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

Abstract

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.

Figure 1.

A–D, Morphological analysis of Purkinje cell dendrites of newborn WT mice treated with vehicle, EB, tamoxifen, or EB plus tamoxifen. Parasagittal sections of cerebella at 6 d of age were immunostained for calbindin (lobe IX). Scale bars, 10 μm. M, Molecular layer; P, Purkinje cell layer. E–H, Ultrastructural analysis of dendritic spines of Purkinje cells of newborn WT mice treated with vehicle, EB, tamoxifen, or EB plus tamoxifen. Parasagittal sections of cerebella at 6 d of age were immunostained for calbindin (lobe IX). Arrowheads indicate presumptive spine structures. Scale bars, 2 μm. PD, Purkinje cell dendrite.

Figure 2.

A, Quantitative analysis of the maximal Purkinje dendritic length of newborn WT mice treated with vehicle, EB, tamoxifen, or EB plus tamoxifen. Each column and error bar represent the mean ± SEM (n = 10 in each group). *p < 0.05 versus vehicle; ^†p < 0.05 EB or tamoxifen vs EB plus tamoxifen; ^☆☆p < 0.01 EB vs tamoxifen (by one-way ANOVA, followed by Duncan's multiple range test). B, Quantitative electron microscopic analysis of Purkinje dendritic spine density per unit area (100 μm² field) of newborn WT mice treated with vehicle, EB, tamoxifen, or EB plus tamoxifen. Each column and error bar represent the mean ± SEM (n = 5 in each group). *p < 0.05 versus vehicle; ^†p < 0.05 and ^††p < 0.01 EB or tamoxifen versus EB plus tamoxifen; ^☆☆p < 0.01 EB versus tamoxifen (by one-way ANOVA, followed by Duncan's multiple range test).

Figure 3.

A, B, Higher magnification of synaptic terminals in the molecular layers of vermal cerebella (lobe IX). Arrows indicate postsynaptic density (psd), synaptic vesicle (sv), synaptic cleft (sc), and presynaptic dense projections (pdp). PD, Purkinje cell dendrite. Scale bars, 500 nm. C, D, Quantitative electron microscopic analysis of Purkinje dendritic synapse density of newborn WT mice treated with vehicle, EB, tamoxifen, or EB plus tamoxifen. C, Axospinous synapse density. D, Shaft synapse density. Ultrathin sections (60 nm in thickness) containing calbindin-immunoreactive Purkinje dendrites in lobe IX at 6 d of age were analyzed. Each column and error bar represent the mean ± SEM (n = 5 in each group). *p < 0.05 and **p < 0.01 versus vehicle; ^†p < 0.05 and ^††p < 0.01 EB or tamoxifen versus EB plus tamoxifen; ^☆☆p < 0.01 EB versus tamoxifen (by one-way ANOVA, followed by Duncan's multiple range test).

Figure 4.

A–C, Morphological analysis of Purkinje cell dendrites of WT (A), ArKO (B), and EB-treated ArKO (C; ArKO + Estradiol benzoate) newborn mice. Parasagittal sections of cerebella at 6 d of age were immunostained for calbindin (lobe IX). Scale bars, 10 μm. M, Molecular layer; P, Purkinje cell layer. D–F, Ultrastructural analysis of dendritic spines of Purkinje cells of WT (D), ArKO (E), and EB-treated ArKO (F) newborn mice. Parasagittal sections of cerebella at 6 d of age were immunostained for calbindin (lobe IX). Arrowheads indicate presumptive spine structures. Scale bars, 2 μm. PD, Purkinje cell dendrite.

Figure 5.

A, Quantitative analysis of the maximal Purkinje dendritic length of WT, ArKO, and EB-treated ArKO (ArKO + Estradiol benzoate) newborn mice. Each column and error bar represent the mean ± SEM (n = 5 in each group). *p < 0.05 versus WT; ^†p < 0.05 ArKO versus ArKO plus EB (by one-way ANOVA, followed by Duncan's multiple range test). B, Quantitative electron microscopic analysis of Purkinje dendritic spine density per unit area (100 μm² field) of WT, ArKO, and EB-treated ArKO newborn mice. Each column and error bar represent the mean ± SEM (n = 5 in each group). **p < 0.01 versus WT; ^††p < 0.01 ArKO versus ArKO plus EB (by one-way ANOVA, followed by Duncan's multiple range test).

Figure 6.

Quantitative electron microscopic analysis of Purkinje dendritic synapse density of WT, ArKO, and EB-treated ArKO (ArKO + Estradiol benzoate) newborn mice. A, Axospinous synapse density. B, Shaft synapse density. Ultrathin sections (60 nm in thickness) containing calbindin-immunoreactive Purkinje dendrites in lobe IX at 6 d of age were analyzed. Each column and error bar represent the mean ± SEM (n = 5 in each group). **p < 0.01 versus WT; ^††p < 0.01 ArKO versus ArKO plus EB (by one-way ANOVA, followed by Duncan's multiple range test).

Figure 7.

Changes in the level of BDNF in the neonatal cerebellum after estrogen manipulation. A, Comparison of the level of BDNF in newborn WT mice treated with vehicle, EB, tamoxifen, or EB plus tamoxifen. Each column and error bar represent the mean ± SEM (n = 8 in each group). *p < 0.05 versus vehicle; ^†p < 0.05 tamoxifen versus EB plus tamoxifen; ^☆☆p < 0.01 EB versus tamoxifen (by one-way ANOVA, followed by Duncan's multiple range test). B, Comparison of the level of BDNF in WT, ArKO, and EB-treated ArKO (ArKO + Estradiol benzoate) newborn mice. Each column and error bar represent the mean ± SEM (n = 8 in each group). **p < 0.01 versus WT; ^†p < 0.05 ArKO versus EB-treated ArKO (by one-way ANOVA, followed by Duncan's multiple range test).

Figure 8.

Changes in the level of NT-3 in the neonatal cerebellum after estrogen manipulation. A, Comparison of the level of NT-3 in newborn WT mice treated with vehicle, EB, tamoxifen, or EB plus tamoxifen. Each column and error bar represent the mean ± SEM (n = 8 in each group). B, Comparison of the level of NT-3 in WT, ArKO, and EB-treated ArKO (ArKO + Estradiol benzoate) newborn mice. Each column and error bar represent the mean ± SEM (n = 8 in each group).

Figure 9.

A–C, Morphological analysis of Purkinje cell dendrites of newborn WT mice treated with vehicle, tamoxifen, or BDNF plus tamoxifen. Parasagittal sections of cerebella at 6 d of age were immunostained for calbindin (lobe IX). Scale bars, 10 μm. M, Molecular layer; P, Purkinje cell layer. D, Quantitative analysis of the maximal Purkinje dendritic length of newborn WT mice treated with vehicle, tamoxifen, or BDNF plus tamoxifen. Each column and error bar represent the mean ± SEM (n = 10 in each group). *p < 0.05 versus vehicle; ^†p < 0.05 tamoxifen versus BDNF plus tamoxifen (by one-way ANOVA, followed by Duncan's multiple range test).