scholarly journals Differential gene expression in populations of sympathetic neurons purified from a neuropeptide Y reporter mouse

2017 ◽  
Author(s):  
Kristine M. Sikora ◽  
Paul H. M. Kullmann ◽  
John P. Horn
Keyword(s):  
Neuropeptide Y ◽  
Candidate Genes ◽  
Fluorescent Protein ◽  
Sympathetic Neurons ◽  
Quantitative Polymerase Chain Reaction ◽  
Promoter Sequences ◽  
Reporter Mouse ◽  
Cervical Ganglion ◽  
Differential Gene ◽  
Green Fluorescent

AbstractUsing gene chip methodology, we identified candidate genes differentially expressed by vasomotor and non-vasomotor sympathetic neurons in the superior cervical ganglion. Groups of 10 neurons were manually sorted after isolation from a transgenic neuropeptide Y (NPY) reporter mouse that expresses the humanized Renilla green fluorescent protein (GFP) under control of Npy promoter sequences. Anatomical analysis of GFP and NPY co-expression showed that 98% of GFP-positive neurons and 16% of GFP-negative neurons express NPY. The probability of contamination in sorted cell samples was therefore high. To minimize this problem, we screened amplified cDNA samples by the quantitative polymerase chain reaction. This approach identified 101 candidate genes preferentially expressed by GFP-positive neurons and 74 candidate genes preferentially expressed by GFP-negative neurons.

scholarly journals Adiponectin receptors are expressed in hypothalamus and colocalized with proopiomelanocortin and neuropeptide Y in rodent arcuate neurons

2008 ◽  
Vol 200 (1) ◽  
pp. 93-105 ◽  
Author(s):  
E Guillod-Maximin ◽  
A F Roy ◽  
C M Vacher ◽  
A Aubourg ◽  
V Bailleux ◽  
...  
Keyword(s):  
Neuropeptide Y ◽  
Energy Homeostasis ◽  
Fluorescent Protein ◽  
Peripheral Tissues ◽  
Rat Hypothalamus ◽  
Ampk Phosphorylation ◽  
Hypothalamic Nuclei ◽  
Green Fluorescent ◽  
Amp Activated Protein Kinase

Adiponectin is involved in the control of energy homeostasis in peripheral tissues through Adipor1 and Adipor2 receptors. An increasing amount of evidence suggests that this adipocyte-secreted hormone may also act at the hypothalamic level to control energy homeostasis. In the present study, we observed the gene and protein expressions of Adipor1 and Adipor2 in rat hypothalamus using different approaches. By immunohistochemistry, Adipor1 expression was ubiquitous in the rat brain. By contrast, Adipor2 expression was more limited to specific brain areas such as hypothalamus, cortex, and hippocampus. In arcuate and paraventricular hypothalamic nuclei, Adipor1, and Adipor2 were expressed by neurons and astrocytes. Furthermore, using transgenic green fluorescent protein mice, we showed that Adipor1 and Adipor2 were present in pro–opiomelanocortin (POMC) and neuropeptide Y (NPY) neurons in the arcuate nucleus. Finally, adiponectin treatment by intracerebroventricular injection induced AMP-activated protein kinase (AMPK) phosphorylation in the rat hypothalamus. This was confirmed byin vitrostudies using hypothalamic membrane fractions. In conclusion, Adipor1 and Adipor2 are both expressed by neurons (including POMC and NPY neurons) and astrocytes in the rat hypothalamic nuclei. Adiponectin is able to increase AMPK phosphorylation in the rat hypothalamus. These data reinforced a potential role of adiponectin and its hypothalamic receptors in the control of energy homeostasis.

scholarly journals Suppression of Basal Spontaneous Gonadotropin-Releasing Hormone Neuronal Activity during Lactation: Role of Inhibitory Effects of Neuropeptide Y

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 333-340 ◽  
Author(s):  
Jing Xu ◽  
Melissa A. Kirigiti ◽  
Michael A. Cowley ◽  
Kevin L. Grove ◽  
M. Susan Smith
Keyword(s):  
Neuropeptide Y ◽  
Neuronal Activity ◽  
Fluorescent Protein ◽  
Resting Membrane Potential ◽  
Inhibitory Effects ◽  
Gnrh Neurons ◽  
Cell Current ◽  
Green Fluorescent ◽  
Hypothalamic Slices

Increased neuropeptide Y (NPY) activity drives the chronic hyperphagia of lactation and may contribute to the suppression of GnRH activity. The majority of GnRH neurons are contacted by NPY fibers, and GnRH cells express NPY Y5 receptor (Y5R). Therefore, NPY provides a neurocircuitry for information about food intake/energy balance to be directly transmitted to GnRH neurons. To investigate the effects of lactation on GnRH neuronal activity, hypothalamic slices were prepared from green fluorescent protein-GnRH transgenic rats. Extracellular loose-patch recordings determined basal GnRH neuronal activity from slices of ovariectomized control and lactating rats. Compared with controls, hypothalamic slices from lactating rats had double the number of quiescent GnRH neurons (14.51 ± 2.86 vs. 7.04 ± 2.84%) and significantly lower firing rates of active GnRH neurons (0.25 ± 0.02 vs. 0.37 ± 0.03 Hz). To study the NPY-postsynaptic Y5R system, whole-cell current-clamp recordings were performed in hypothalamic slices from control rats to examine NPY/Y5R antagonist effects on GnRH neuronal resting membrane potential. Under tetrodotoxin treatment, NPY hyperpolarized GnRH neurons from −56.7 ± 1.94 to −62.1 ± 1.83 mV; NPY’s effects were blocked by Y5R antagonist. To determine whether increased endogenous NPY tone contributes to GnRH neuronal suppression during lactation, hypothalamic slices were treated with Y5R antagonist. A significantly greater percentage of GnRH cells were activated in slices from lactating rats (52%) compared with controls (28%). These results suggest that: 1) basal GnRH neuronal activity is suppressed during lactation; 2) NPY can hyperpolarize GnRH neurons via postsynaptic Y5R; and 3) increased inhibitory NPY tone during lactation is a component of the mechanisms responsible for suppression of GnRH neuronal activity. Neuropeptide Y (NPY) directly hyperpolarizes GnRH neurons via postsynaptic NPY Y5 receptor. Increased inhibitory NPY tone during lactation is an important component of the suppression of GnRH neuronal activity.

Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cryptococcus Neoformans ◽  
Differential Gene Expression ◽  
Fluorescent Protein ◽  
Differential Gene ◽  
Green Fluorescent

Elucidation of the distal convoluted tubule transcriptome identifies new candidate genes involved in renal Mg2+ handling

2013 ◽  
Vol 305 (11) ◽  
pp. F1563-F1573 ◽  
Author(s):  
Jeroen H. F. de Baaij ◽  
Marian J. Groot Koerkamp ◽  
Marla Lavrijsen ◽  
Femke van Zeeland ◽  
Hans Meijer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Candidate Genes ◽  
Transient Receptor Potential ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Distal Convoluted Tubule ◽  
Fine Tuning ◽  
Complex Object ◽  
New Genes ◽  
Green Fluorescent

The kidney plays a key role in the maintenance of Mg2+ homeostasis. Specifically, the distal convoluted tubule (DCT) is instrumental in the fine-tuning of renal Mg2+ handling. In recent years, hereditary Mg2+ transport disorders have helped to identify important players in DCT Mg2+ homeostasis. Nevertheless, several proteins involved in DCT-mediated Mg2+ reabsorption remain to be discovered, and a full expression profile of this complex nephron segment may facilitate the discovery of new Mg2+-related genes. Here, we report Mg2+-sensitive expression of the DCT transcriptome. To this end, transgenic mice expressing enhanced green fluorescent protein under a DCT-specific parvalbumin promoter were subjected to Mg2+-deficient or Mg2+-enriched diets. Subsequently, the Complex Object Parametric Analyzer and Sorter allowed, for the first time, isolation of enhanced green fluorescent protein-positive DCT cells. RNA extracts thereof were analyzed by DNA microarrays comparing high versus low Mg2+ to identify Mg2+ regulatory genes. Based on statistical significance and a fold change of at least 2, 46 genes showed differential expression. Several known magnesiotropic genes, such as transient receptor potential cation channel, subfamily M, member 6 ( Trpm6), and Parvalbumin, were upregulated under low dietary Mg2+. Moreover, new genes were identified that are potentially involved in renal Mg2+ handling. To confirm that the selected candidate genes were regulated by dietary Mg2+ availability, the expression levels of solute carrier family 41, member 3 ( Slc41a3), pterin-4 α-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1α ( Pcbd1), TBC1 domain family, member 4 ( Tbc1d4), and uromodulin ( Umod) were determined by RT-PCR analysis. Indeed, all four genes show significant upregulation in the DCT of mice fed a Mg2+-deficient diet. By elucidating the Mg2+-sensitive DCT transcriptome, new candidate genes in renal Mg2+ handling have been identified.

scholarly journals Pseudorabies Virus Us9 Directs Axonal Sorting of Viral Capsids

2007 ◽  
Vol 81 (20) ◽  
pp. 11363-11371 ◽  
Author(s):  
M. G. Lyman ◽  
B. Feierbach ◽  
D. Curanovic ◽  
M. Bisher ◽  
L. W. Enquist
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Sympathetic Neurons ◽  
Anterograde Transport ◽  
Viral Capsids ◽  
Green Fluorescent ◽  
Cultured Sympathetic Neurons ◽  
Retrograde Spread ◽  
Axonal Sorting

ABSTRACT Pseudorabies virus (PRV) mutants lacking the Us9 gene cannot spread from presynaptic to postsynaptic neurons in the rat visual system, although retrograde spread remains unaffected. We sought to recapitulate these findings in vitro using the isolator chamber system developed in our lab for analysis of the transneuronal spread of infection. The wild-type PRV Becker strain spreads efficiently to postsynaptic neurons in vitro, whereas the Us9-null strain does not. As determined by indirect immunofluorescence, the axons of Us9-null infected neurons do not contain the glycoproteins gB and gE, suggesting that their axonal sorting is dependent on Us9. Importantly, we failed to detect viral capsids in the axons of Us9-null infected neurons. We confirmed this observation by using three different techniques: by direct fluorescence of green fluorescent protein-tagged capsids; by transmission electron microscopy; and by live-cell imaging in cultured, sympathetic neurons. This finding has broad impact on two competing models for how virus particles are trafficked inside axons during anterograde transport and redefines a role for Us9 in viral sorting and transport.

scholarly journals Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820 ◽  
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Cns Infection ◽  
Gfp Expression ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
Differential Gene ◽  
Green Fluorescent

ABSTRACT Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungusCryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFα1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFα1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFα1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFα1 promoter is induced by nutrient deprivation and the MATα locus containing the MFα1 gene has been linked with virulence, yeast cells containing the pMFα1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFα1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFα1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.

scholarly journals Necrosis-Inducing Proteins of Rhynchosporium commune, Effectors in Quantitative Disease Resistance

2012 ◽  
Vol 25 (10) ◽  
pp. 1314-1325 ◽  
Author(s):  
S. Kirsten ◽  
A. Navarro-Quezada ◽  
D. Penselin ◽  
C. Wenzel ◽  
A. Matern ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Quantitative Polymerase Chain Reaction ◽  
Fungal Spores ◽  
Growth Patterns ◽  
Effector Proteins ◽  
Defense Gene Expression ◽  
Host Genotype ◽  
Barley Cultivars ◽  
Green Fluorescent ◽  
The Impact

The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an “active” NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner.

scholarly journals Properties of Wild-Type and Fluorescent Protein-Tagged Mouse Tetrodotoxin-Resistant Sodium Channel (NaV1.8) Heterologously Expressed in Rat Sympathetic Neurons

2008 ◽  
Vol 99 (4) ◽  
pp. 1917-1927 ◽  
Author(s):  
Geoffrey G. Schofield ◽  
Henry L. Puhl ◽  
Stephen R. Ikeda
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Sympathetic Neurons ◽  
Functional Expression ◽  
Nodose Ganglion Neurons ◽  
Similar Expression Pattern ◽  
Cervical Ganglion ◽  
Clonal Cell Lines ◽  
Mouse Dorsal Root Ganglion ◽  
Ganglion Neurons

The tetrodotoxin (TTX)-resistant Na+ current arising from NaV1.8-containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. Here, we show that injection of cDNA encoding mouse NaV1.8 into the nuclei of rat superior cervical ganglion (SCG) neurons results in TTX-resistant Na+ currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion (DRG) neurons. The activation and inactivation properties of the heterologously expressed NaV1.8 Na+ channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed NaV1.8 channels was shifted about 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of NaV1.8 did not substantially affect functional expression in SCG neurons. Unexpectedly, fluorescence was not concentrated at the plasma membrane but found throughout the interior of the neuron in a granular pattern. A similar expression pattern was observed in nodose ganglion neurons expressing the tagged channels. In contrast, expression of tagged NaV1.8 in HeLa cells revealed a fluorescence pattern consistent with sequestration in the endoplasmic reticulum, thus providing a basis for poor functional expression in clonal cell lines. Our results establish SCG neurons as a favorable surrogate for the expression and study of molecularly defined NaV1.8-containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of NaV1.8 with a biophysical phenotype identical to that found in sensory neurons.

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