María Julia Cambiasso

Sexual differentiation of the brain: genes, estrogen, and neurotrophic factors.

Based on evidence obtained during the past 50 years, the current hypothesis to explain the sexual dimorphism of structure and function in the brain of vertebrates maintains that these differences are produced by the epigenetic action of gonadal hormones. However, evidence has progressively accumulated suggesting that genetic mechanisms controlling sexual-specific neuronal characteristics precede, or occur in parallel with, hormonal effects.1. In cultures of hypothalamic neurons taken from gestation day 16 (GD16) embryos, treatment of sexually segregated cultures with estradiol (E2) induces axon growth in neurons from male neurons, but not from female neurons. In these cultures treatment with E2 increased the levels of tyrosine kinase type B (TrkB) and insulin-like growth factor I (IGF-I) receptors in male but not in female neurons. This and other sex differences cannot be explained by differences in hormonal environment, because the donor embryos were obtained when gonadal secretion of steroids is just beginning, before the perinatal surge of testosterone that determines development of the male brain beginning at GD17/18.2. The response to estrogen is contingent upon coculture with heterotopic glia (mostly astrocytes) from a target region (amygdala) harvested from same-sex fetuses at GD16, whereas in the presence of homotopic glia or in cultures without glia, E2 had no effect. It was concluded that the axogenic effect of E2 depends on interaction between neurons and glia from a target region and that neurons from fetal male donors appear to mature earlier than neurons from females, a differentiated response that takes place prior to divergent exposure to gonadal secretions.3. The effects of target and nontarget glia-conditioned media (CM) on the E2-induced growth of neuronal processes of hypothalamic neurons obtained from sexually segregated fetal donors were also studied. Estrogen added to media conditioned by target glia modified the number of primary neurites and the growth of axons of hypothalamic neurons of males but not of females.4. Neither the Type III steroidal receptor blocker tamoxifen nor Type I antiestrogen ICI 182,780 prevented the axogenic effects of the hormone. Estradiol made membrane-impermeable by conjugation to a protein of high molecular weight (E2-BSA) preserved its axogenic capacity, suggesting the possibility of a membrane effect responsible for the action of E2.5. Western blot analysis of the tyrosine kinase type A (TrkA), type B (TrkB), type C (TrkC), and insulin-like growth factor (IGF-I R) receptors in extracts from homogenates of cultured hypothalamic neurons showed that in cultures of male-derived neurons grown with E2 and CM from target glia, the amounts of TrkB and IGF-I R increased notably. Densitometric quantification showed that these cultures had more TrkB than cultures with CM alone or E2 alone. On the contrary, in cultures of female-derived neurons, the presence of CM alone induced maximal levels of TrkB, which were not further increased by E2; female-derived neurons in all conditions did not contain IGF-I R. Levels of TrkC were not modified by any experimental condition in male- or female-derived cultures and Trk A was not found in the homogenates.These results are compared with similar data from other laboratories and integrated in a model for the confluent interaction of estrogen and neurotrophic factors released by glia that may contribute to the sexual differentiation of the brain.

Differential effect of oestradiol and astroglia-conditioned media on the growth of hypothalamic neurons from male and female rat brains.

To determine whether soluble products from different CNS regions differ in their ability to support oestrogen‐stimulated neurite growth, hypothalamic neurons from sexually segregated embryos were cultured with astroglia‐conditioned medium (CM) derived from cortex, striatum and mesencephalon, with or without 17‐β‐oestradiol 100u2003n m added to the medium. After 48u2003h in vitro, neurite outgrowth was quantified by morphometric analysis. Astroglia‐CM from mesencephalon (a target for the axons of hypothalamic neurons) induced the greatest axogenic response in males and in this case only a neuritogenic effect could be demonstrated for oestradiol. On the other hand, astroglia‐CM from regions that do not receive projections from ventromedial hypothalamus inhibited axon growth. A sexual difference in the response of hypothalamic neurons to astroglia‐CM and oestradiol was found; growth of neurons from female foetuses was increased by astroglia‐CM from mesencephalon, but no neuritogenic effect could be demonstrated for oestradiol in these cultures. Blot immunobinding demonstrated the presence of receptors for neurotrophic factors in cultures of hypothalamic neurons; Western blot analysis of these cultures demonstrated that oestradiol increased the concentration of trkB and IGF‐I Rβ, whereas trkA was not detected and the concentration of trkC was not modified. These results support the hypothesis that target regions produce some factor(s) that stimulate the growth of axons from projecting neurons and further indicate that in the case of males this effect is modulated by oestradiol, perhaps mediated through the upregulation of trkB and IGF‐I receptors.

Effects of estrogen on neuronal growth and differentiation

Previous work from our laboratory has shown that in cultures of hypothalamic neurons obtained from male fetuses at embryonic day 16 the axogenic response to estradiol (E2) is contingent upon culture with medium conditioned by astroglia from a target region for hypothalamic axons. E2 also induced increased levels of TrkB that were necessary for the axonal growth to occur. This convergence between estrogenic and neurotrophic signals prompted investigation of the mitogen activated protein kinase (MAPK) cascade. Analysis of the temporal course of MAPK activation showed increased levels of phosphorylated ERK up to 60 min after E2 exposure, with a maximal response at 5-15 min. UO126 (specific inhibitor of MEK 1/2) blocked E2 induced axonal elongation and ERK phosphorylation, confirming the involvement of ERK in the neuritogenic effect of E2. The membrane impermeable construct E2-BSA proved as effective as free E2 to induce axon elongation, suggesting that E2 exerted its effect through a membrane-associated receptor. This possibility received additional support from experiments showing that E2-BSA also increased ERK phosphorylation with the same time course than E2. These results indicate that ERK signaling is necessary for E2 to induce axon growth and this activation is mediated by a membrane bound estrogen receptor.

Molecular mechanisms involved in the regulation of neuritogenesis by estradiol: Recent advances.

This review analyzes the signaling mechanisms activated by estradiol to regulate neuritogenesis in several neuronal populations. Estradiol regulates axogenesis by the activation of the mitogen activated protein kinase (MAPK) cascade through estrogen receptor α located in the plasma membrane. In addition, estradiol regulates MAPK signaling via the activation of protein kinase C and by increasing the expression of brain derived neurotrophic factor and tyrosine kinase receptor B. Estradiol also interacts with the signaling of insulin-like growth factor-I receptor through estrogen receptor α, modulating the phosphoinositide-3 kinase signaling pathway, which contributes to the stabilization of microtubules. Finally, estradiol modulates dendritogenesis by the inhibition of Notch signaling, by a mechanism that, at least in hippocampal neurons, is mediated by G-protein coupled receptor 30. This article is part of a Special Issue entitled Neurosteroids.

Neurotrophic Factors and Estradiol Interact To Control Axogenic Growth in Hypothalamic Neurons

Abstract: Previous work from our laboratory has shown that in cultures of hypothalamic neurons obtained from male fetuses at embryonic day 16, the axogenic response to estrogen (E2) is contingent on coculture with target glia or target glia‐conditioned media (CM). Neither the estrogen receptor blockers tamoxifen nor ICI 182,780 prevented the axogenic effects of the hormone. Estradiol made membrane‐impermeable by conjugation to a protein of high molecular weight (E2‐BSA) preserved its axogenic capacity, suggesting the possibility of a membrane effect responsible for the action of E2. Western blot analysis of extracts from homogenates of cultured neurons grown with E2 and CM from target glia had more TrkB than cultures with CM alone or E2 alone. To further investigate the interaction between E2 and the neurotrophin receptors, we used a specific antisense oligonucleotide (AS) to prevent the estradiol‐induced increase of TrkB. The effect of E2 was suppressed in cultures in which TrkB was down‐regulated by the AS, showing decreased axonal elongation when compared with neurons treated with E2 without AS or with sense TrkB. In cultures grown with AS, the axonal length of E2‐treated cultures was not different from cultures without E2. Evidence suggesting cross‐talk between E2 and neurotrophic factor(s) prompted investigation of signaling along the MAPK cascade. Immuno blotting of E2‐treated cultures showed increased levels of phosphorylated ERK1 and ERK2. UO126 but not LY294002 blocked E2‐induced axonal elongation, suggesting that the MAPKs are involved in this response.

Oestrogens and Progestagens: Synthesis and Action in the Brain.

When steroids, such as pregnenolone, progesterone and oestrogen, are synthesised de novo in neural tissues, they are more specifically referred to as neurosteroids. These neurosteroids bind specific receptors to promote essential brain functions. Pregnenolone supports cognition and protects mouse hippocampal cells against glutamate and amyloid peptide‐induced cell death. Progesterone promotes myelination, spinogenesis, synaptogenesis, neuronal survival and dendritic growth. Allopregnanolone increases hippocampal neurogenesis, neuronal survival and cognitive functions. Oestrogens, such as oestradiol, regulate synaptic plasticity, reproductive behaviour, aggressive behaviour and learning. In addition, neurosteroids are neuroprotective in animal models of Alzheimers disease, Parkinsons disease, brain injury and ageing. Using in situ hybridisation and/or immunohistochemistry, steroidogenic enzymes, including cytochrome P450 side‐chain cleavage, 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 isomerase, cytochrome P450arom, steroid 5α‐reductase and 3α‐hydroxysteroid dehydrogenase, have been detected in numerous brain regions, including the hippocampus, hypothalamus and cerebral cortex. In the present review, we summarise some of the studies related to the synthesis and function of oestrogens and progestagens in the central nervous system.

Inhibition of tyrosine kinase receptor type B synthesis blocks axogenic effect of estradiol on rat hypothalamic neurones in vitro

17‐β‐estradiol (E2) increases axonal growth and tyrosine kinase receptor (Trk)B levels of male‐derived hypothalamic neurones in vitro. To investigate whether the axogenic response depends on the upregulation of TrkB, we analysed neuritic growth and neuronal polarization in cultures treated with an antisense oligonucleotide against TrkB mRNA. In cultures without E2, treatment with 7.5 or 10u2003µm antisense reduced TrkB levels and the percentage of neurones showing an identifiable axon; the number and length of minor processes were increased. In cultures treated with 5u2003µm antisense, morphometric parameters were normal although total TrkB levels were reduced. The same dose prevented the E2‐dependent increase of TrkB levels and suppressed the axogenic effect of E2. These results indicate that TrkB is necessary for normal neuronal growth and maturation and further suggest that an increase in TrkB is necessary for E2 to exert its axogenic effect in male‐derived neurones.

Sex Chromosome Complement Contributes to Sex Differences in Bradycardic Baroreflex Response

To investigate whether sex chromosome complement modulates bradycardic baroreflex response and contributes to the angiotensin II–bradycardic baroreflex sex differences, we used the four core genotype mouse model in which the effect of gonadal sex and sex chromosome complement is dissociated, allowing comparisons of sexually dimorphic traits among XX and XY females, as well as in XX and XY males. In conscious gonadectomized (GDX) MF1 transgenic mice we evaluated baroreflex regulation of heart rate in response to changes in blood pressure evoked by phenylephrine (1 mg/mL), angiotensin II (100 &mgr;g/mL), and sodium nitroprusside (1 mg/mL). The administration of phenylephrine in GDX-XY females resulted in a significantly lower baroreflex response when compared with the other genotypes (in beats · min−1 · mm Hg−1 [slopes of regression lines for GDX-XY females −3.56±0.37 versus −6.06±0.38, −6.37±0.54 and −6.70±0.34 for GDX-XY male, GDX-XX female, and GDX-XX male mice, respectively]) {F(1,19)=9.63; P<0.01}. In addition, in both GDX-XY males and females, the angiotensin II-bradycardic baroreflex response was attenuated when compared with heart rate changes in GDX-XX male and female mice (in beats · min−1 · mm Hg−1 [slopes of regression lines: −2.83±0.28 versus −5.76±0.26 in GDX-XY and GDX-XX mice, respectively]) {F(1,19)=13.91; P<0.005}. In contrast, reflex tachycardic responses to sodium nitroprusside were comparable in all of the genotypes. These data support the hypothesis that sex chromosome complement modulates reflex inhibition of heart rate to phenylephrine and angiotensin II. Elucidating the foundational sources of sexually dimorphic traits in the regulation of baroreceptor reflex may enable the design of more appropriate sex-tailored therapeutic treatments in the future.

Erk activation in the amygdala and hippocampus induced by fear conditioning in ethanol withdrawn rats: Modulation by mk-801

The extracellular signal-regulated kinase (ERK) pathway, which can be activated by NMDA receptor stimulation, is involved in fear conditioning and drug addiction. We have previously shown that withdrawal from chronic ethanol administration facilitated the formation of contextual fear memory. In order to explore the neural substrates and the potential mechanism involved in this effect, we examined: 1) the ERK1/2 activation in the central (CeA) and basolateral (BLA) nuclei of the amygdala and in the dorsal hippocampus (dHip), 2) the effect of the NMDA receptor antagonist MK-801 on fear conditioning and ERK activation and 3) the effect of the infusion of U0126, a MEK inhibitor, into the BLA on fear memory formation in ethanol withdrawn rats. Rats made dependent via an ethanol-containing liquid diet were subjected to contextual fear conditioning on day 3 of ethanol withdrawal. High basal levels of p-ERK were found in CeA and dHip from ethanol withdrawn rats. ERK activation was significantly increased both in control (60min) and ethanol withdrawn rats (30 and 60min) in BLA after fear conditioning. Pre-training administration of MK-801, at a dose that had no effect on control rats, prevented the increase in ERK phosphorylation in BLA and attenuated the freezing response 24h later in ethanol withdrawn rats. Furthermore, the infusion of U0126 into the BLA, but not the CeA, before fear conditioning disrupted fear memory formation. These results suggest that the increased fear memory can be linked to changes in ERK phosphorylation, probably due to NMDA receptor activation in BLA in ethanol withdrawn rats.

17β-Estradiol stimulates the translocation of endogenous estrogen receptor α at the plasma membrane of normal anterior pituitary cells.

In the present work we aimed at identifying ERα in the plasma membrane of normal anterior pituitary cells and investigated if 17β-estradiol was able to induce their subcellular redistribution. Our results show that about 8% of anterior pituitary cells expressed ERα in the plasma membrane, with the geometrical mean fluorescence intensity being increased after steroid hormone treatment. 17β-Estradiol and the selective ERα agonist PPT induced an increase of ERα expression in the plasma membrane and activated the PKCα/ERK 1/2 pathway in a time-course not compatible with genomic actions, thus supporting the notion of membrane-initiated effects. These findings suggest that 17β-estradiol stimulates the translocation of endogenous ERα to the plasma membrane, consequently modulating this ER pool and leading to cellular biological effects in normal anterior pituitary gland.