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

Hemoprotein H-450 was purified from rat liver cytosol to homogeneity by an improved procedure. The purified H-450 showed a subunit molecular weight of 64,000 daltons and contained 0.7-0.9 mol of protoheme per mol subunit. Among rat tissues examined, liver and kidney contained significant amounts of H-450 in the cytosol. Oxidized H-450 showed a Soret peak at 428 nm and a broad beta band at around 550 nm. Reduced H-450 was found to exist in two interconvertible forms, alkaline and acid forms. The alkaline form showed Soret, beta, and alpha peaks at 448, 540, and 571 nm, whereas the acid form showed Soret, beta, and alpha peaks at 425, 530, and 558 nm. The spectral properties of both oxidized and reduced H-450 in alkaline medium resemble those of cytochrome P-450 having a nitrogenous ligand at the 6th coordination position of the heme. Upon addition of low concentrations of HgCl2, H-450 was converted to a denatured form both in the oxidized and the reduced states and lost its unique spectral characteristics. Addition of carbon monoxide to reduced H-450 produced a new spectral species which resembled that of the reduced carbon monoxide complex of P-420, a denatured form of cytochrome P-450. Comparison of the EPR signal of oxidized H-450 with those of a cytochrome P-450, P-450(PB-1), and several model compounds indicated the presence of a thiolate anion at the 5th coordination position of the heme of H-450. Judging from EPR data, oxidized H-450 also converts between acid and alkaline forms, whose signals were observed at g = 1.867, 2.31, and 2.507 and at g = 1.910, 2.28, and 2.424, respectively. These lines of evidence indicate that the 5th and 6th coordination positions of the heme of H-450 are a thiolate and a nitrogenous group, respectively. With respect to the heme environments, H-450 is a member of the cytochrome P-450 family, and has a nitrogenous ligand at the 6th coordination position of the heme.