Malcolm V. Lane

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.

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.

Distribution of estrogen receptor α and β in the mouse central nervous system: In vivo autoradiographic and immunocytochemical analyses

Although the distribution of estrogen receptor β (ERβ) 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 125I‐estrogen that has equal binding affinity for both receptors as well as immunohistochemistry for ERβ and ERα, to investigate and compare the distribution of the two ERs in the mouse CNS. The use specific antisera against ERα and ERβ allowed us to evaluate the contribution of these receptors to the binding detected with autoradiography. In addition, data were collected in ovariectomized wildtype and ERα KO (knockout) mice to examine developmental regulation of ERβ expression by ERα. These studies revealed that in the mouse CNS, combining immunoreactivity for ERα with that for ERβ 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 ERα and ERβ. 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 ERα KO mice, these studies suggest that the lack of ERα does not influence the expression of ERβ in the central nervous system. J. Comp. Neurol. 473:270–291, 2004.

The Distribution of Estrogen Receptor-β mRNA in Forebrain Regions of the Estrogen Receptor-α Knockout Mouse

Neurons in the hypothalamus of estrogen receptor alpha-knockout (ER alphaKO) mice have been shown to concentrate radiolabeled estrogen and estrogen treatment regulates the expression of progesterone receptor mRNA. The purpose of the present study was to utilize in situ hybridization histochemistry to determine the anatomical distribution of ER beta mRNA in ER alphaKO mouse forebrain. The results of these studies revealed an extensive distribution of ER beta mRNA in the hypothalamic regions including medial preoptic area, suprachiasmatic nucleus, paraventricular nucleus, dorsomedial nucleus, medial tuberal nucleus, and the premammillary nuclei. Additional labeled perikarya were also detected in the glomerular layer of the olfactory bulb; tenia tecta; anterior septum; bed nucleus of the stria terminalis; medial, basolateral and cortical nuclei of the amygdala; cerebral and entorhinal cortex; the septohippocampal nucleus; Ammons horn of the hippocampus and the dorsal raphe. The results of these in situ hybridization histochemical studies have provided novel information about the distribution of ER beta mRNA in the ER alphaKO mouse forebrain. In addition, these morphological data provides evidence that estrogen may exert its actions in the ER alphaKO mouse brain via ER beta and thereby maintain organizational and activational effects.

In situ hybridization analysis of the distribution of neurokinin-3 mRNA in the rat central nervous system

The tachykinin family of neuropeptides, which includes substance P, neurokinin A, and neurokinin B, have three distinct receptors; NK‐1, NK‐2, and NK‐3. With the cloning of the rat NK‐3 cDNA, it is now possible to evaluate the distribution of NK‐3 mRNA in the rat brain. Female rat brains were sectioned and hybridized with a riboprobe complimentary to NK‐3 mRNA. The results of these studies revealed an extensive distribution of NK‐3 mRNA throughout the rostral‐caudal extent of the brain, spinal cord, and retina. In agreement with previous binding studies, we observed NK‐3 mRNA in the cortex, the amygdala, the hippocampus, the medial habenula, the zona incerta, the paraventricular and supraoptic nuclei of the hypothalamus, the substantia nigra, the ventral tegmental area, the interpeduncular nucleus, the raphe nuclei, the dorsal tegmental nucleus, and the nucleus of the solitary tract. In contrast with binding data, only a few NK‐3 mRNA cells were detected in the striatum. In addition, the present study detected NK‐3 mRNA in the olfactory bulb, the dentate gyrus and subiculum, the medial septum, the diagonal band of Broca, the ventral pallidum, the globus pallidus, the bed nucleus of the stria terminalis, the arcuate, the premammillary and mammillary nuclei, the dorsal and lateral regions of the posterior hypothalamus, the central gray, the cerebellum, the parabrachial nuclei, the nucleus of the spinal trigeminal tract, the dorsal horn of the spinal cord, and the retina. The results of these in situ hybridization histochemical studies have provided detailed and novel information about the distribution of NK‐3 mRNA and have elucidated the putative sites of neurokinin B action in the rat central nervous system.

Biologically Active Estrogen Receptor-β: Evidence from in Vivo Autoradiographic Studies with Estrogen Receptor α-Knockout Mice

Estrogen receptor-β (ERβ) messenger RNA (mRNA) has been detected in the brain of wild-type and estrogen receptor-α knockout (ERαKO) mice. The present study used in vivo autoradiography to evaluate the binding of 125I-estrogen, a compound with a similar affinity for both ERs to ascertain whether ERβ mRNA is translated into biologically active receptor. Mice were injected with 125I-estrogen, and sections were mounted on slides and opposed to emulsion. After exposure, labeled cells were seen in ERαKO brain regions where ERβ is expressed (preoptic and paraventricular nuclei of the hypothalamus; bed nucleus of the stria terminalis; amygdala; entorhinal cortex; and dorsal raphe). Competition studies with 17β-estradiol eliminated binding in the ERαKO brain, whereas 16αIE2, an ERα selective agonist and dihydrotestosterone had no effect. In contrast, competition studies with 16αIE2 in wild-type mice eliminated 125I-estrogen binding to ERα and resulted in a pattern of residual binding comparable to that seen in the...Estrogen receptor-1 (ER beta) messenger RNA (mRNA) has been detected in the brain of wild-type and estrogen receptor-alpha knockout (ER alphaKO) mice. The present study used in vivo autoradiography to evaluate the binding of 125I-estrogen, a compound with a similar affinity for both ERs to ascertain whether ER beta mRNA is translated into biologically active receptor. Mice were injected with 125I-estrogen, and sections were mounted on slides and opposed to emulsion. After exposure, labeled cells were seen in ER alphaKO brain regions where ER beta is expressed (preoptic and paraventricular nuclei of the hypothalamus; bed nucleus of the stria terminalis; amygdala; entorhinal cortex; and dorsal raphe). Competition studies with 17beta-estradiol eliminated binding in the ER alphaKO brain, whereas 16alphaIE2, an ER alpha selective agonist and dihydrotestosterone had no effect. In contrast, competition studies with 16alphaIE2 in wild-type mice eliminated 125I-estrogen binding to ER alpha and resulted in a pattern of residual binding comparable to that seen in the ER alphaKO brain. The results demonstrate that residual estrogen binding sites are present in regions of the ER alphaKO brain where ER beta is expressed, brain regions that were also seen after eliminating binding to ER alpha in wild-type mice. These data provide the first evidence that ER beta mRNA is translated into a biologically active protein in the rodent brain.

Estrogen Promotes Germ Cell and Seminiferous Tubule Development in the Baboon Fetal Testis

Abstract The foundation for development of the male reproduction system occurs in utero, but relatively little is known about the regulation of primate fetal testis maturation. Our laboratories have shown that estrogen regulates key aspects of the physiology of pregnancy and fetal development. Therefore, in the present study, we characterized and quantified germ cells and Sertoli cells in the fetal baboon testis in late normal gestation (i.e., Day 165; term is 184 days) and in baboons administered the aromatase inhibitor letrozole throughout the second half of gestation to assess the impact of endogenous estrogen on fetal testis development. In untreated baboons, the seminiferous cords were comprised of undifferentiated (i.e., type A) spermatogonia classified by their morphology as dark (Ad) or pale (Ap), gonocytes (precursors of type A spermatogonia), unidentified cells (UI), and Sertoli cells. In letrozole-treated baboons, serum estradiol levels were decreased by 95%. The number per milligram of fetal testis (×104) of Ad spermatogonia (0.42 ± 0.11) was 45% lower (P = 0.03), and that of gonocytes (0.58 ± 0.06) and UI (0.45 ± 0.12) was twofold greater (P < 0.01 and P = 0.06, respectively), than in untreated baboons. Moreover, in the seminiferous cords of estrogen-deprived baboons, the basement membrane appeared fragmented, the germ cells and Sertoli cells appeared disorganized, and vacuoles were present. We conclude that endogenous estrogen promotes fetal testis development and that the changes in the germ cell population in the estrogen-deprived baboon fetus may impair spermatogenesis and fertility in adulthood.

Carboxypeptidase E Protects Hippocampal Neurons During Stress in Male Mice by Up-regulating Pro-survival BCL2 Protein Expression

Prolonged chronic stress causing elevated plasma glucocorticoids leads to neurodegeneration. Adaptation to stress (allostasis) through neuroprotective mechanisms can delay this process. Studies on hippocampal neurons have identified carboxypeptidase E (CPE) as a novel neuroprotective protein that acts extracellularly, independent of its enzymatic activity, although the mechanism of action is unclear. Here, we aim to determine if CPE plays a neuroprotective role in allostasis in mouse hippocampus during chronic restraint stress (CRS), and the molecular mechanisms involved. Quantitative RT-PCR/in situ hybridization and Western blots were used to assay for mRNA and protein. After mild CRS (1 h/d for 7 d), CPE protein and mRNA were significantly elevated in the hippocampal CA3 region, compared to naïve littermates. In addition, luciferase reporter assays identified a functional glucocorticoid regulatory element within the cpe promoter that mediated the up-regulation of CPE expression in primary hippocampal neurons following dexamethasone treatment, suggesting that circulating plasma glucocorticoids could evoke a similar effect on CPE in the hippocampus in vivo. Overexpression of CPE in hippocampal neurons, or CRS in mice, resulted in elevated prosurvival BCL2 protein/mRNA and p-AKT levels in the hippocampus; however, CPE(-/-) mice showed a decrease. Thus, during mild CRS, CPE expression is up-regulated, possibly contributed by glucocorticoids, to mediate neuroprotection of the hippocampus by enhancing BCL2 expression through AKT signaling, and thereby maintaining allostasis.

Functional G-protein-coupled receptor 35 is expressed by neurons in the CA1 field of the hippocampus

The G-protein-coupled receptor 35 (GPR35) was de-orphanized after the discovery that kynurenic acid (KYNA), an endogenous tryptophan metabolite, acts as an agonist of this receptor. Abundant evidence supports that GPR35 exists primarily in peripheral tissues. Here, we tested the hypothesis that GPR35 exists in the hippocampus and influences the neuronal activity. Fluorescence immunohistochemical staining using an antibody anti-NeuN (a neuronal marker), an antibody anti-GFAP (a glial marker), and an antibody anti-GPR35 revealed that neurons in the stratum oriens, stratum pyramidale, and stratum radiatum of the CA1 field of the hippocampus express GPR35. To determine the presence of functional GPR35 in the neurocircuitry, we tested the effects of various GPR35 agonists on the frequency of spontaneous action potentials recorded as fast current transients (CTs) from stratum radiatum interneurons (SRIs) under cell-attached configuration in rat hippocampal slices. Bath application of the GPR35 agonists zaprinast (1-10 μM), dicumarol (50-100 μM), pamoic acid (500-1000 μM), and amlexanox (3 μM) produced a concentration- and time-dependent reduction in the frequency of CTs. Superfusion of the hippocampal slices with the GPR35 antagonist ML145 (1 μM) increased the frequency of CTs and reduced the inhibitory effect of zaprinast. Bath application of phosphodiesterase 5 inhibitor sildenafil (1 or 5 μM) was ineffective, whereas a subsequent application of zaprinast was effective in reducing the CT frequency. The present results demonstrate for the first time that functional GPR35s are expressed by CA1 neurons and suggest that these receptors can be molecular targets for controlling neuronal activity in the hippocampus.