Genetic Targeting of Green Fluorescent Protein to Gonadotropin-Releasing Hormone Neurons: Characterization of Whole-Cell Electrophysiological Properties and Morphology

Suter, Kelly J.; Song, Walter J.; Sampson, Traci L.; Wuarin, Jean Pierre; Saunders, Justin T.; Dudek, F. Edward; Moenter, Suzanne M.

doi:10.1210/en.141.1.412

Cited by 190 publications

(184 citation statements)

References 40 publications

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“…The data obtained by RT-PCR and in situ hybridization studies strongly indicate that 3 kb of the rat GnRH gene 5Ј f lanking regions specifically target expression to GnRH neurons. Although no previous studies had been performed in transgenic rats, this same promoter specifically directed expression of green f luorescent protein to GnRH neurons in transgenic mice (38). The infertility of the transgenic rats supports the use of overexpression of a phosphodiesterase (PDE) as a strategy to manipulate cAMP responses in vivo.…”

Section: Discussionmentioning

confidence: 98%

Phosphodiesterase expression targeted to gonadotropin-releasing hormone neurons inhibits luteinizing hormone pulses in transgenic rats

Paruthiyil

Majdoubi

Conti

et al. 2002

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

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Experiments in the GT1 gonadotropin-releasing hormone (GnRH) cell line have shown that the cAMP signaling pathway plays a central role in regulating the excitability of the cells. Lowering cAMP levels by expressing the constitutively active cAMP-specific phosphodiesterase PDE4D1 in GT1 cells inhibited spontaneous Ca 2؉ oscillations and intrinsic pulsatile GnRH secretion. To address the role of cAMP levels in endogenous GnRH neurons, we genetically targeted expression of PDE4D1 (P) to GnRH neurons in transgenic rats (R) by using the GnRH gene promoter͞enhancer regions (G). Three lines of transgenic rats, GPR-2, -4, and -5, were established. In situ hybridization and RT-PCR studies demonstrated that transgene expression was specifically targeted to GnRH neurons. Decreased fertility was observed in female but not in male rats from all three lines. The mean luteinizing hormone (LH) levels in ovariectomized rats were significantly reduced in the GPR-4 and -5 lines but not in the GPR-2 line. In castrated male and female GPR-4 rats, the LH pulse frequency was dramatically reduced. Six of twelve GPR-4 females studied did not ovulate and had polycystic ovaries. The remaining six females ovulated, but the magnitude of the preovulatory LH surge was inhibited by 63%. These findings support the hypothesis that cAMP signaling may play a central role in regulating excitability of GnRH neurons in vivo. The GPR-4 line of transgenic rats provides a genetic model for the understanding of the role of pulsatile gonadotropin release in follicular development.T he pulsatile release of gonadotropin-releasing hormone (GnRH) underlies reproductive function in male and female mammals. Understanding the molecular mechanisms regulating pulsatile GnRH secretion has been hampered by the low number of GnRH neurons and their scattered localization throughout the preoptic area in rodents. Further complicating the understanding of these questions is the complexity of the neural connectivity of GnRH neurons. The development of the highly differentiated GT1 GnRH neuronal cell lines (1) and the demonstration that pulsatile release was an intrinsic property of the cells provided a model to study molecular events underlying pulsatile GnRH release (2-4). However, one is always faced with the problem of translating findings from an immortalized cell line to the physiological setting. We have attempted to bridge observations made in GT1 cells regarding signaling events regulating GnRH pulsatility to the physiological setting by developing a transgenic rat model. We used an established genetic approach to alter the activity of a signaling pathway that could be used in parallel experiments in GT1 cells and transgenic rats. Transgenic rats, unlike transgenic mice, permit the reliable analysis of pulsatile luteinizing hormone (LH) release that closely mimics pulsatile GnRH release in castrated animals (5).The secretion of GnRH in GT1 neurons is stimulated by elevations in cAMP levels (6-8). The stimulation of GnRH secretion by elevated cAMP levels appears to...

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Section: Discussionmentioning

confidence: 98%

Phosphodiesterase expression targeted to gonadotropin-releasing hormone neurons inhibits luteinizing hormone pulses in transgenic rats

Paruthiyil

Majdoubi

Conti

et al. 2002

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

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“…The latter findings combined with recent technological advances, such as the development of ER mutants and transgenic animals expressing green fluorescent protein (GFP) in GnRH neurons, have greatly facilitated studies to understand the cellular mechanisms by which GnRH neurons are regulated by estrogen (Spergel et al, 1999, Suter et al, 2000, Kato et al, 2003, Han et al, 2005, Abraham et al, 2003, Smith et al, 2006, Wintermantel et al, 2006. A series of recent publications show that in mouse hypothalamic neuronal explants and in primate nasal placode cultures, E2 augments synchronous intracellular Ca 2+ oscillations in GnRH neurons (Temple et al, 2004, Temple and Wray, 2005, Abe et al, 2007.…”

Section: β-Estradiol and Gnrh Neurosecretionmentioning

confidence: 99%

Membrane-initiated estrogen signaling in hypothalamic neurons

Kelly

Rønnekleiv

2008

Molecular and Cellular Endocrinology

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SummaryIt is well known that many of the actions of 17β-estradiol (E2) in the central nervous system are mediated via intracellular receptor/transcription factors that interact with steroid response elements on target genes. However, there is compelling evidence for membrane steroid receptors for estrogen in hypothalamic and other brain neurons. But it is not well understood how estrogen signals via membrane receptors, and how these signals impact not only membrane excitability but also gene transcription in neurons. Indeed, it has been known for sometime that E2 can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane delimited events. In addition, E2 can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell signaling. Therefore, this review will consider our current knowledge of rapid membrane-initiated and intracellular signaling by E2 in the hypothalamus, the nature of receptors involved and how they contribute to homeostatic functions. KeywordsERα; ERβ; GPCR (G protein-coupled receptor); mER (membrane estrogen receptor that is a GPCR) Electrophysiological studies in the 1960's and 1970's suggested that gonadal steroids could directly modulate the electrical activity of hypothalamic neurons

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“…It appears that correcting for protein content did not qualitatively change the target faithfully reporter expression to GnRH neurons. The Ϫ3446/ϩ23 bp mGnRH promoter fragment was used to generate transgenic mice in which green fluorescent protein (GFP) was targeted to GnRH neurons (23). In the GFP mice, 99.5% of GFP-expressing neurons were found in association with immunodetectable GnRH peptide.…”

Section: Figmentioning

confidence: 99%

Promoter Sequences Targeting Tissue-specific Gene Expression of Hypothalamic and Ovarian Gonadotropin-releasing Hormone in Vivo

Kim

Wolfe

Smith

et al. 2002

Journal of Biological Chemistry

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Molecular mechanisms directing tissue-specific expression of gonadotropin-releasing hormone (GnRH) are difficult to study due to the paucity and scattered distribution of GnRH neurons. To identify regions of the mouse GnRH (mGnRH) promoter that are critical for appropriate tissue-specific gene expression, we generated transgenic mice with fragments (؊3446/؉23 bp, ؊2078/؉23 bp, and ؊1005/؉28 bp) of mGnRH promoter fused to the luciferase reporter gene. The pattern of mGnRH promoter activity was assessed by measuring luciferase activity in tissue homogenates. All three 5-fragments of mGnRH promoter targeted hypothalamic expression of the luciferase transgene, but with the exception of the ovary, luciferase expression was absent in non-neural tissues. High levels of ovarian luciferase activity were observed in mice generated with both ؊2078 and ؊1005 bp of promoter. Our study is the first to define a region of the GnRH gene promoter that directs expression to both neural and non-neural tissues in vivo. We demonstrate that DNA sequences contained within the proximal ؊1005 bp of the mGnRH promoter are sufficient to direct mGnRH gene expression to both the ovary and hypothalamus. Our results also suggest that DNA sequences distal to ؊2078 bp mediate the repression of ovarian GnRH.

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Genetic Targeting of Green Fluorescent Protein to Gonadotropin-Releasing Hormone Neurons: Characterization of Whole-Cell Electrophysiological Properties and Morphology

Cited by 190 publications

References 40 publications

Phosphodiesterase expression targeted to gonadotropin-releasing hormone neurons inhibits luteinizing hormone pulses in transgenic rats

Phosphodiesterase expression targeted to gonadotropin-releasing hormone neurons inhibits luteinizing hormone pulses in transgenic rats

Membrane-initiated estrogen signaling in hypothalamic neurons

Promoter Sequences Targeting Tissue-specific Gene Expression of Hypothalamic and Ovarian Gonadotropin-releasing Hormone in Vivo

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