scholarly journals Characterization of Leptin-Responsive Neurons in the Caudal Brainstem

Endocrinology ◽  
2006 ◽  
Vol 147 (7) ◽  
pp. 3190-3195 ◽  
Author(s):  
Kate L. J. Ellacott ◽  
Ilia G. Halatchev ◽  
Roger D. Cone
Keyword(s):  
Green Fluorescent Protein ◽  
Energy Homeostasis ◽  
Leptin Receptor ◽  
Fluorescent Protein ◽  
Physiological Role ◽  
Cell Group ◽  
Melanocortin System ◽  
Peptide Precursor ◽  
Green Fluorescent

The central melanocortin system plays a key role in the regulation of energy homeostasis. Neurons containing the peptide precursor proopiomelanocortin (POMC) are found at two sites in the brain, the arcuate nucleus of the hypothalamus (ARC) and the caudal region of the nucleus of the solitary tract (NTS). ARC POMC neurons, which also express cocaine- and amphetamine-regulated transcript (CART), are known to mediate part of the response to factors regulating energy homeostasis, such as leptin and ghrelin. In contrast, the physiological role(s) of the POMC neurons in the caudal brainstem are not well characterized. However, development of a transgenic mouse expressing green fluorescent protein under the control of the POMC promoter [POMC-enhanced green fluorescent protein (EGFP) mouse] has aided the study of these neurons. Indeed, recent studies have shown significant activation of NTS POMC-EGFP cells by the gut released satiety factor cholecystokinin (CCK). Here we show that peripheral leptin administration induces the expression of phospho-signal transducer and activator of transcription 3 immunoreactivity (pSTAT3-IR), a marker of leptin receptor signaling, in more than 50% of NTS POMC-EGFP neurons. Furthermore, these POMC-EGFP neurons comprise 30% of all pSTAT3-IR cells in the NTS. Additionally, we also show that in contrast to the ARC population, NTS POMC-EGFP neurons do not coexpress CART immunoreactivity. These data suggest that NTS POMC neurons may participate with ARC POMC cells in mediating some of the effects of leptin and thus comprise a novel cell group regulated by both long-term adipostatic signals and satiety factors such as CCK.

scholarly journals Isolation and Immortalization of MIP-GFP Neurons From the Hypothalamus

Endocrinology ◽  
2014 ◽  
Vol 155 (6) ◽  
pp. 2314-2319 ◽  
Author(s):  
Zi Chen Wang ◽  
Michael B. Wheeler ◽  
Denise D. Belsham
Keyword(s):  
Green Fluorescent Protein ◽  
Energy Homeostasis ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Unintended Effects ◽  
Hypothalamic Neurons ◽  
Related Peptide ◽  
Promoter Construct ◽  
Insulin Responsiveness ◽  
Green Fluorescent

The mouse insulin I promoter (MIP) construct was developed to eliminate the promoter activity detected with the rat insulin II promoter in specific hypothalamic neurons that may have unintended effects on glucose and energy homeostasis in transgenic models. Thus, the specificity of this novel construct must be validated prior to the widespread availability of derived Cre models. Although limited validation efforts have indicated a lack of MIP activity within neuronal tissue, the global immunohistochemical methodology used may not be specific enough to rule out the possibility of specific populations of neurons with MIP activity. To investigate possible MIP activity within the hypothalamus, primary hypothalamic isolates from MIP-green fluorescent protein reporter mice were analyzed after fluorescent-activated cell sorting. Primary hypothalamic neurons isolated from the MIP-green fluorescent protein mice were immortalized. Characterization detected the presence of hypothalamic neuropeptide Y (NPY) and agouti-related peptide, involved in the control of energy homeostasis, as well as confirmed insulin responsiveness in the cell lines. Moreover, because insulin was demonstrated to differentially regulate NPY expression within these MIP neurons, the promoter construct may be active in multiple hypothalamic NPY/agouti-related peptide subpopulations with unique physiological functions. MIP transgenic animals may therefore face similar limitations seen previously with rat insulin II promoter-based models.

scholarly journals Retrograde Transport of Transcription Factor NF-κB in Living Neurons

2000 ◽  
Vol 276 (15) ◽  
pp. 11821-11829 ◽  
Author(s):  
Henning Wellmann ◽  
Barbara Kaltschmidt ◽  
Christian Kaltschmidt
Keyword(s):  
Transcription Factor ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Receptor Activation ◽  
Transcriptional Changes ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Living Neurons

The mechanism by which signals such as those produced by glutamate are transferred to the nucleus may involve direct transport of an activated transcription factor to trigger long-term transcriptional changes. Ionotropic glutamate receptor activation or depolarization activates transcription factor NF-κB and leads to translocation of NF-κB from the cytoplasm to the nucleus. We investigated the dynamics of NF-κB translocation in living neurons by tracing the NF-κB subunit RelA (p65) with jellyfish green fluorescent protein. We found that green fluorescent protein-RelA was located in either the nucleus or cytoplasm and neurites, depending on the coexpression of the cognate inhibitor of NF-κB, IκB-α. Stimulation with glutamate, kainate, or potassium chloride resulted in a redistribution of NF-κB from neurites to the nucleus. This transport depended on an intact nuclear localization signal on RelA. Thus, in addition to its role as a transcription factor, NF-κB may be a signal transducer, transmitting transient glutamatergic signals from distant sites to the nucleus.

scholarly journals Leptin receptor neurons in the mouse hypothalamus are colocalized with the neuropeptide galanin and mediate anorexigenic leptin action

2013 ◽  
Vol 304 (9) ◽  
pp. E999-E1011 ◽  
Author(s):  
Amanda Laque ◽  
Yan Zhang ◽  
Sarah Gettys ◽  
Tu-Anh Nguyen ◽  
Kelly Bui ◽  
...  
Keyword(s):  
Energy Homeostasis ◽  
Leptin Receptor ◽  
Fluorescent Protein ◽  
Inhibitory Function ◽  
Reporter Mice ◽  
Brainstem Nucleus ◽  
Melanin Concentrating Hormone ◽  
Regulate Food Intake ◽  
Receptor Neurons ◽  
Green Fluorescent

Leptin acts centrally via leptin receptor (LepRb)-expressing neurons to regulate food intake, energy expenditure, and other physiological functions. LepRb neurons are found throughout the brain, and several distinct populations contribute to energy homeostasis control. However, the function of most LepRb populations remains unknown, and their contribution to regulate energy homeostasis has not been studied. Galanin has been hypothesized to interact with the leptin signaling system, but literature investigating colocalization of LepRb and galanin has been inconsistent, which is likely due to technical difficulties to visualize both. We used reporter mice with green fluorescent protein expression from the galanin locus to recapitulate the colocalization of galanin and leptin-induced p-STAT3 as a marker for LepRb expression. Here, we report the existence of two populations of galanin-expressing LepRb neurons (Gal-LepRb neurons): in the hypothalamus overspanning the perifornical area and adjacent dorsomedial and lateral hypothalamus [collectively named extended perifornical area (exPFA)] and in the brainstem (nucleus of the solitary tract). Surprisingly, despite the known orexigenic galanin action, leptin induces galanin mRNA expression and stimulates LepRb neurons in the exPFA, thus conflicting with the expected anorexigenic leptin action. However, we confirmed that intra-exPFA leptin injections were indeed sufficient to mediate anorexic responses. Interestingly, LepRb and galanin-expressing neurons are distinct from orexin or melanin-concentrating hormone (MCH)-expressing neurons, but exPFA galanin neurons colocalized with the anorexigenic neuropeptides neurotensin and cocaine- and amphetamine-regulated transcript (CART). Based on galanin's known inhibitory function, we speculate that in exPFA Gal-LepRb neurons galanin acts inhibitory rather than orexigenic.

scholarly journals Transgenic Expression of Enhanced Green Fluorescent Protein Enables Direct Visualization for Physiological Studies of Vasopressin Neurons and Isolated Nerve Terminals of the Rat

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 406-413 ◽  
Author(s):  
Yoichi Ueta ◽  
Hiroaki Fujihara ◽  
Ryota Serino ◽  
Govindan Dayanithi ◽  
Hitoshi Ozawa ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Physiological Role ◽  
Transgenic Rats ◽  
Patch Clamp Technique ◽  
Axon Terminals ◽  
Green Fluorescent ◽  
Vasopressin Neurons

We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.

scholarly journals Cellular Leptin Resistance Impairs the Leptin-Mediated Suppression of Neuropeptide Y Secretion in Hypothalamic Neurons

Endocrinology ◽  
2011 ◽  
Vol 152 (11) ◽  
pp. 4138-4147 ◽  
Author(s):  
Sandeep S. Dhillon ◽  
Sean A. McFadden ◽  
Jennifer A. Chalmers ◽  
Maria-Luisa Centeno ◽  
Ginah L. Kim ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Neuropeptide Y ◽  
Energy Homeostasis ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Leptin Resistance ◽  
Cellular Processes ◽  
Compound C ◽  
Green Fluorescent ◽  
Induced Changes

Evidence shows that neuropeptide Y (NPY) neurons are involved in mediating the anorexigenic action of leptin via neuronal circuits in the hypothalamus. However, studies have produced limited data on the cellular processes involved and whether hypothalamic NPY neurons are susceptible to cellular leptin resistance. To investigate the direct regulation of NPY secretion by leptin, we used novel NPY-synthesizing, immortalized mHypoA-NPY/green fluorescent protein and mHypoA-59 hypothalamic cell lines derived from adult hypothalamic primary cultures. We report that leptin treatment significantly suppressed NPY secretion in the cells by approximately 20%. We found a decrease in c-fos expression upon leptin exposure, indicating deactivation or hyperpolarization of the neurons. Protein analysis indicated that leptin inhibits AMP-activated protein kinase (AMPK) activity and activates acetyl-coenzyme A carboxylase in NPY neurons, supporting the hypothesis of an AMPK-dependent mechanism. Inhibiting both AMPK with Compound C or phosphatidylinositol 3 kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-1(4H)-1-benzopyran-4-one hydrochloride prevented the leptin-mediated decrease in NPY secretion, indicating both AMPK- and PI3K-mediated mechanisms. Further, NPY secretion was stimulated by 30% by the AMPK activator, aminoimidazole carboxamide ribonucleotide. Importantly, prolonged leptin exposure in the mHypoA-NPY/green fluorescent protein cells prevented leptin-induced changes in AMPK phosphorylation and suppression of NPY secretion, indicating that NPY neurons are susceptible to leptin resistance. Our studies indicate that AMPK and PI3K pathways are involved in leptin action in NPY neurons and that leptin resistance blocks the feedback response likely required to maintain energy homeostasis.

Long-Term, Stable Expression of Green Fluorescent Protein in Mammalian Cells

1997 ◽  
Vol 236 (2) ◽  
pp. 347-350 ◽  
Author(s):  
Alexander N. Gubin ◽  
Bindu Reddy ◽  
Joyce M. Njoroge ◽  
Jeffery L. Miller
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Stable Expression ◽  
Green Fluorescent
Sign in / Sign up

Export Citation Format

Share Document