Istvan Merchenthaler

COMPARATIVE DISTRIBUTION OF ESTROGEN RECEPTOR-ALPHA AND -BETA MRNA IN THE RAT CENTRAL NERVOUS SYSTEM

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‐β). To ascertain which ER is involved in the regulation of different brain regions, the present study compared the distribution of the classical (ER‐α) and novel (ER‐β) 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‐α or ER‐β mRNA, stringently washed, and opposed to emulsion. The results of these studies revealed the presence of ER‐α and ER‐β 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‐β mRNA. In contrast, only ER‐α 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‐α mRNA was very weak compared with ER‐β mRNA. The results of these in situ hybridization studies provide detailed information about the distribution of ER‐α and ER‐β mRNAs in the rat CNS. In addition, this comparative study provides evidence that the region‐specific expression of ER‐α, ER‐β, or both may be important in determining the physiological responses of neuronal populations to estrogen action. J. Comp. Neurol. 388:507–525, 1997.

Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system

Glucagon‐like peptide‐1 (GLP‐1) is derived from the peptide precursor pre‐pro‐glucagon (PPG) by enzymatic cleavage and acts via its receptor, glucagon‐like peptide‐1 receptor (GLP‐1R). By using riboprobes complementary to PPG and GLP‐1R, we described the distribution of PPG and GLP‐1R messenger RNAs (mRNAs) in the central nervous system of the rat. PPG mRNA‐expressing perikarya were restricted to the nucleus of the solitary tact or to the dorsal and ventral medulla and olfactory bulb. GLP‐1R mRNA was detected in numerous brain regions, including the mitral cell layer of the olfactory bulb; temporal cortex; caudal hippocampus; lateral septum; amygdala; nucleus accumbens; ventral pallium; nucleus basalis Meynert; bed nucleus of the stria terminalis; preoptic area; paraventricular, supraoptic, arcuate, and dorsomedial nuclei of the hypothalamus; lateral habenula; zona incerta; substantia innominata; posterior thalamic nuclei; ventral tegmental area; dorsal tegmental, posterodorsal tegmental, and interpeduncular nuclei; substantia nigra, central gray; raphe nuclei; parabrachial nuclei; locus ceruleus, nucleus of the solitary tract; area postrema; dorsal nucleus of the vagus; lateral reticular nucleus; and spinal cord. These studies, in addition to describing the sites of GLP‐1 and GLP‐1R synthesis, suggest that the efferent connections from the nucleus of the solitary tract are more widespread than previously reported. Although the current role of GLP‐1 in regulating neuronal physiology is not known, these studies provide detailed information about the sites of GLP‐1 synthesis and potential sites of action, an important first step in evaluating the function of GLP‐1 in the brain. The widespread distribution of GLP‐1R mRNA‐containing cells strongly suggests that GLP‐1 not only functions as a satiety factor but also acts as a neurotransmitter or neuromodulator in anatomically and functionally distinct areas of the central nervous system. J. Comp. Neurol. 403:261–280, 1999.

Distribution of estrogen receptor β immunoreactivity in the rat central nervous system

The discovery of estrogen receptor β (ERβ) 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 ERβ is demonstrating that the mRNA is translated into functional protein. The present study used a new ERβ‐specific polyclonal antiserum (Z8P) and immunocytochemistry (ICC) to investigate the distribution of ERβ 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 ERβ 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 ERβ, but not ERα, as well as a 60‐kDa protein from rat granulosa cells and ovary extracts. The results of these studies have demonstrated that (1) ERβ mRNA is translated into immunoreactive protein throughout the rat brain, and (2) ERβ 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. J. Comp. Neurol. 436:64–81, 2001.

The Estrogen Receptor β Subtype: A Novel Mediator of Estrogen Action in Neuroendocrine Systems☆

The recent discovery that an additional estrogen receptor (ERbeta) subtype is present in many rat, mouse, and human tissues has advanced our understanding of the mechanisms underlying estrogen signalling. Ligand-binding experiments have shown specific binding of 17beta-estradiol by ERbeta with an affinity similar to that of ERalpha. The rat tissue distribution and/or the relative level of ERalpha and ERbeta expression seems to be quite different, i.e., moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ERalpha and prostate, ovary, lung, bladder, brain, bone, uterus, and testis for ERbeta. Within the same organ it often appears that the ER subtypes are expressed in different cell types, supporting the hypothesis that the ERs may have different biological functions. The cell type-specific expression of ERalpha and ERbeta in rat prostate, testis, uterus, ovary, and brain and the distribution of ERbeta mRNA in the ERalpha knock-out mouse brain are discussed. The discovery of ERbeta suggests the existence of two previously unrecognized pathways of estrogen signalling; via the ERbeta subtype in tissues exclusively expressing this subtype and via the formation of heterodimers in tissues expressing both ER subtypes. The existence of two ER subtypes, their differential expression pattern, and different actions on certain response elements could provide explanations for the striking species-, cell-, and promoter-specific actions of estrogens and antiestrogens. The challenge for the future is to unravel the detailed physiological role of each subtype and to use this knowledge to develop the next generation of ER-targeted drugs with improved therapeutic profiles in the treatment or prevention of osteoporosis, cardiovascular system disorders, Alzheimers disease, breast cancer, and disorders of the urogenital tract.

Anatomy and physiology of central galanin-containing pathways

C O N T E N T S Abbreviations

The distribution of estrogen receptor-β mRNA in the rat hypothalamus

Abstract The recent cloning of a second form of the estrogen receptor (ER-β) has made it possible to map the distribution of ER-β mRNA-containing perikarya in the rat hypothalamus. The present in situ hybridization histochemical studies have detected ER-β mRNA in the medial preoptic area; the anterior periventricular, paraventricular, supraoptic, arcuate, medial tuberal and medial mammillary nuclei; the bed nucleus of the stria terminalis, and zona incerta. As previously described for the classical ER (ER-α) mRNA, a dense accumulation of ER-β mRNA-expressing perikarya is present in the medial preoptic area and bed nucleus of the stria terminalis. In contrast, ER-β mRNA was also concentrated in the paraventricular and supraoptic nuclei, brain regions which contain little or no ER-α mRNA. Moreover, the arcuate and ventromedial nuclei, areas with abundant ER-α, contain only a weak level of ER-β hybridization signal. The description of ER-β mRNA-containing perikarya in the rat hypothalamus provides a foundation for further morphological and physiological studies aimed at elucidating the role of ER-β in the hypothalamus.

Comparative distribution of estrogen receptor-α (ER-α) and β (ER-β) mRNA in the rat pituitary, gonad, and reproductive tract

Abstract The present study used in situ hybridization histochemistry to compare the distribution of estrogen receptor (ER)-α and ER-β mRNA-containing cells in rat pituitary, gonads, uterus, and prostate of intact animals or after hormonal manipulations. Cryostat tissue sections were hybridized with 35S-labeled antisense riboprobes complimentary to ER-α or ER-β mRNA, stringently washed and apposed to emulsion. The results of these studies indicate that the expression of the two receptors is tissue and region specific, with estrogen target tissues specifically expressing ER-α, ER-β, or both forms of ER. In the intact rat, ER-α and ER-β mRNA were both seen in the pituitary, although more cells expressed ER-α than ER-β mRNA. The distribution of the two transcripts in the ovary was qualitatively different, with ER-α being primarily localized in the stromal cells, while ER-β mRNA was concentrated in the granulosa cells of developing follicles. In the uterus, ER-α mRNA was abundant in the stromal and epithelial cells of the endometrium, while only very weak ER-β hybridization signal was detected in these cells. ER-β mRNA- expressing cells, but not ER-α, were also detected in the prostate and in the Sertoli cells, and the large, round spermatocytes of the testis. Gonadectomy markedly attenuated the expression of ER-β mRNA in the peripheral tissues, with the level of ER-β mRNA in the uterus and prostate reduced to non-detectable levels. The results of these in situ hybridization studies demonstrate that the distribution and regulation of ER-β mRNA expression is tissue specific and different from ER-α mRNA. The differential expression of ERs in these tissues may explain in part the tissue selective activity of estrogenic compounds.

Estrogen Receptor-β Immunoreactivity in Luteinizing Hormone-Releasing Hormone Neurons of the Rat Brain

Abstract Feedback regulation of luteinizing hormone-releasing hormone (LHRH) neurons by estradiol plays important roles in the neuroendocrine control of reproduction. Recently, we found that the majority of LHRH neurons in the rat contain estrogen receptor-β (ER-β) mRNA, whereas, they seemed to lack ER-α mRNA expression. In addition, we observed nuclear uptake of 125I-estrogen by a subset of these cells. These data suggest that ER-β is the chief receptor isoform mediating direct estrogen effects upon LHRH neurons. To verify the translation of ER-β protein within LHRH cells, the present studies applied dual-label immunocytochemistry (ICC) to free-floating sections obtained from the preoptic area of rats. The improved ICC method using the silver-gold intensification of nickel-diaminobenzidine chromogen, enabled the observation of nuclear ER-β-immunoreactivity in the majority of LHRH cells. The incidence of ER-β expression was similarly high in LHRH neurons of ovariectomized female (87.8 ± 2.3%, mean ± SEM),...

Detection of Estrogen Receptor-β Messenger Ribonucleic Acid and 125I-Estrogen Binding Sites in Luteinizing Hormone-Releasing Hormone Neurons of the Rat Brain

Luteinizing hormone-releasing hormone (LHRH) neurons of the forebrain play a pivotal role in the neuroendocrine control of reproduction. Although serum estrogen levels influence many aspects of LHRH neuronal activity in the female, earlier studies were unable to detect estrogen receptors (ERs) within LHRH neurons, thus shaping a consensus view that the effects of estradiol on the LHRH neuronal system are mediated by interneurons and/or the glial matrix. The present studies used dual-label in situ hybridization histochemistry (ISHH) and combined LHRH-immunocytochemistry/125I-estrogen binding to readdress the estrogen-receptivity of LHRH neurons in the female rat. In ISHH experiments we found that the majority of LHRH neurons exhibited hybridization signal for the “β” form of ER (ER-β). The degree of colocalization was similar in topographically distinct populations of LHRH neurons and was not significantly altered by estradiol (67.2±1.8 % in ovariectomized and 73.8±4.2 % in ovariectomized and estradiol-tre...

Evidence of the Colocalization of Estrogen Receptor-β mRNA and Estrogen Receptor-α Immunoreactivity in Neurons of the Rat Forebrain

Estrogen receptor β (ERβ) mRNA is expressed in several rat brain regions where ERα is abundant. In vitro studies have shown that ERα and ERβ can heterodimerize and that the activity of this complex may be different than an ERα or ERβ homodimer complex. The purpose of the present study was to ascertain if ERα and ERβ are co-expressed by certain neuronal populations using a double label in situ hybridization/immunocytochemistry method. The results revealed that neurons in the bed nucleus of the stria terminalis, medial amygdala and preoptic area contain both ERs, with the vast majority of the neurons being double labeled. In other brain regions including the arcuate nucleus, cortical amygdaloid nuclei and ventromedial nucleus, only a few double-labeled cells were detected, while neurons in the paraventricular nucleus, supraoptic nucleus, and cerebral cortex expressed only ERβ mRNA. The results of these double label experiments provide the first evidence that ERα and ERβ coexist in neurons under in vivo cond...