scholarly journals Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

10.3791/50672 ◽  
2015 ◽  
Author(s):  
Gustavo Arriaga ◽  
Joshua J. Macopson ◽  
Erich D. Jarvis
Keyword(s):  
Cholera Toxin ◽  
Pseudorabies Virus ◽  
Double Labeling ◽  
Transsynaptic Tracing ◽  
Biotinylated Dextran

scholarly journals Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells.

1983 ◽  
Vol 3 (1) ◽  
pp. 91-101 ◽  
Author(s):  
N K Gonatas ◽  
A Stieber ◽  
J Gonatas ◽  
T Mommoi ◽  
P H Fishman
Keyword(s):  
Cholera Toxin ◽  
Neuroblastoma Cells ◽  
Double Labeling ◽  
Electron Microscope Autoradiography ◽  
Density Distributions ◽  
Quantitative Studies ◽  
Covalently Linked ◽  
The Relationship ◽  
Cytochemical Marker ◽  
In Cells

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.

Transneuronal retrograde transport of attenuated pseudorabies viruses within central visual pathways

2001 ◽  
Vol 18 (4) ◽  
pp. 633-640 ◽  
Author(s):  
RODNEY J. MOORE ◽  
SHERRY VINSANT ◽  
ANITA K. McCAULEY ◽  
NUWAN C. KURUKULASURIYA ◽  
DWAYNE W. GODWIN
Keyword(s):  
Visual Cortex ◽  
Pseudorabies Virus ◽  
Retrograde Transport ◽  
Visual Pathways ◽  
Thalamic Reticular Nucleus ◽  
Reticular Nucleus ◽  
Gamma Aminobutyric Acid ◽  
Anterograde Transport ◽  
Double Labeling ◽  
The Central Nervous System

Pseudorabies virus (PRV) has been shown to be an effective transneuronal tracer within both the peripheral and the central nervous system. The only investigations of this virus in the visual system have examined anterograde transport of PRV from injection sites in the retina. In the present study, we injected attenuated forms of PRV into the primary visual cortex of both rats and cats to determine whether transneuronal retrograde infection would occur back to the retina. In rats, we made small injections into visual cortex of a strain of PRV (Bartha Blu) that contained a β-galactosidase promoter insert. In cats, we injected PRV-M201 into area V1 of visual cortex. After a 2- to 4-day incubation period, we examined tissue from these animals for the presence of the β-galactosidase marker (rats) or the virus itself (cats). Cortical PRV injections resulted in transneuronal retrograde infection of the lateral geniculate nucleus (LGN), thalamic reticular nucleus (TRN), and retina. PRV was retinotopically distributed in the pathway. In addition, double-labeling experiments in cats using an antibody against gamma-aminobutyric acid (GABA) were conducted to reveal PRV-labeled interneurons within the LGN and TRN. All TRN neurons were GABA+, as was a subset of LGN neurons. Only the subset of TRN neurons adjacent to the PRV-labeled sector of LGN was labeled with PRV. In addition, a subset of GABA+ interneurons in LGN was also labeled with PRV. We processed some tissue for electron microscopy to examine the morphology of the virus at various replication stages. No mature virions were detected in terminals from efferent pathways, although forms consistent with retrograde infection were encountered. We conclude that the PRV strains we have used produce a local infection that progresses primarily in the retrograde direction in the central visual pathways. The infection is transneuronal and viral replication maintains the intensity of the label throughout the chain of connected neurons, providing a means of examining detailed circuitry within the visual pathway.

Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells

1983 ◽  
Vol 3 (1) ◽  
pp. 91-101
Author(s):  
N K Gonatas ◽  
A Stieber ◽  
J Gonatas ◽  
T Mommoi ◽  
P H Fishman
Keyword(s):  
Cholera Toxin ◽  
Neuroblastoma Cells ◽  
Double Labeling ◽  
Electron Microscope Autoradiography ◽  
Density Distributions ◽  
Quantitative Studies ◽  
Covalently Linked ◽  
The Relationship ◽  
Cytochemical Marker ◽  
In Cells

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.

Anterograde transsynaptic tracing in the murine somatosensory system using Pseudorabies virus (PrV): A “live-cell”-tracing tool for analysis of identified neurons in vitro

2007 ◽  
Vol 13 (6) ◽  
pp. 579-585 ◽  
Author(s):  
Markus Rothermel ◽  
Nicole Schöbel ◽  
Nils Damann ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Somatosensory System ◽  
Live Cell ◽  
Identified Neurons ◽  
Cell Tracing ◽  
Transsynaptic Tracing

Characterization of transsynaptic tracing with central application of pseudorabies virus

1999 ◽  
Vol 838 (1-2) ◽  
pp. 171-183 ◽  
Author(s):  
Sheng Chen ◽  
Ming Yang ◽  
Richard R. Miselis ◽  
Gary Aston-Jones
Keyword(s):  
Pseudorabies Virus ◽  
Transsynaptic Tracing
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