Simultaneous visualization of two cellular mRNA species in individual neurons by use of a new double in Situ hybridization method

Marks, Daniel L.; Wiemann, J; Burton, Kimberly A.; Lent, Karin; Clifton, Donald K.; Steiner, Robert A.

doi:10.1016/1044-7431(92)90051-3

Cited by 59 publications

(36 citation statements)

References 14 publications

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“…The incidence of colocalization exhibits a striking sex difference-i.e., in females =80% of the LHRH neurons contain GAL, whereas in males only 10-15% of these neurons synthesize this peptide (2). In situ hybridization studies have corroborated our earlier findings since GAL mRNA is colocalized with the decapeptide LHRH in LHRH neurons (3). It has been reported that estradiol has a profound effect on GAL gene expression in the pituitary and the hypothalamus (see ref.…”

supporting

confidence: 78%

Neonatal imprinting predetermines the sexually dimorphic, estrogen-dependent expression of galanin in luteinizing hormone-releasing hormone neurons.

Merchenthaler

Lennard

López

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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The incidence of colocalization of galanin (GAL) in luteinizing hormone-releasing hormone (LHRH) neurons is 4-to 5-fold higher in female than male rats. This fact and the finding that the degree of colocalization parallels estradiol levels during the estrous cycle suggest that GAL is an estrogen-inducible product in a subset of LHRH neurons. To analyze further this paradigm we evaluated the effects of gonadectomy and steroid replacement therapy in male and female rats. Ovariectomy resulted in a significant decrease in the number of cells colocalizing LHRH and GAL, whereas estradiol replacement to such animals restored the incidence of colocalization to that observed in controls. In males, however, estradiol treatment failed to enhance the incidence of colocalization of GAL and LHRH, indicating, therefore, that the colocalization of these peptides is gender-determined. This possibility-i.e., gender-specific determination of LHRH neurons coexpressing GAL-was evaluated by neonatal manipulation of hypothalamic steroid imprinting. As mentioned above, male rats did not respond to estrogen or testosterone by increasing GAL/LHRH colocalization as females did. Neonatally orchidectomized rats, whose hypothalami have not been exposed to testosterone during the critical period, when treated with estrogen in adulthood showed an increase in colocalization of GAL and LHRH similar to that seen in female animals. These observations indicate that the colocalization of LHRH/GAL is neonatally determined by an epigenetic mechanism that involves the testis. In summary, this sex difference in the incidence of colocalization of GAL and LHRH represents a unique aspect of sexual differentiation in that only certain phenotypic characteristics of a certain cellular lineage are dimorphic. The subpopulation of LHRH neurons that also produces GAL represents a portion of the LHRH neuronal system that is sexually differentiated and programed to integrate, under steroidal control, a network of LHRH neurons that could synchronize their activity to control the estrous cycle in rats.Recent observations from our laboratory indicate that a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons in the rat brain also expresses galanin (GAL) (1). The incidence of colocalization exhibits a striking sex difference-i.e., in females =80% of the LHRH neurons contain GAL, whereas in males only 10-15% of these neurons synthesize this peptide (2). In situ hybridization studies have corroborated our earlier findings since GAL mRNA is colocalized with the decapeptide LHRH in LHRH neurons (3). It has been reported that estradiol has a profound effect on GAL gene expression in the pituitary and the hypothalamus (see ref. 4 for a review). In this respect, we have observed differential coexpression ofGAL in LHRH neuronsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.during the estrous c...

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supporting

confidence: 78%

Neonatal imprinting predetermines the sexually dimorphic, estrogen-dependent expression of galanin in luteinizing hormone-releasing hormone neurons.

Merchenthaler

Lennard

López

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…Sections were next incubated overnight in sheep antidigoxigenin antiserum conjugated to alkaline phosphatase(1:1,000), followed by overnight incubation in a nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate chromagen solution (21). Sections were dipped in 3% parlodion in isoamyl acetate, dried, and then dipped in NTB2 photographic emulsion (Eastman Kodak Co. Scientific Imaging Systems (New Haven, Connecticut, USA), stored at 4°C for 2-4 weeks and developed with D-19 developer (Eastman Kodak Co. Scientific Imaging Systems).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling

Harris¹,

Aschkenasi²,

Elias³

et al. 2001

J. Clin. Invest.

307

221

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IntroductionThe hypothalamic peptide, thyrotropin-releasing hormone (TRH), is essential for the normal production of thyroid-stimulating hormone in the pituitary and thyroid hormones in the thyroid gland (1). Within the paraventricular hypothalamic nucleus (PVH), TRH is regulated at the transcriptional level by thyroid hormone (T3), such that in hyperthyroid states TRH expression is reduced and in hypothyroid states its expression is increased (2). Recently, it has become clear that TRH is also regulated by nutritional states. To conserve energy during periods of food deprivation, rodents dramatically reduce their thyroid hormone levels, which in turn allows reductions in their metabolic rate (3). This adaptation to starvation is accomplished through a reduction in the expression of TRH in the PVH, indicating that a central process contributes to the regulation of this physiological adaptation. The mechanism governing the nutritional regulation of TRH has been clarified by Ahima et al., who reversed starvation-induced suppression of thyroid hormone levels in mice by administering leptin during starvation (4). This finding was extended by Legradi et al., who further demonstrated that leptin's effects were due to upregulation of TRH gene expression (5).Although leptin can regulate TRH gene expression in the PVH, it remains unclear whether this effect is mediated by direct actions of leptin on the TRH neuron in the PVH or indirectly through leptin effects on other neurons, which then project to the TRH neuron.Recently Legradi et al. demonstrated that chemical ablation of the arcuate nucleus blocks leptin's effect on TRH expression, suggesting that an indirect pathway is involved (6). Given that leptin activates pro-opiomelanocortin (POMC) gene expression in the arcuate nucleus, POMC-derived α-melanocyte stimulating hormone (α-MSH) is a candidate hormone that may be responsible for the regulation of TRH expression (7,8). Starvation causes a rapid reduction in thyroid hormone levels in rodents. This adaptive response is caused by a reduction in thyrotropin-releasing hormone (TRH) expression that can be reversed by the administration of leptin. Here we examined hypothalamic signaling pathways engaged by leptin to upregulate TRH gene expression. As assessed by leptin-induced expression of suppressor of cytokine signaling-3 (SOCS-3) in fasted rats, TRH neurons in the paraventricular nucleus are activated directly by leptin. To a greater degree, they also contain melanocortin-4 receptors (MC4Rs), implying that leptin can act directly or indirectly by increasing the production of the MC4R ligand, α-melanocyte stimulating hormone (α-MSH), to regulate TRH expression. We further demonstrate that both pathways converge on the TRH promoter. The melanocortin system activates the TRH promoter through the phosphorylation and DNA binding of the cAMP response element binding protein (CREB), and leptin signaling directly regulates the TRH promoter through the phosphorylation of signal transducer and activator of transcription 3 (St...

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“…Slides were processed for double-label in situ hybridization as previously described, with some modi®cations. 21 Slides were processed initially in a similar fashion to those in experiments 1 and 2. They were then hybridized overnight at 608C with the 33 P-labeled mouse Ob-Rb riboprobe, which was used at 2.5 pmol/ml, and the digoxigenin-labeled rat galanin riboprobe, which was used at 1:300 dilution.…”

Section: S-labeled Uridinementioning

confidence: 99%

Distribution of galanin messenger RNA-expressing cells in murine brain and their regulation by leptin in regions of the hypothalamus

et al. 2001

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AbstractÐGalanin is widely distributed throughout the mammalian brain and has been implicated in the regulation of food intake, metabolism and reproductionÐfunctions that are also thought to be under the control of leptin. To investigate the possible role of galanin in mediating the physiological effects of leptin in the mouse, we had three experimental objectives: ®rst, to map the distribution of galanin messenger RNA-expressing cells in the brain of the mouse; second, to assess the effects of leptin on galanin gene expression in areas of the brain thought to be involved in the regulation of body weight and reproduction; and third, to determine whether galanin neurons in these regions express leptin receptor messenger RNA. We found the pattern of galanin messenger RNA expression in the mouse brain to be similar, but not identical, to that in the rat. Leptin treatment (2 mg/g for six days) signi®cantly reduced cellular levels of galanin messenger RNA in the hypothalamic periventricular nucleus of leptin-de®cient obese (ob/ob) mice (P , 0.01) by approximately 30%; however, leptin did not appear to in¯uence the expression of galanin in the arcuate or dorsomedial nucleus of the hypothalamus. Galanin-producing neurons in the arcuate, dorsomedial and periventricular nuclei did not appear to express leptin receptor messenger RNA (P . 0.05).These results demonstrate that galanin distribution patterns in the mouse brain are comparable to other species and, yet, possess unique features. In addition, galanin-expressing neurons in the hypothalamic periventricular nucleus are targets for regulation by leptin; however, the effect of leptin on galanin gene expression is likely to be mediated indirectly, perhaps through either proopiomelanocortin-or neuropeptide Y-expressing cells in the hypothalamus. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.Key words: neuropeptide, gene regulation, neuroanatomy, in situ hybridization, leptin receptor.Galanin is a 29 amino acid neuropeptide that was originally isolated from the porcine gut. 42 In mammals, galanin is widely distributed throughout the central and peripheral nervous systems. 26 One curious observation about galanin's expression in the brain of mammals is its species speci®city, which is particularly evident in the rat and monkey. For example, in the rat there is little or no expression of galanin in the hippocampus but there are abundant galanin-containing cells in the dorsal raphe. 23,34 In the monkey, however, galanin producing cells are found throughout the hippocampus, yet are absent from the dorsal raphe. 17 To date, there has been no detailed mapping of the distribution of galanin in the mouse brain. Because of the growing importance of the mouse as a genetic model for understanding the brain, it is vital that we know the comparative anatomy of neuropeptide expression in the murine brain and use this information to shed light on the differences and similarities between species. The ®rst objective of this study was to complete a detailed map of the...

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Simultaneous visualization of two cellular mRNA species in individual neurons by use of a new double in Situ hybridization method

Cited by 59 publications

References 14 publications

Neonatal imprinting predetermines the sexually dimorphic, estrogen-dependent expression of galanin in luteinizing hormone-releasing hormone neurons.

Neonatal imprinting predetermines the sexually dimorphic, estrogen-dependent expression of galanin in luteinizing hormone-releasing hormone neurons.

Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling

Distribution of galanin messenger RNA-expressing cells in murine brain and their regulation by leptin in regions of the hypothalamus

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