Estradiol promotes purkinje dendritic growth, spinogenesis, and synaptogenesis during neonatal life by inducing the expression of BDNF.

Neurosteroids are synthesized de novo from cholesterol in the brain. In rodents, the Purkinje cell actively produces several kinds of neurosteroids including estradiol during neonatal life, when cerebellar neuronal circuit formation occurs. Estradiol may be involved in cerebellar neuronal circuit formation through promoting neuronal growth and synaptic contact, because the Purkinje cell expresses estrogen receptor-ß. To test this hypothesis, in this study we examined the effect of estradiol on dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell using neonatal wild-type (WT) mice or cytochrome P450 aromatase knock-out (ArKO) mice. Administration of estradiol to neonatal WT or ArKO mice increased dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell. In contrast, WT mice treated with tamoxifen, an ER antagonist, or ArKO mice exhibited decreased Purkinje dendritic growth, spinogenesis, and synaptogenesis at the same neonatal period. Estrogen administration to neonatal WT or ArKO mice increased the expression of brain-derived neurotrophic factor (BDNF) in the cerebellum, whereas tamoxifen decreased the BDNF level in WT mice similar to ArKO mice. BDNF administration to tamoxifen-treated WT mice increased Purkinje dendritic growth. These results indicate that estradiol induces dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell via BDNF action during neonatal life.

Mode of action and functional significance of estrogen-inducing dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell.

Neurosteroids are synthesized de novo from cholesterol in the brain. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. Recently, we identified the Purkinje cell as an active neurosteroidogenic cell. In rodents, this neuron actively produces several neurosteroids including estradiol during neonatal life, when cerebellar neuronal circuit formation occurs. Estradiol may be involved in cerebellar neuronal circuit formation through promoting neuronal growth and neuronal synaptic contact, because the Purkinje cell expresses estrogen receptor-beta (ERbeta). To test this hypothesis, in this study we examined the effects of estradiol on dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell using neonatal wild-type (WT) mice or cytochrome P450 aromatase knock-out (ArKO) mice. Administration of estradiol to neonatal WT or ArKO mice increased dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell. In contrast, WT mice treated with tamoxifen, an ER antagonist, or ArKO mice exhibited decreased Purkinje dendritic growth, spinogenesis, and synaptogenesis at the same neonatal period. To elucidate the mode of action of estradiol, we further examined the expression of brain-derived neurotrophic factor (BDNF) in response to estrogen actions in the neonate. Estrogen administration to neonatal WT or ArKO mice increased the BDNF level in the cerebellum, whereas tamoxifen decreased the BDNF level in WT mice similar to ArKO mice. BDNF administration to tamoxifen-treated WT mice increased Purkinje dendritic growth. These results indicate that estradiol induces dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell via BDNF action during neonatal life.

Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell.

Neurosteroids are synthesized de novo in the brain, and the cerebellar Purkinje cell is a major site for neurosteroid formation. We have demonstrated that the Purkinje cell possesses intranuclear receptor for progesterone and actively produces progesterone de novo from cholesterol only during rat neonatal life, when cerebellar cortical formation occurs dramatically. We have further demonstrated that progesterone promotes dendritic growth, spinogenesis, and synaptogenesis via its receptor in this neuron in the neonate. On the other hand, estrogen may also play an important role in the process of cerebellar cortical formation, because the neonatal rat Purkinje cell possesses estrogen receptor (ER)beta. However, estrogen formation in the neonatal cerebellum is still unclear. In this study, we therefore analyzed the biosynthesis and action of estrogen in Purkinje cells during neonatal life. RT-PCR-Southern and in situ hybridization analyses showed that Purkinje cells expressed the key enzyme of estrogen formation, cytochrome P450 aromatase, in neonatal rats. A specific enzyme immunoassay for estradiol further indicated that cerebellar estradiol concentrations in the neonate were significantly higher than those in the prepuberty and adult. Both in vitro and in vivo studies with newborn rats showed that estradiol promoted dose-dependent dendritic growth of Purkinje cells. Estradiol also increased the density of Purkinje dendritic spines. These effects were inhibited by the ER antagonist tamoxifen. These results suggest that estradiol in the developing Purkinje cell promotes dendritic growth and spinogenesis via ERbeta in this neuron. Estradiol as well as progesterone may contribute to the growth of Purkinje cells during the cerebellar cortical formation.

Organizing actions of neurosteroids in the Purkinje neuron.

It is becoming clear that steroids can be synthesized de novo by the brain of vertebrates. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To understand neurosteroid actions in the brain, we need data on the specific biosynthesis in particular sites of the brain at particular times. Therefore our studies for this exciting area of neuroscience research have focused on the biosynthesis and action of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This neuron actively synthesizes progesterone and estradiol de novo from cholesterol only during neonatal life, when cerebellar cortical formation occurs dramatically. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently the actions of progesterone and estradiol during cerebellar development have become clear by a series of our studies using an excellent Purkinje cellular model. These neurosteroids promote dendritic growth, spinogenesis and synaptogenesis via each receptor in the Purkinje cell. Here we summarize the advances made in our understanding of organizing actions of neurosteroids in the Purkinje cell, an important brain neuron.

Neonatal expression of progesterone receptor isoforms in the cerebellar Purkinje cell in rats.

The cerebellar Purkinje cell (PC) is a typical site for neurosteroid formation. Progesterone synthesized de novo in developing PCs participates in the promotion of dendritic growth, spinogenesis and synaptogenesis in this neuron and such organizing actions may contribute to the formation of the cerebellar neuronal circuit during rat neonatal life. Progesterone receptors (PR) occur as two isoforms (PR-A and PR-B) derived from a single gene. To clarify the mode of organizing actions of progesterone, therefore, we examined the expression of these PR isoforms in the rat cerebellum during development. Western immunoblot analysis revealed that both PR isoforms were expressed highly in the cerebellum during neonatal life and the expression decreased thereafter. PR-like immunoreactivity was localized primarily in PCs in the neonatal cerebellum. Thus, progesterone may act directly on PCs via PR isoforms to promote its dendritic growth, spinogenesis and synaptogenesis.

Dendritic growth in response to environmental estrogens in the developing Purkinje cell in rats.

The cerebellar Purkinje cell is a major site for neurosteroid formation. We have demonstrated recently that the Purkinje cell actively produces sex steroids, such as estradiol and progesterone, de novo from cholesterol only during rat neonatal life, when cerebellar cortical formation occurs. We have further demonstrated that both estradiol and progesterone promote the growth of Purkinje cells through intranuclear receptor-mediated mechanisms during cerebellar development. On the other hand, environmental estrogens, such as octylphenol (OP), bisphenol A (BPA), and nonylphenol (NP) are thought to mimic the action of estrogen in the developing central nervous system. Therefore, in this study, the effect of these environmental estrogens on the growth of Purkinje cells was examined in vivo using newborn rats. OP and BPA promoted a dose-dependent dendritic outgrowth of the Purkinje cell but did not affect its soma and cell number. The stimulatory effect of OP and BPA on Purkinje dendritic growth was induced by an injection of 500 microg/day into the cerebrospinal fluid for 4 days and blocked by the estrogen receptor antagonist tamoxifen. However, there was no significant effect of NP on any Purkinje cell morphology. These results suggest that the environmental estrogens, OP and BPA, promote Purkinje dendritic growth during neonatal life. This effect may be mediated by estrogen receptor in the Purkinje cell.