Estradiol and the developing brain

Abstract

Estradiol is the most potent and ubiquitous member of a class of steroid hormones called estrogens. Fetuses and newborns are exposed to estradiol derived from their mother, their own gonads, and synthesized locally in their brains. Receptors for estradiol are nuclear transcription factors that regulate gene expression but also have actions at the membrane, including activation of signal transduction pathways. The developing brain expresses high levels of receptors for estradiol. The actions of estradiol on developing brain are generally permanent and range from establishment of sex differences to pervasive trophic and neuroprotective effects. Cellular end points mediated by estradiol include the following: 1) apoptosis, with estradiol preventing it in some regions but promoting it in others; 2) synaptogenesis, again estradiol promotes in some regions and inhibits in others; and 3) morphometry of neurons and astrocytes. Estradiol also impacts cellular physiology by modulating calcium handling, immediate-early-gene expression, and kinase activity. The specific mechanisms of estradiol action permanently impacting the brain are regionally specific and often involve neuronal/glial cross-talk. The introduction of endocrine disrupting compounds into the environment that mimic or alter the actions of estradiol has generated considerable concern, and the developing brain is a particularly sensitive target. Prostaglandins, glutamate, GABA, granulin, and focal adhesion kinase are among the signaling molecules co-opted by estradiol to differentiate male from female brains, but much remains to be learned. Only by understanding completely the mechanisms and impact of estradiol action on the developing brain can we also understand when these processes go awry.

FIG 1

A multiplicity of estradiol actions. Receptors for estradiol are members of a nuclear targeted transcription factor superfam-ily. Upon binding, the receptors dimerize and are translocated to the nucleus where they associate with coactivators such as steroid receptor coactivator 1 (SRC-1) and CREB binding protein (CBP) and form part of a preinitiation complex for inducing transcription. A critical function of this complex is the acetylation of histones on the chromatin to relax the DNA helix and allow for association with the palindromic sequences that constitute the estrogen response element (ERE). In addition to this long-term genomic effect of estradiol, the ER has been associated with the cell membrane of neurons where it can interact directly with signal transduction pathways such as that involving mitogen-activated protein kinase. Estradiol has also been reported to directly affect ion channels and G proteincoupled receptors, independent of the estrogen receptor (ER), and there may be a novel membrane-bound receptor for estradiol distinct from the classic ER’s, but this remains unsettled.

FIG 2

The organizational/activational hypothesis of estradiol action on the developing brain. Originally proposed in 1959 by Phoenix et al. (164), the organizational/activational hypothesis codified the principle that early hormone effects organize the brain such that adult hormonal effects are constrained by that prior exposure. The establishment of sex differences in physiology and behavior is a function of differential gonadal steroid synthesis during a perinatal sensitive period. In rats, the production of testicular androgens begins prenatally, around embryonic day 18, and defines the onset of the sensitive period. The female ovary remains quiescent, and a lack of exposure to androgens, and the aromatized product estradiol, is essential for normal female brain development. Treatment of females with exogenous testosterone results in its aromatization to estradiol and masculinization of adult brain and behavior. The developmental time point in which the female becomes insensitive to the masculinizing effects of exogenous testosterone operationally defines the end of the sensitive period. As adults, males show only pulsatile release of the gonadotropin luteinizing hormone (LH) from the pituitary, while females exhibit a large surge in LH release to induce ovulation at the midpoint of the estrus cycle. Likewise, only adult males exhibit the masculine pattern of sexual behavior of mounting a female, while only females adopt the sexually receptive posture termed lordosis. Exposure of developing females to testosterone, which is aromatized to estradiol, during the sensitive period will render them both sterile and sexually unreceptive. Both the male and female adult patterns are determined by hormonal organization during development but are dependent on adult sex-specific hormones to be activated in the adult.

FIG 3

Mechanisms of estradiol action for establishing a sexually dimorphic projection from the principle nucleus bed nucleus of the stria terminals (BSTp) to the anteroventral periventricular nucleus (AVPV). The AVPV is notable both for its central role in the control of the female-specific LH surge and for being larger in females than males. There is also a substantive sex difference in the size of the afferent projection from the BSTp to the AVPV, being up to 10-fold larger in males. To distinguish whether this sex difference in innervation arises in the BSTp or the AVPV, Simerly and colleagues (103)developed mixed-sex cocultures of the two nuclei. The BSTp is labeled with DiI (pseudo-colored red), and the AVPV is visualized with a Hoeschst stain (visualized here as green). Note the neuronal processes originating from the BSTp explant and extending toward the AVPV (left panel). The use of mixed sex cultures is illustrated with representative photomicrographs in the top panel and graphically by fiber density in the bottom panel. When a male-derived BSTp was paired with a male-derived AVPV (M/M), there were significantly more neurites extending toward the AVPV compared with a male-derived BSTp cocultured with a female-derived AVPV (M/F), and was not different from female-female cocultures (F/F). A female-derived BSTp innervated a male-derived AVPV (F/M) to the same degree as a M/M coculture, and treatment of females with testosterone before coculturing with a male BSTp resulted in a neurite growth rate identical to that of M/M cocultures. These data demonstrate that a hormonally determined target derived factor in the AVPV directs the innervation by the BSTp to produce a sexually dimorphic neural circuit. [From Ibanez et al. (103), copyright 2001 by Society for Neuroscience.]

FIG 4

Working model of estradiol action establishing sexually dimorphic astrocyte morphology and synaptic patterning in the arcuate nucleus. The arcuate nucleus is located in the dorsomedial hypothalamus and exerts a regulatory subluence over the anterior pituitary and adjacent hypothalamic nuclei to regulate such diverse functions as feeding, growth, stress responding, and reproduction. There are half as many dendritic spine synapses on neurons in the neonatal male arcuate as there are on female dendrites (126). This sex difference is entirely dependent on elevated estradiol in the male brain during the perinatal sensitive period. Conversely, the astrocytes in the male arcuate are far more stellate, with more processes that frequently branch, compared with females. Mong et al. (144) determined that estradiol increases the synthesis of the inhibitory neurotransmitter GABA by neurons, which then acts on neighboring astrocytes to induce stellation. It is speculated that the increased complexity of the astrocytes in males suppresses the formation of dendritic spine synapses, but the mechanism of how that is achieved is currently unknown.

FIG 5

Working model of mechanism of estradiol action establishing sexually dimorphic synaptic patterning in the preoptic area. The preoptic area (POA) is the critical brain region controlling expression of male sexual behavior and exhibits some of the most robust sex differences in the brain. In addition to the sexually dimorphic nucleus, male POA neurons have about twice as many dendritic spines as females, and this level of spines can be induced in females by treatment with estradiol during the perinatal sensitive period. Dendritic spines are the primary site for excitatory synapses. Astrocytes are also more complex in the male POA, with longer and more frequently branching processes. Both of these morphological sex differences are the result of estradiol action in the neonatal brain (4). The initiating event is the induction of COX-2, a pivotal enzyme in the production of prostanoids and specifically linked to an increased synthesis and release of prostaglandin E₂ (PGE₂). Receptors for PGE₂ are G protein linked and can be found on astrocytes. Activation of EP receptors can induce glutamate release from astrocytes in a calcium-dependent manner, and glutamate induces the formation of dendritic spines. In this system, application of PGE₂ induces a 2- to 3-fold increase in the density of dendritic spines on POA dendrites, and this effect can be blocked by antagonists to the glutamate AMPA receptor. Thus, in this model, a critical neuronal/astrocytic cross-talk is believed to be essential for PGE₂ to induce a sexually dimorphic synaptic pattern determined by estradiol.

FIG 6

Working model of mechanism of estradiol action establishing sexually dimorphic dendritic morphology in the ventromedial nucleus (VMN) of the hypothalamus. The VMN is the critical brain region controlling expression of female sexual behavior. Dendrites on neurons in this nucleus branch more frequently in males, and also have an overall greater number of spine synapses (210). This sex difference is established during the perinatal sensitive period and is a function of estradiol action in the male brain. The estradiol-induced increase in dendritic spines can be blocked by antagonism of the NMDA glutamate receptor and mimicked by application of an NMDA receptor-specific agonist. Downstream to activation of glutamate receptors is activation of the mitogen-activated protein kinase pathway, leading to spine formation (Schwarz et al., unpublished observations) and possibly dendritic branching. The fundamental effect of estradiol is to promote the release of glutamate from nerve terminals. Unlike the arcuate nucleus and the POA, no definitive role for astrocytes has been established in this system. However, since the primary effect of estradiol is presynaptic, there is a requirement for cell-to-cell communication to permanently organize this brain region into the masculine phenotype.

FIG 7

Masculinization, defeminization, and feminization of sex behavior. The embryonic brain is bipotential in its ability to adopt a masculine or feminine phenotype, but this potentiality is lost after a perinatal sensitive period. The reproductive behavior of the laboratory rodent provides a valuable model system for fundamental questions about how a sex phenotype is permanently imprinted on the immature brain. In this context, masculinization refers to the organization of the brain such that it will support the expression of male sexual behavior in adulthood. Defeminization is the removal of the default phenotype, the feminine. Defeminization is an active hormonally driven process that organizes the brain to preclude the expression of female sexual behavior in adulthood. Both masculinization and defeminization are induced by estradiol. Feminization occurs in the absence of high levels of estradiol. A: there are two opposing views of how these processes could be codified in the brain. Option one is a continuum; masculinization and feminization are opposite ends of the spectrum, and defeminization is an obligate component of masculinization. In this scenario, one could experimentally manipulate animals during the sensitive period such that in adulthood they could fall anywhere along the spectrum. In option two, masculinization and defeminization are viewed as two separate processes. In this scenario, it should be possible to create animals that show robust male and female sex behavior and animals that are essentially asexual, exhibiting neither male nor female sexual behavior. B: the discovery that PGE₂ is a fundamental determinant of behavioral masculinization allowed for a new examination of these two options by circumventing the need for estradiol to induce masculinization. By inducing masculinization with PGE₂, it could be determined if defeminization was also affected. Treatment of neonatal females with PGE₂ resulted in animals that would show either male or female sexual behavior as adults if provided the appropriate activational hormonal milieu. Likewise, treatment of newborn males with a cyclooxygenase (COX)-2 inhibitor to block the endogenous production of PGE₂ eliminated all sex behavior, regardless of the adult hormonal milieu. From these observations it is concluded that masculinization and defeminization are mechanistically distinct processes regulated by estradiol. How this steroid hormone mediates defeminization has yet to be determined.

FIG 8

Estradiol enhancement of depolarizing GABA action. The normally inhibitory transmitter GABA is predominantly excitatory in the developing brain due to a shift in the reversal potential for chloride so that activation of GABA_A receptors results in chloride efflux and membrane depolarization as opposed to hyperpolarization. The membrane depolarization induced by GABA is sufficient to activate voltage-gated calcium channels and promote calcium influx. This excitatory effect of GABA is linked to trophic actions and promotes the maturation of synapses. Estradiol enhances the excitatory actions of depolarizing GABA by increasing the magnitude of the calcium influx, increasing the number of neurons that respond to GABA as depolarizing and extending the developmental duration of depolarizing GABA. The principle action of estradiol is to increase the amount and activity of the sodiumpotassium-chloride cotransporter NKCC1 that maintains intracellular chloride. This transporter is expressed at high levels in mature neurons but gradually declines as development proceeds and is superceded by another transporter, KCC2, which transports chloride out of the cell. A potential deleterious consequence of the estradiol-induced enhancement of depolarizing GABA is a lowering of the threshold to excitotoxicity in the immature brain.

FIG 9

Estradiol provides neuroprotection from glutamate excitotoxicity in immature brain. Excessive calcium influx following overactivation of glutamate receptors is the gateway to adult neuronal excitotoxicity. NMDA and AMPA receptors are primary routes for calcium influx into mature neurons. In immature hippocampal neurons, glutamate-mediated excitotoxicity is determined by the mGluR class of glutamate receptors and involves release of calcium from internal stores. A: neurons imaged with the calcium-sensitive dye fura 2-AM can be visualized over time to reveal the dynamics of calcium handling. Bath application of kanic acid (KA) versus glutamate (Glu) reflects the differential sources of calcium. KA activates AMPA, and then NMDA receptors and calcium influx into the cell with a slow upward slope that terminates gradually. In contrast, glutamate activates metabotropic glutamate receptors which release calcium from internal stores in a rapid surge that quickly depletes the reserve before the drug application is complete. B: treatment of cultured immature hippocampal neurons with glutamate significantly increases cell death, as indicated by TUNEL assay; pretreatment with estradiol completely prevents the glutamate-induced cell death and is neuroprotective against spontaneous cell death as well. C: pseudo-colored image of neurons being imaged for free calcium shows the control condition in panel 1, revealing a high level of calcium, whereas in panel 2, a decrease in the peak amplitude of the calcium is observed following pretreatment with estradiol. This reduction in the amplitude of the calcium transient correlates with an observed decrease in the amount of metabotropic glutamate receptor following estradiol treatment. [From Hilton et al. (93), copyright 2006 Wiley-Blackwell Publishing.]