In adult mammalian brain, occurrence of the synthesis of estradiol from endogenous cholesterol has been doubted because of the inability to detect dehydroepiandrosterone synthase, P45017␣. In adult male rat hippocampal formation, significant localization was demonstrated for both cytochromes P45017␣ and P450 aromatase, in pyramidal neurons in the CA1-CA3 regions, as well as in the granule cells in the dentate gyrus, by means of immunohistochemical staining of slices. Only a weak immunoreaction of these P450s was observed in astrocytes and oligodendrocytes. ImmunoGold electron microscopy revealed that P45017␣ and P450 aromatase were localized in pre-and postsynaptic compartments as well as in the endoplasmic reticulum in principal neurons. The expression of these cytochromes was further verified by using Western blot analysis and RT-PCR. Stimulation of hippocampal neurons with N-methyl-D-aspartate induced a significant net production of estradiol. Analysis of radioactive metabolites demonstrated the conversion from [ 3 H]pregnenolone to [ 3 H]estradiol through dehydroepiandrosterone and testosterone.This activity was abolished by the application of specific inhibitors of cytochrome P450s. Interestingly, estradiol was not significantly converted to other steroid metabolites. Taken together with our previous finding of a P450scc-containing neuronal system for pregnenolone synthesis, these results imply that 17␤-estradiol is synthesized by P45017␣ and P450 aromatase localized in hippocampal neurons from endogenous cholesterol. This synthesis may be regulated by a glutamate-mediated synaptic communication that evokes Ca 2؉ signals.

Rapid modulation of hippocampal synaptic plasticity by estrogen has long been a hot topic, but analysis of molecular mechanisms via synaptic estrogen receptors has been seriously difficult. Here, two types of independent synaptic plasticity, long-term depression (LTD) and spinogenesis, were investigated, in response to 17b-estradiol and agonists of estrogen receptors using hippocampal slices from adult male rats. Multi-electrode investigations demonstrated that estradiol rapidly enhanced LTD not only in CA1 but also in CA3 and dentate gyrus. Dendritic spine morphology analysis demonstrated that the density of thin type spines was selectively increased in CA1 pyramidal neurons within 2 h after application of 1 nM estradiol. This enhancement of spinogenesis was completely suppressed by mitogen-activated protein (MAP) kinase inhibitor. Only the estrogen receptor (ER) alpha agonist, (propyl-pyrazole-trinyl)tris-phenol (PPT), induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. The ERbeta agonist, (4-hydroxyphenyl)-propionitrile (DPN), suppressed LTD and did not affect spinogenesis. Because the mode of synaptic modulations by estradiol was mostly the same as that by the

Estradiol (E2) and other sex steroids play essential roles in the modulation of synaptic plasticity and neuroprotection in the hippocampus. To clarify the mechanisms for these events, it is important to determine the respective role of circulating vs. locally produced sex steroids in the male hippocampus. Liquid chromatography-tandem mass spectrometry in combination with novel derivatization was employed to determine the concentration of sex steroids in adult male rat hippocampus. The hippocampal levels of 17beta-E2, testosterone (T), and dihydrotestosterone (DHT) were 8.4, 16.9, and 6.6 nm, respectively, and these levels were significantly higher than circulating levels. The hippocampal estrone (E1) level was, in contrast, very low around 0.015 nm. After castration to deplete circulating high level T, hippocampal levels of T and DHT decreased considerably to 18 and 3%, respectively, whereas E2 level only slightly decreased to 83%. The strong reduction in hippocampal DHT resulting from castration implies that circulating T may be a main origin of DHT. In combination with results obtained from metabolism analysis of [(3)H]steroids, we suggest that male hippocampal E2 synthesis pathway may be androstenedione --> T --> E2 or dehydroepiandrosterone --> androstenediol --> T --> E2 but not androstenedione --> E1 --> E2.

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In neuroendocrinology, it is believed that steroid hormones are synthesized in the gonads and/or adrenal glands, and reach the brain via the blood circulation. In contrast to this view, we are in progress of demonstrating that estrogens and androgens are also synthesized locally by cytochrome P450s in the hippocampus, and that these steroids act rapidly to modulate neuronal synaptic plasticity. We demonstrated that estrogens were locally synthesized in the adult hippocampal neurons. In the pathway of steroidogenesis, cholesterol is converted to pregnenolone (by P450scc), dehydroepiandrosterone [by P450(17α)], androstenediol (by 17β-hydroxysteroid dehydrogenase, 17β-HSD), testosterone (by 3β-HSD) and finally to estradiol (by P450arom) and dihydrotestosterone (by 5α-reductase). The basal concentration of estradiol in the hippocampus was approximately 1 nM, which was greater than that in blood plasma. Significant expression of mRNA for P450scc, P450(17α), P450arom, 17β-HSD, 3β-HSD and 5α-reductase was demonstrated by RT-PCR. Their mRNA levels in the hippocampus were 1/200–1/5,000 of those in the endocrine organs. Localization of P450(17α) and P450arom was observed in synapses in addition to endoplasmic reticulum of principal neurons using immunoelectron microscopy. Different from slow action of gonadal estradiol which reaches the brain via the blood circulation, hippocampal neuron-derived estradiol may act locally and rapidly within the neurons. For example, 1 nM 17β-estradiol rapidly enhanced the long-term depression (LTD) not only in CA1 but also in CA3 and dentate gyrus. The density of thin spines was selectively increased within 2 h upon application of 1 nM estradiol in CA1 pyramidal neurons. Only ERα agonist propyl-pyrazole-trinyl-phenol induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. ERβ agonist hydroxyphenyl-propionitrile suppressed LTD and did not affect spinogenesis. Localization of estrogen receptor ERα in spines in addition to nuclei of principal neurons implies that synaptic ERα can drive rapid modulation of synaptic plasticity by endogenous estradiol.

The hippocampus is essentially involved in learning and memory processes. Its functions are affected by various neuromodulators, including 17beta-estradiol, testosterone, and retinoid. Brain-synthesized steroid hormones act as autocrine and paracrine modulators. The regulatory mechanism underlying brain steroidogenesis has not been fully elucidated. Synthesis of sex steroids in the gonads is stimulated by retinoic acids. Therefore, we examined the effects of retinoic acids on estradiol and testosterone biosynthesis in the rat hippocampus. We used cultured hippocampal slices from 10- to 12-d-old male rats to investigate de novo steroidogenesis. The infant rat hippocampus possesses mRNAs for steroidogenic enzymes and retinoid receptors. Slices were used after 24 h of preculture to obtain maximal steroidogenic activity because steroidogenesis in cultured slices decreases with time. The mRNA levels for P450(17alpha), P450 aromatase and estrogen receptor-beta in the slices were increased by treatment with 9-cis-retinoic acid but not by all-trans-isomer. The magnitude of stimulation and the shape of the dose-response curve for the mRNA level for P450(17alpha) were similar to those for cellular retinoid binding protein type 2, the transcription of which is activated by retinoid X receptor signaling. 9-cis-Retinoic acid also induced a 1.7-fold increase in the protein content of P450(17alpha) and a 2-fold increase in de novo synthesis of 17beta-estradiol and testosterone. These steroids may be synthesized from a steroid precursor(s), such as pregnenolone or other steroids, or from cholesterol, as so-called neurosteroids. The stimulation of estradiol and testosterone synthesis by 9-cis-retinoic acid might be caused by activation of P450(17alpha) transcription via retinoid X receptor signaling.