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Microarrays comprise an efficient approach to discovering large numbers of differentially expressed mRNA transcripts in the CNS resulting from changes in hormonal milieu. We used high-density oligonucleotide microarrays to examine the short-and long-term actions of estradiol (E2) on the transcriptomes from the medial basal hypothalamus and other brain regions of E2-treated (10 g) adult female mice. Our results have revealed several unanticipated gene regulations. Most striking is lipocalin prostaglandin D2 synthase (L-PGDS), which catalyzes the conversion of prostaglandin (PG) H2 to PGD2, a neuromodulator involved in a variety of functions, including sleep, pain, and odor responses. In situ hybridization revealed significant increases in L-PGDS expression in the arcuate and ventromedial nucleus of the medial basal hypothalamus compared with vehicle controls. The magnitude of these changes is Ϸ2-fold and suggests a modulatory role for PGD2 in E2-controlled neuroendocrine secretions and behaviors. Surprisingly, L-PGDS gene expression is reduced 2-fold after E2 treatment in the ventrolateral preoptic area (VLPO), the suspected site of action for the sleep-promoting effects of PGD2. Finally, whereas L-PGDS has been reported to be expressed primarily in oligodendrocytes of the adult rodent brain, we demonstrate, immunocytochemically, that L-PGDS is also expressed in a population of VLPO neurons. Thus, our data suggest the intriguing possibility that E2 modulation of L-PGDS plays a role in the regulation of sleep-wake states through hitherto unknown mechanisms in VLPO neurons and through hormone-dependent neuronal-glial cooperation.microarrays ͉ preoptic area ͉ oligodendrocytes ͉ estrogens ͉ arousal G onadal estrogens have wide-ranging effects in the central nervous system (CNS) of most adult mammals, including rodents and humans. These effects include but are not limited to the regulation of endocrine secretions from the anterior pituitary (1-4), sex-specific behaviors in rodents (5, 6), learning and memory (7-9), and neuroprotection (10-12). Estrogens are also known to regulate the expression of a number of different genes in the CNS (6). Despite this knowledge, we lack a complete understanding of estrogen-targeted genes in the CNS and how changes in their transcript levels may contribute to the myriad of neurobiological functions affected by estrogens.Microarray technology is a powerful method for assessing the changes simultaneously in thousands of gene transcripts in neuroendocrine systems (reviewed in ref. 13). It can reveal the orchestration of genomic changes taking place within the cells of the CNS and can lead to unanticipated discoveries of genomic alterations. We have been using high-density oligonucleotide arrays to elucidate the short-and long-term actions of estrogens on the transcriptomes from the medial basal hypothalamus (MBH) and other brain regions of female adult mice. Because the functional consequences of estrogens acting at the level of the MBH are well documented (see above), this region is an...

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Our understanding of myelination has been greatly enhanced via the study of spontaneous mutants that harbor a defect in a gene encoding one of the major myelin proteins (myelin mutants). In this study, we describe a unique genetic defect in a new myelin mutant called the Long Evans shaker (les) rat that causes severe dysmyelination of the CNS. Myelin deficits result from disruption of the myelin basic protein (Mbp) gene caused by the insertion of an endogenous retrotransposon [early transposons (ETn) element] into a noncoding region (intron 3) of the gene. The ETn element alters the normal splicing dynamics of MBP mRNA, leading to a dramatic reduction in the levels of full-length isoforms (<5% of normal) and the appearance of improperly spliced, chimeric transcripts. Although these aberrant transcripts contain proximal coding regions of the MBP gene (exons 1-3), they are unable to encode functional proteins required to maintain the structural integrity of the myelin sheath. These chimeric transcripts seem capable, however, of producing the necessary signal to initiate and coordinate myelin gene expression because normal numbers of mature oligodendrocytes synthesizing abundant levels of other myelin proteins are present in the mutant CNS. The les rat is thus an excellent model to study alternative functions of MBP beyond its well characterized role in myelin compaction.

To understand how the differentiation of stem cells to oligodendroglial progenitors is regulated, we established cultures of neural stem cells from neonatal rat striatum in the presence of epidermal growth factor (EGF) as free-floating neurospheres that were then exposed to an increasing amount of B104 cell-conditioned medium (B104CM). The resultant cells proliferated in response to B104CM but no longer to EGF. In vitro analysis and transplantation studies indicated that these cells were committed to the oligodendroglial lineage, and they were thus referred to as oligospheres. Further characterization of their expression of early markers, cell cycle, migration, and self-renewal suggests that they were pre-O2A progenitors. RT-PCR analysis indicated that the oligosphere cells expressed mRNAs of platelet-derived growth factor alpha receptor in addition to fibroblast growth factor receptor but not EGF receptor; the latter two receptor mRNAs were expressed by neurosphere cells. Thus, the progression of stem cells to oligodendroglial progenitors is likely induced by factors in B104CM.

The ability of estrogens to produce rapid changes in cellular function has been firmly established. The question remains whether these changes are mediated by a modified form of the nuclear estrogen receptor (ER) that is associated with the plasma membrane (mER) or by a completely novel membrane receptor. Therefore, we characterized the biochemical properties of the nuclear and membrane-associated ERs expressed endogenously in a rat hypothalamic endothelial cell line (D12). Radioligand binding experiments using D12 membrane fractions showed that these cells exhibit properties consistent with a binding site specific for estrogens (mER). Equilibrium binding assays using [125I]16-alpha-iodo-3,17- beta-estradiol revealed saturable binding to mER, an affinity value similar to nuclear ER, with differing receptor expression levels. Competition assays revealed that 9 of 12 ER ligands tested had comparable affinities for mER and ER. For example, 17-alpha-estradiol and estrone had similar binding characteristics for both receptors while differences were noted for raloxifene, 17beta-estradiol (E2), and genistein. Western blot and immunocytochemical analyses using antibodies specific for ERalpha confirmed that D12 cells expressed a membrane-associated protein with a molecular mass (67 kDa) similar to that of ERalpha that colocalized with caveolae-enriched membranes. A rapid increase in intracellar Ca2+ levels in the presence of E2 suggests that mER can mediate physiologic changes through calcium mobilization. These data support the expression of mER in these brain-derived endothelial cells that is similar to, but biochemically distinguishable from, nuclear ERalpha.

Using in situ hybridization and immunochemical methods, we have observed an increase in the expression of SNS/PN3 sodium channel mRNA and protein in cerebellar Purkinje cells of the taiep rat. These changes are present in taiep rats at 12 months of age, following loss of myelin, but not at one month, prior to loss of myelin. Increased SNS/PN3 expression is not associated with aging per se, because it was not observed in control rats at 12 months of age. These results suggest that altered sodium channel expression in Purkinje cells may contribute to the ataxia that occurs in taiep rats.

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