurones use to navigate. Unexpectedly, we found further that NPY neurones from obese pregnancies had a reduced responsiveness to Netrin-1, raising the possibility that ARN NPY neurones from foetuses developing in obese dams were phenotypically different from normal NPY neurones. Finally, we observed that IL-6 treatment of normal NPY neurones in vitro led to a reduced growth cone responsiveness to Netrin-1, essentially causing them to behave similarly to NPY neurones from obese pregnancies. These results support the hypothesis that IL-6 can disrupt the normal process of axon growth from NPY neurones, and suggest one possible mechanism for how the body weight regulating circuitry fails to develop properly in the offspring of obese dams.

The LIM-homeobox transcription factor Isl1 plays crucial roles in the development of multiple arcuate nucleus neurons.

Neurons in the hypothalamic arcuate nucleus relay and translate important cues from the periphery into the central nervous system. However, the gene regulatory program directing their development remains poorly understood. Here, we report that the LIM-homeodomain transcription factor Isl1 is expressed in several subpopulations of developing arcuate neurons and plays crucial roles in their fate specification. Mice with conditional deletion of the Isl1 gene in developing hypothalamus display severe deficits in both feeding and linear growth. Consistent with these results, their arcuate nucleus fails to express key fate markers of Isl1-expressing neurons that regulate feeding and growth. These include the orexigenic neuropeptides AgRP and NPY for specifying AgRP-neurons, the anorexigenic neuropeptide αMSH for POMC-neurons, and two growth-stimulatory peptides, growth hormone-releasing hormone (GHRH) for GHRH-neurons and somatostatin (Sst) for Sst-neurons. Finally, we show that Isl1 directly enhances the expression of AgRP by cooperating with the key orexigenic transcription factors glucocorticoid receptor and brain-specific homeobox factor. Our results identify Isl1 as a crucial transcription factor that plays essential roles in the gene regulatory program directing development of multiple arcuate neuronal subpopulations.

Rbpj-κ mediated Notch signaling plays a critical role in development of hypothalamic Kisspeptin neurons.

The mammalian arcuate nucleus (ARC) houses neurons critical for energy homeostasis and sexual maturation. Proopiomelanocortin (POMC) and Neuropeptide Y (NPY) neurons function to balance energy intake and Kisspeptin neurons are critical for the onset of puberty and reproductive function. While the physiological roles of these neurons have been well established, their development remains unclear. We have previously shown that Notch signaling plays an important role in cell fate within the ARC of mice. Active Notch signaling prevented neural progenitors from differentiating into feeding circuit neurons, whereas conditional loss of Notch signaling lead to a premature differentiation of these neurons. Presently, we hypothesized that Kisspeptin neurons would similarly be affected by Notch manipulation. To address this, we utilized mice with a conditional deletion of the Notch signaling co-factor Rbpj-κ (Rbpj cKO), or mice persistently expressing the Notch1 intracellular domain (NICD tg) within Nkx2.1 expressing cells of the developing hypothalamus. Interestingly, we found that in both models, a lack of Kisspeptin neurons are observed. This suggests that Notch signaling must be properly titrated for formation of Kisspeptin neurons. These results led us to hypothesize that Kisspeptin neurons of the ARC may arise from a different lineage of intermediate progenitors than NPY neurons and that Notch was responsible for the fate choice between these neurons. To determine if Kisspeptin neurons of the ARC differentiate similarly through a Pomc intermediate, we utilized a genetic model expressing the tdTomato fluorescent protein in all cells that have ever expressed Pomc. We observed some Kisspeptin expressing neurons labeled with the Pomc reporter similar to NPY neurons, suggesting that these distinct neurons can arise from a common progenitor. Finally, we hypothesized that temporal differences leading to premature depletion of progenitors in cKO mice lead to our observed phenotype. Using a BrdU birthdating paradigm, we determined the percentage of NPY and Kisspeptin neurons born on embryonic days 11.5, 12.5, and 13.5. We found no difference in the timing of differentiation of either neuronal subtype, with a majority occurring at e11.5. Taken together, our findings suggest that active Notch signaling is an important molecular switch involved in instructing subpopulations of progenitor cells to differentiate into Kisspeptin neurons.

In Utero Development of Kisspeptin/GnRH Neural Circuitry in Male Mice.

The neuropeptide kisspeptin is a major regulator of the hypothalamus-pituitary-gonadal axis. Although it has long been known that kisspeptin and its receptor G protein-coupled receptor 54 (GPR54) are expressed in the developing brain well before puberty onset, the potential role of kisspeptin/GPR54 signaling in the embryonic brain has remained mysterious. Recent studies in female mice have shown that kisspeptin neurons in the arcuate nucleus of the hypothalamus (ARC) already communicate with a subset of GnRH neurons in utero. Whether this specific neural circuit is also formed in the developing male brain is not known. Here, we used a combination of different genetic strategies to analyze the ontogeny and development of the kisspeptin/GPR54 system in the male mouse brain. We demonstrate orchestrated onset of kisspeptin and GPR54 expression in the male embryonic mouse brain and find that androgen receptor and estrogen receptor-α immunoreactivity within the male brain delineate the birthplace of kisspeptin neurons in the ARC. Using conditional transsynaptic tracing from kisspeptin neurons, we find that ARC kisspeptin neurons already communicate with a subset of GnRH neurons in utero and that the neural circuits between ARC kisspeptin and GnRH neurons in the male mouse brain are established before birth. Furthermore, we also show that the connectivity between kisspeptin and GnRH neurons does not depend on the spatial position of GnRH neurons. Our data delineate the maturing neural circuits underlying control of the reproductive axis in the male embryonic mouse brain.

Maternal obesity and IL-6 lead to aberrant developmental gene expression and deregulated neurite growth in the fetal arcuate nucleus.

Maternal obesity during pregnancy increases the risk of obesity in the offspring. Several observations have pointed to a causative role for the proinflammatory cytokine IL-6, but whether it is present in the fetal circulation and how it acts on the developing fetus are unclear. We first observed that postnatal day 0 offspring from obese mothers had significantly reduced neuropeptide Y (NPY) innervation of the paraventricular nucleus (PVN) compared with that for offspring of normal-weight controls. Thus, the growth of NPY neurites from the arcuate nucleus (ARC) was impaired in the fetal brain by maternal obesity. The neurite growth regulator, Netrin-1, was expressed in the ARC and PVN and along the pathway between the two at gestational day (GD) 17.5 in normal animals, making it likely to be involved in the development of NPY ARC-PVN projections. In addition, the expression of Dcc and Unc5d, receptors for Netrin-1, were altered in the GD17.5 ARC in obese but not normal weight pregnancies. Thus, this important developmental pathway is perturbed by maternal obesity and may explain the defect in NPY innervation of the PVN that occurs in fetuses developing in obese mothers. To investigate whether IL-6 may play a role in these developmental changes, we found first that IL-6 was significantly elevated in the fetal and maternal circulation in pregnancies of obese mice compared with those of normal-weight mice. In addition, treatment of GD17.5 ARC tissue with IL-6 in vitro significantly reduced ARC neurite outgrowth and altered developmental gene expression similar to maternal obesity in vivo. These findings demonstrate that maternal obesity may alter the way in which fetal ARC NPY neurons respond to key developmental signals that regulate normal prenatal neural connectivity and suggest a causative role for elevated IL-6 in these changes.

Murine arcuate nucleus kisspeptin neurons communicate with GnRH neurons in utero.

Puberty is a transition period of reproductive development from juvenile stages to adulthood and depends upon the activity of gonadotropin-releasing hormone (GnRH) neurons. GnRH neurons are initially activated in utero but remain quiescent throughout the juvenile period. Premature reactivation of GnRH neurons results in precocious puberty in mice and humans, but the mechanisms underlying developmental control of GnRH neuron activity remain unknown. The neuropeptide kisspeptin, a potent activator of GnRH neurons that is implicated as a critical permissive signal triggering puberty and a major regulator of the adult female hypothalamus-pituitary-gonadal axis, is paradoxically produced by neurons in the developing brain well before puberty onset. Thus, the neural circuits controlling the timing of reproductive maturation remain elusive. Here, we delineate the underlying neural circuitry using conditional genetic transsynaptic tracing in female mouse embryos. We find that kisspeptin-producing neurons in the arcuate nucleus (ARC) already communicate with a specific subset of GnRH neurons in utero. We show that ARC kisspeptin neurons are upstream of GnRH neurons, and that GnRH neuron connectivity to ARC kisspeptin neurons does not depend on their spatial position in the brain. Furthermore, we demonstrate that the neural circuits between ARC kisspeptin and GnRH neurons are fully established and operative before birth. Finally, we find that most GnRH neurons express the kisspeptin receptor GPR54 upon circuit formation, suggesting that the signaling system implicated in gatekeeping puberty becomes operative in the embryo.

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.

Role of homeobox genes in the hypothalamic development and energy balance.

Homeobox genes contribute to the regionalization, patterning and cell differentiation during embryogenesis and organ development. During mammalian embryonic development, homeobox genes, including orthopedia (Otp), a brain-specific homeobox transcription factor (Bsx) and a thyroid transcription factor-1 (TTF-1), are expressed in the hypothalamus. The genetic ablation of these genes indicated that Otp and TTF-1 are essential for the normal morphological development of the hypothalamus, including the arcuate nucleus (ARC), whereas Bsx is not required. In the adult stage, Bsx and TTF-1 continue to be expressed in the hypothalamus, including the ARC, and serve as transcription factors of neuropeptide Y and agouti-related protein. The expression of hypothalamic Bsx and TTF-1 can be altered by the feeding state and appetite regulatory hormones such as ghrelin and leptin. Although Bsx and TTF-1 are essential for normal feeding behavior in adult mice, they exert different effects on the expression of hypothalamic pro-opiomelanocortin (POMC) and body weight homeostasis. Thus, the hypothalamic homeobox genes may contribute to the dissociation of food intake and body weight via AgRP-POMC neurons.

Fetal hypothalamic neuroprogenitor cell culture: preferential differentiation paths induced by leptin and insulin.

In response to temporally orchestrated growth factor stimulation, developing neural stem/progenitor cells undergo extensive self-renewal and then generate neurons and astrocytes. Fetal neonatal leptin and insulin deficiency results in reduced hypothalamic axonal pathways regulating appetite, which may predispose to offspring hyperphagia and obesity. Neural development of the arcuate nucleus, a key target of adiposity signals, leptin and insulin, is immature at birth. Hence, to explore proximate effects of leptin/insulin on hypothalamic development, we determined trophic and differentiation effects on neural stem/progenitor cells using a model of fetal hypothalamic neurospheres (NS). NS cultures were produced from embryonic d 20 fetal rats and passage 1 and passage 2 cells examined for proliferation and differentiation into neurons (neuronal nuclei, class IIIß-tubulin, and doublecortin) and astrocytes (glial fibrillary acidic protein). Leptin-induced NS proliferation was significantly greater than that induced by insulin, although both effects were blocked by Notch, extracellular signal-regulated kinase, or signal transducer and activator of transcription 3 inhibition. Leptin preferentially induced neuronal, whereas insulin promoted astrocyte differentiation. Extracellular signal-regulated kinase inhibition suppressed both leptin and insulin-mediated differentiation, whereas signal transducer and activator of transcription inhibition only affected leptin-mediated responses. These findings demonstrate preferential and disparate differentiation paths induced by leptin and insulin. Altered fetal exposure to leptin or insulin, resulting from fetal growth restriction, macrosomia, or maternal diabetes, may potentially have marked effects on fetal brain development.

Kisspeptins in reproductive biology: consensus knowledge and recent developments.

Kisspeptins, a family of neuropeptides encoded by the Kiss1 gene that are mainly expressed in discrete neuronal populations of the hypothalamus, have recently emerged as essential upstream regulatory elements of GnRH (gonadotropin-releasing hormone) neurons and, thereby, potent elicitors of gonadotropin secretion. Indeed, kisspeptins are now recognized as important regulators of key aspects of the maturation and function of the reproductive axis, including the sexual differentiation of the brain, the timing of puberty, the adult regulation of gonadotropin secretion by gonadal hormones, and the control of fertility by metabolic and environmental (e.g., photoperiod) cues. Appreciation of these fundamental biological features has led to the contention that kisspeptins are indispensable elements of the reproductive brain whose relevance goes beyond their crucial physiological roles and may pose potential pathophysiological and therapeutic interest. In spite of such a consensus, recent developments in the field have helped to expand, and somewhat challenged, our current understanding of the neuroendocrine and molecular mechanisms whereby some of the effects of kisspeptins are conducted. This review aims to provide a synoptic and balanced account of the consensus knowledge and recent findings in the field of kisspeptin physiology, which we predict will be crucial in shaping the progress of our understanding of the roles played by this family of neuropeptides in reproductive biology.

Development of leptin-sensitive circuits.

Energy homeostasis is achieved by the integration of peripheral metabolic signals by neural circuits. The organisation and function of neural circuits regulating energy homeostasis has been the subject of intense investigation and has led to the definition of a core circuitry in the hypothalamus that interacts with key regions in the brain stem, which appear to mediate many of the effects of the adipocyte-derived hormone leptin on feeding and energy balance. Recent data on the ontogeny of these pathways indicate that, in rodents, these feeding circuits primarily form during neonatal life and remain structurally and functionally immature until 3 weeks of life. Our understanding of the mechanisms promoting the formation of these critical circuits has been advanced significantly by recent evidence showing that neonatal leptin acts as a neurotrophic factor promoting the development of projections from the arcuate nucleus of the hypothalamus. Together with an expanding literature on the role of nutritional factors to affect health, these discoveries may contribute to our understanding on perinatally acquired predisposition to later disease, such as obesity and diabetes.

Expression of the transcriptional coactivator CITED1 in the adult and developing murine brain.

The transcription coactivator CITED1 is an important mediator of transcriptional events regulated by estrogen or TGF-beta. We used in situ hybridization to delineate the distribution of CITED1 mRNA in the adult and developing murine brain and found robust CITED1 expression in ventral hypothalamus and midbrain raphe. The distribution of CITED1 in these regions overlapped the reported expression of estrogen receptors alpha and beta. Less intense expression of CITED1 was also evident in medial preoptic area, subfornical organ, thalamus and cerebral cortex. CITED1 mRNA in the arcuate nucleus (an area of active transcriptional modulation by TGF-beta) was evident in postmigratory neurons as early as embryonic day 16. Expression of CITED1 in arcuate continued throughout postnatal development. CITED1 in developing cerebellum was first evident in external granule cells and was transiently expressed in the Purkinje cell/granule cell layer in a temporal pattern similar to estrogen receptor-beta. The spatial and temporal distribution of CITED1 mRNA reported here is consistent with a role for CITED1 in the modulation of transcriptional events mediated by steroid hormone and cytokine signaling pathways.

Prenatal development of hypothalamic neuropeptide systems in the nonhuman primate.

In the rodent, arcuate nucleus of the hypothalamus (ARH)-derived neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons have efferent projections throughout the hypothalamus that do not fully mature until the second and third postnatal weeks. Since this process is likely completed by birth in primates we characterized the ontogeny of NPY and melanocortin systems in the fetal Japanese macaque during the late second (G100), early third (G130) and late third trimesters (G170). NPY mRNA was expressed in the ARH, paraventricular nucleus (PVH), and dorsomedial nucleus of the hypothalamus (DMH) as early as G100. ARH-derived NPY projections to the PVH were initiated at G100 but were limited and variable; however, there was a modest increase in density and number by G130. ARH-NPY/agouti-related peptide (AgRP) fiber projections to efferent target sites were completely developed by G170, but the density continued to increase in the postnatal period. In contrast to NPY/AgRP projections, alphaMSH fibers were minimal at G100 and G130 but were moderate at G170. This study also revealed several significant species differences between rodent and the nonhuman primate (NHP). There were few NPY/catecholamine projections to the PVH and ARH prior to birth, while projections were increased in the adult. A substantial proportion of the catecholamine fibers did not coexpress NPY. In addition, cocaine and amphetamine-related transcript (CART) and alpha-melanocyte stimulating hormone (alphaMSH) were not colocalized in fibers or cell bodies. As a consequence of the prenatal development of these neuropeptide systems in the NHP, the maternal environment may critically influence these circuits. Additionally, because differences exist in the neuroanatomy of NPY and melanocortin circuitry the regulation of these systems may be different in primates than in rodents.

Evidence of altered brain sexual differentiation in mice exposed perinatally to low, environmentally relevant levels of bisphenol A.

Humans are routinely exposed to bisphenol A (BPA), an estrogenic chemical present in food and beverage containers, dental composites, and many products in the home and workplace. BPA binds both classical nuclear estrogen receptors and facilitates membrane-initiated estrogenic effects. Here we explore the ability of environmentally relevant exposure to BPA to affect anatomical and functional measures of brain development and sexual differentiation. Anatomical evidence of alterations in brain sexual differentiation were examined in male and female offspring born to mouse dams exposed to 0, 25, or 250 ng BPA/kg body weight per day from the evening of d 8 of gestation through d 16 of lactation. These studies examined the sexually dimorphic population of tyrosine hydroxylase (TH) neurons in the rostral periventricular preoptic area, an important brain region for estrous cyclicity and estrogen-positive feedback. The significant sex differences in TH neuron number observed in control offspring were diminished or obliterated in offspring exposed to BPA primarily because of a decline in TH neuron number in BPA-exposed females. As a functional endpoint of BPA action on brain sexual differentiation, we examined the effects of perinatal BPA exposure on sexually dimorphic behaviors in the open field. Data from these studies revealed significant sex differences in the vehicle-exposed offspring that were not observed in the BPA-exposed offspring. These data indicate that BPA may be capable of altering important events during critical periods of brain development.