Estrogen plays a profound role in regulating the structure and function of many neuronal systems in the adult rat brain. The actions of estrogen were thought to be mediated by a single nuclear estrogen receptor (ER) until the recent cloning of a novel ER (ER-beta). To ascertain which ER is involved in the regulation of different brain regions, the present study compared the distribution of the classical (ER-alpha) and novel (ER-beta) forms of ER mRNA-expressing neurons in the central nervous system (CNS) of the rat with in situ hybridization histochemistry. Female rat brain, spinal cord, and eyes were frozen, and cryostat sections were collected on slides, hybridized with [35S]-labeled antisense riboprobes complimentary to ER-alpha or ER-beta mRNA, stringently washed, and opposed to emulsion. The results of these studies revealed the presence of ER-alpha and ER-beta mRNA throughout the rostral-caudal extent of the brain and spinal cord. Neurons of the olfactory bulb, supraoptic, paraventricular, suprachiasmatic, and tuberal hypothalamic nuclei, zona incerta, ventral tegmental area, cerebellum (Purkinje cells), laminae III-V, VIII, and IX of the spinal cord, and pineal gland contained exclusively ER-beta mRNA. In contrast, only ER-alpha hybridization signal was seen in the ventromedial hypothalamic nucleus and subfornical organ. Perikarya in other brain regions, including the bed nucleus of the stria terminalis, medial and cortical amygdaloid nuclei, preoptic area, lateral habenula, periaqueductal gray, parabrachial nucleus, locus ceruleus, nucleus of the solitary tract, spinal trigeminal nucleus and superficial laminae of the spinal cord, contained both forms of ER mRNA. Although the cerebral cortex and hippocampus contained both ER mRNAs, the hybridization signal for ER-alpha mRNA was very weak compared with ER-beta mRNA. The results of these in situ hybridization studies provide detailed information about the distribution of ER-alpha and ER-beta mRNAs in the rat CNS. In addition, this comparative study provides evidence that the region-specific expression of ER-alpha, ER-beta, or both may be important in determining the physiological responses of neuronal populations to estrogen action.

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The discovery of estrogen receptor beta (ER beta) and subsequent localization of its mRNA in the rat central nervous system (CNS) has provided new insights about estrogen action in brain. A critical step in understanding the role of ER beta is demonstrating that the mRNA is translated into functional protein. The present study used a new ER beta-specific polyclonal antiserum (Z8P) and immunocytochemistry (ICC) to investigate the distribution of ER beta in the rat CNS. Ovariectomized female rats were perfusion fixed, and free-floating sections were incubated with Z8P. After visualization with a standard ABC method, nuclear immunoreactivity was seen in neurons throughout the brain, including the olfactory nuclei, laminae IV-VI of the cerebral cortex, medial septum, preoptic area, bed nucleus of the stria terminalis, supraoptic nucleus, paraventricular nucleus, zona incerta, medial and cortical amygdaloid nuclei, cerebellum, nucleus of the solitary tract, ventral tegmental area, and spinal trigeminal nucleus. Moreover, the results of a double-label ICC/ in situ hybridization study revealed that ER beta mRNA and immunoreactivity were colocalized in neurons of the brain, thus confirming the specificity of the antiserum. Through the use of Western blot analysis, Z8P was shown to recognize in vitro translated ER beta, but not ER alpha, as well as a 60-kDa protein from rat granulosa cells and ovary extracts. The results of these studies have demonstrated that (1) ER beta mRNA is translated into immunoreactive protein throughout the rat brain, and (2) ER beta resides in the cell nucleus. Together, these data provide an anatomic foundation for future studies and advance our understanding of estrogen action in hypothalamic and extrahypothalamic brain regions.

We have shown that physiological levels of estradiol exert profound protective effects on the cerebral cortex in ischemia induced by permanent middle cerebral artery occlusion. The major goal of this study was to begin to elucidate potential mechanisms of estradiol action in injury. Bcl-2 is a proto-oncogene that promotes cell survival in a variety of tissues including the brain. Because estradiol is known to promote cell survival via Bcl-2 in non-neural tissues, we tested the hypothesis that estradiol decreases cell death by influencing bcl-2 expression in ischemic brain injury. Furthermore, because estradiol may protect the brain through estrogen receptor-mediated mechanisms, we examined expression of both receptor subtypes ERalpha and ERbeta in the normal and injured brain. We analyzed gene expression by RT-PCR in microdissected regions of the cerebral cortex obtained from injured and sham female rats treated with estradiol or oil. We found that estradiol prevented the injury-induced downregulation of bcl-2 expression. This effect was specific to bcl-2, as expression of other members of the bcl-2 family (bax, bcl-x(L), bcl-x(S), and bad) was unaffected by estradiol treatment. We also found that estrogen receptors were differentially modulated in injury, with ERbeta expression paralleling bcl-2 expression. Finally, we provide the first evidence of functional ERbeta protein that is capable of binding ligand within the region of the cortex where estradiol-mediated neuroprotection was observed in cerebral ischemia. These findings indicate that estradiol modulates the expression of bcl-2 in ischemic injury. Furthermore, our data suggest that estrogen receptors may be involved in hormone-mediated neuroprotection.

Estradiol protects against brain injury, neurodegeneration, and cognitive decline. Our previous work demonstrates that physiological levels of estradiol protect against stroke injury and that this protection may be mediated through receptor-dependent alterations of gene expression. In this report, we tested the hypothesis that estrogen receptors play a pivotal role in mediating neuroprotective actions of estradiol and dissected the potential biological roles of each estrogen receptor (ER) subtype, ER␣ and ER␤, in the injured brain. To investigate and delineate these mechanisms, we used ER␣-knockout (ER␣KO) and ER␤-knockout (ER␤KO) mice in an animal model of stroke. We performed our studies by using a controlled endocrine paradigm, because endogenous levels of estradiol differ dramatically among ER␣KO, ER␤KO, and wild-type mice. We ovariectomized ER␣KO, ER␤KO, and the respective wild-type mice and implanted them with capsules filled with oil (vehicle) or a dose of 17␤-estradiol that produces physiological hormone levels in serum. One week later, mice underwent ischemia. Our results demonstrate that deletion of ER␣ completely abolishes the protective actions of estradiol in all regions of the brain; whereas the ability of estradiol to protect against brain injury is totally preserved in the absence of ER␤. Thus, our results clearly establish that the ER␣ subtype is a critical mechanistic link in mediating the protective effects of physiological levels of estradiol in brain injury. Our discovery that ER␣ mediates protection of the brain carries far-reaching implications for the selective targeting of ERs in the treatment and prevention of neural dysfunction associated with normal aging or brain injury. Menopause marks the end of female reproduction and is accompanied by a dramatic and permanent decrease in estrogen levels. Although the life span of women has increased significantly in the past century, the average age of menopause has remained constant. Consequently, women may now spend more than one-third of their lives in a chronic hypoestrogenic postmenopausal state. Because estradiol is an important trophic and protective factor in the adult brain (1, 2), hypoestrogenic postmenopausal women may be more vulnerable to brain injury and dysfunction caused by neurodegenerative conditions and cognitive decline. It is, therefore, crucial that we gain a complete understanding of the mechanisms underlying the neuroprotective actions of estradiol.A growing body of evidence has begun to reveal that estrogen replacement therapy may ameliorate neural dysfunctions resulting from Alzheimer's disease (3-5) and stroke (6, 7) through multiple and complex cellular and molecular mechanisms of action. The protective role of estrogen in brain function has been examined by using a variety of in vivo and in vitro models of brain injury that mimic neurotoxic environments found in Alzheimer's disease, stroke, and other neurodegenerative conditions (8-13). These studies demonstrate that physiological and pharmacological concentrations of...

Although the distribution of estrogen receptor beta (ERbeta) immunoreactivity in the rat central nervous has been reported, no such data are available in the mouse. The present study used in vivo autoradiography utilizing a (125)I-estrogen that has equal binding affinity for both receptors as well as immunohistochemistry for ERbeta and ERalpha, to investigate and compare the distribution of the two ERs in the mouse CNS. The use specific antisera against ERalpha and ERbeta allowed us to evaluate the contribution of these receptors to the binding detected with autoradiography. In addition, data were collected in ovariectomized wildtype and ERalpha KO (knockout) mice to examine developmental regulation of ERbeta expression by ERalpha. These studies revealed that in the mouse CNS, combining immunoreactivity for ERalpha with that for ERbeta accounted for all regions where binding was seen using autoradiography. Therefore, these data strongly suggest that the major contributors of estrogen binding in the mouse CNS are ERalpha and ERbeta. Together, these data provide an anatomical foundation for future studies and advance our understanding of estrogen action in the CNS. Moreover, since the immunocytochemical images were similar in wildtype and ERalpha KO mice, these studies suggest that the lack of ERalpha does not influence the expression of ERbeta in the central nervous system.

Binge drinking (blood-alcohol levels ≥ 0.08 g% in a 2-h period), is a significant public health burden in need of improved treatment. Gene therapy may offer beneficial alternatives to current psychosocial and pharmacotherapeutic interventions, but identification of the target genes is a clinical challenge. We report that a GABA A α2 siRNA vector (pHSVsiLA2) infused into the central nucleus of the amygdala (CeA) of alcohol-preferring (P) rats caused profound and selective reduction of binge drinking associated with inhibition of α2 expression, decreased GABA A receptor density, and inhibition of Toll-like receptor 4 (TLR4). CeA infusion of a TLR4 siRNA vector (pHSVsiLTLR4a) also inhibited binge drinking, but neither vector functioned when infused into the ventral pallidum. Binge drinking was inhibited by a GABA A α1 siRNA vector (pHSVsiLA1) infused into the ventral pallidum, unrelated to TLR4. The vectors did not alter sucrose intake and a scrambled siRNA vector was negative. The data indicate that GABA A α2-regulated TLR4 expression in the CeA contributes to binge drinking and may be a key early neuroadaptation in excessive drinking.alcoholism | HSV vector | reinforcing effects | ethanol | innate immunity

Abstract. Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (S, e, e', and~) as well as the calcium-dependent isoforms (a, 01, ßn, and -y).These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised . Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide . PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography . Peak I reacted exclusively with antisera to PKCy, peak II with PKCß, and -ßu, and peak III with PKCa. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinc-P ROTEIN kinase C (PKC)' plays a major role in transmembrane signal transduction (35). PKC is activated by diacylglycerol, which is generated from membrane phospholipids upon stimulation of cells with various agonists (4) . PKC also serves as the receptor for phorbol esters and related tumor promoters (34), which activate the kinase by substituting for endogenous diacylglycerol (8) . Because of the pleiotropic actions of diacylglycerol and phorbol esters, PKC has been implicated in the regulation ofa variety of cellular processes, including proliferation, differentiation, and release of hormones and neurotransmitters (35,36).Molecular cloning studies have revealed that PKC consists of a large family of at least eight different isoenzymes (36, 37) that can be divided into two major groups. Initially, four isoforms of PKC were described . This group consists of PKCca, -01, -ß, 1, and -y, with PKCß, and -ß arising via alternate splicing of the same gene transcript and resulting in distinct carboxy-terminal regions . All four of these isoen-1. Abbreviations used in this paper: PKC, protein kinase C.O The Rockefeller University Press,