Microglia play a critical role in neurodegenerative diseases and in the brain aging process. Yet, little is known about the functional dynamics of microglia during aging. Thus, using young and aging transgenic mice expressing enhancedgreen fluorescent protein (EGFP) under the promoter of the c-fms gene for macrophage-colony stimulating factor receptor, we evaluated in vivo-induced inflammatory responses of EGFP-expressing microglia sorted by flow cytometry. Aging microglia were characterized by the presence of lipofuscin granules, decreased processes complexity, altered granularity, and increased mRNA expression of both proinflammatory (TNFa, IL-1b, IL-6) and anti-inflammatory (IL-10, TGFb1) cytokines. Following lipopolysaccharide (LPS) challenge (1 mg/kg, 3 h), aging microglia exhibit increased basal expression of TNFa, IL-1b, IL-6, and IL-10. Yet, the foldover-basal LPS response remained constant across age, implying that the inflammatory machinery in aging microglia is functional and adjusted to the basal state. Gender differences were not overall observed across the treatments (age, LPS). The low but sustained production of pro-inflammatory cytokines by aging microglia may have a profound impact in the brain aging process. V V C 2006 Wiley-Liss, Inc.

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Steroid hormones such as glucocorticoids and estrogens are well-known regulators of peripheral immune responses and also show anti-inflammatory properties in the brain. However, the expression of steroid hormone receptors in microglia, the pivotal immune cell that coordinates the brain inflammatory response, is still controversial. Here we use real time RT-PCR to show that microglia, isolated from adult fms-EGFP mice by FACS, express glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and estrogen receptor alpha (ERa). GR was the most abundant steroid hormone receptor transcript in microglia. The presence of GR and ERa immunoreactivity was further confirmed in vivo at the ultrastructural level. To understand the role of steroid hormone receptors during the inflammation process, we evaluated the expression of steroid hormone receptors after inflammatory challenge and found a significant down-regulation of GR, MR, and ERa in microglia. Finally, we tested the immunomodulatory properties of estrogens and glucocorticoids. Estradiol benzoate did not have any significant impact on the inflammatory profile of ex vivo sorted microglia, either in resting conditions or after challenge. Furthermore, corticosterone was a more consistent anti-inflammatory agent than 17b-estradiol in vitro. Our results support the hypothesis that adult microglia are a direct target of steroid hormones and that glucocorticoids, through the predominant expression of GR and MR, are the primary steroid hormone regulators of microglial inflammatory activity. The down-regulation of steroid hormone receptors after LPS challenge may serve as a prerequisite to suppressing the antiinflammatory actions of endogenous steroid hormones on the immune system, and contribute to a sustained activation of microglia. V V C

Ovarian steroids have many effects on the brain throughout the lifespan, beginning during gestation and continuing into senescence. These hormones affect areas of the brain that are not primarily involved in reproduction, such as the basal forebrain, hippocampus, caudate putamen, midbrain raphe, and brainstem locus coeruleus. Here we discuss three effects of estrogens and progestins that are especially relevant to memory processes and identify hormonal alterations associated with aging and neurodegenerative diseases. First, estrogens and progestins regulate synaptogenesis in the CA1 region of the hippocampus during the 4- to 5-day estrous cycle of the female rat. Formation of new excitatory synapses is induced by estradiol and involves N-methyl-D-aspartate (NMDA) receptors, whereas synaptic downregulation involves intracellular progestin receptors. Second, there are developmentally programmed sex differences in the hippocampal structure that mat help explain why male and female rats use different strategies to solve spatial navigation problems. During the period of development when testosterone is elevated in the male, aromatase and estrogen receptors are transiently expressed in the hippocampus. Recent data on behavior and synapse induction strongly suggest that this pathway is involved in the masculinization or defeminization of hippocampal structure and function. Third, ovarian steroids have effects throughout the brain, including effects on brainstem and midbrain catecholaminergic neurons, midbrain serotonergic pathways, and the basal forebrain cholinergic system. Regulation of the serotonergic system appears to be linked to the presence of estrogen- and progestin-sensitive neurons in the midbrain raphe, whereas the ovarian steroid influence on cholinergic function involves induction of choline acetyltransferase and acetylcholinesterase according to a sexually dimorphic pattern. Because of these widespread influences on these various neuronal systems, it is not surprising that ovarian steroids produce measurable cognitive effects after ovariectomy and during aging.

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-D-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ER␣) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ER␣ and ER␤ mRNA. At the light microscopic level, autoradiography shows cell nuclear [ 3 H]estrogen and [ 125 I]estrogen uptake according to a distribution that primarily reflects the localization of ER␣-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ER␣ immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation. T he brain is widely responsive to gonadal hormones. Not only is the hypothalamus regulated by these hormones in relation to reproductive behavior and neuroendocrine physiology, but also structures like the hippocampus and midbrain serotonin system undergo sexual differentiation during perinatal development and are hormone responsive in maturity (1, 2). One of the processes regulated by ovarian hormones is the cyclic formation and breakdown of excitatory synapses on dendritic spines in the hippocampus (3). This finding was surprising because, until recently, the hippocampus was known as a brain region in which cell nuclear estrogen receptors (ER) are present in scattered inhibitory interneurons but not in principal neurons where spine formation occurs (4). Yet the effects of ovarian hormones on synaptic turnover were as impressive in the hippocampus as those in the ventromedial hypothalamus (5-7), a classic estrogen (E) target area of the brain for female sexual behavior (8). Moreover, effects of estrogens on hippocampal-dependent cognitive function are now recognized in rodents (9) and humans (10).Recent electron microscopic studies have revealed that ERs are expressed in hippocampus in non-nuclear locations within principal cells (11). This fact, along with the discovery that ER can couple to second messenger systems (12-14), has raised the possibility that ER may be involved in local signaling within neurons as well as regulating expression of genes via nuclear receptors i...