br Activity and expression of

Activity and expression of aromatase in the hippocampus The brain, like the adrenals, gonads and the placenta, is a steroidogenic organ. This paradigm emerged from studies carried out as early as the 1980s by Baulieu and co-workers; these studies showed that steroids such as pregnenolone and dehydroepiandrosterone were present in higher concentrations in the AZD-5438 than in plasma (for review see: [30], [31], [32], [33], [34]). Furthermore, it was demonstrated that steroids persisted in the nervous system even after gonadectomy or adrenalectomy. Neurosteroids are defined as steroids that accumulate in the brain even in the absence of steroidogenic glands; they are synthesized in the brain from endogenous precursors by enzymes that are present in situ[35]. In this context, it is interesting that sex steroids, such as estradiol and testosterone, are not classically considered as brain-derived neurosteroids. The substrate of all steroids, as well as neurosteroids, is cholesterol, and the equipment of the brain with enzymes of steroidogenesis makes a de novo synthesis possible [4], [32], [33], [35]. In 1971, Roselli et al. [36] demonstrated the presence of aromatase, the final enzyme of estradiol synthesis, in the diencephalon for the first time. While others [37], using an in vitro radiometric assay, were unable to detect activity of aromatase in the rat hippocampus. MacLusky et al. [38] found activity of aromatase in explant cultures from mouse, rat, and in rhesus monkeys [39]. Beyer et al. [40] demonstrated immunoreactivity of aromatase in the rat hippocampus for the first time, followed by Beyer in chickens [41], and by Yague et al. in monkeys and humans [42], [43]. On the mRNA level, Wehrenberg et al. [44] detected the expression of aromatase in rat hippocampus. Despite these early results, it still took some time before the synthesis and release was finally demonstrated [3], [9], [14], [15]. Steroid Acute Regulatory Protein (StAR) is required to transport cholesterol into the mitochondria, where steroidogenesis starts by the conversion of cholesterol to pregnenolone. Using in situ hybridization and immunohistochemistry we demonstrated StAR mRNA expression, as well as StAR protein, in the hippocampus [45], thus confirming a previous study by Garcia-Segura et al. [46]. Although glial cells [47] and neurons express aromatase in the hippocampus (for review see: [48]), the coexpression of both proteins in neurons, which we found in our study, made de novo synthesis of estradiol in hippocampal neurons very likely. In fact, we were able to demonstrate a de novo synthesis of estradiol in dissociated cell cultures and in hippocampal slice cultures using the aromatase inhibitor letrozole, and by knockdown of StAR [14], [15]. Studies by Kawato and coworkers [4], [35] supported the hypothesis of a de novo synthesis of estradiol in the hippocampus. In addition, based on differences between StAR [45] and aromatase expression in hippocampal tissue sections, stronger nuclear expression of ERα in CA3 pyramidal neurons than in CA1 pyramidal neurons, and region-specific downregulation of synaptic proteins strongly demonstrate the autocrine/paracrine function of locally synthesized estradiol.
Regulation of hippocampal estradiol synthesis Local concentrations of estrogens and aromatase may be classically regulated by gene transcription and enhanced protein synthesis [49], [50], [51], [52], [53], [54], [55]. We have previously shown that substrate availability and gonadotropins are potent regulators [8], [56] of estradiol synthesis. Application of testosterone, the direct substrate of aromatase, and in particular cholesterol, tremendously upregulated estradiol synthesis in hippocampal cultures [8], [57]. As a result, synapse density was increased in response to cholesterol and testosterone treatment of hippocampal slice cultures. The effect was abolished in the presence of letrozole, the aromatase inhibitor, showing the specificity of the estrogenic effect. Most importantly, gonadotropin releasing hormone (GnRH), of which the receptors are abundantly expressed in the hippocampus, upregulates estradiol synthesis dose-dependently in dissociated hippocampal cultures and in hippocampal slice cultures of female animals. As expected, treatment of hippocampal cultures with GnRH resulted in a consistent dose-dependent increase in the number of synapses and in the expression of synaptic proteins. Similar to our results on substrate availability, the increase in synapse density and in estradiol synthesis was abolished when aromatase was inhibited by simultaneous application of letrozole to the cultures [56].