Visualizing the enteric nervous system using genetically engineered double reporter mice: Comparison with immunofluorescence.
OBJECTIVE
The enteric nervous system (ENS) plays a crucial role in the control of gastrointestinal motility, secretion and absorption functions. Immunohistochemistry has been widely used to visualize neurons of the ENS for more than two decades. Genetically engineered mice that report specific proteins can also be used to visualize neurons of the ENS. The goal of our study was to develop a mouse that expresses fluorescent neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT), the two proteins expressed in 95% of the ENS neurons. We compared ENS neurons visualized in the reporter mouse with the wild type mouse stained using classical immunostaining techniques.


METHODS
Mice hemizygous for ChAT-ChR2-YFP BAC transgene with expression of the mhChR2:YFP fusion protein directed by ChAT promoter/enhancer regions on the BAC transgene were purchased commercially. The Cre/LoxP technique of somatic recombination was used to construct mice with nNOS positive neurons. The two mice were crossbred and tissues were harvested and examined using fluorescent microscopy. Immunostaining was performed in the wild type mice, using antibodies to nNOS, ChAT, Hu and PGP 9.5.


RESULTS
Greater than 95% of the ENS neurons were positive for either nNOS or ChAT or both. The nNOS and ChAT neurons and their processes in the ENS were well visualized in all the regions of the GI tract, i.e., esophagus, small intestine and colon. The number of nNOS and ChAT neurons was approximately same in the reporter mouse and immunostaining method in the wild type mouse. The nNOS fluorescence in the reporter mouse was seen in both cytoplasm as well as nucleus but in the immunostained specimens it was seen only in the cytoplasm.


CONCLUSIONS
We propose that the genetically engineered double reporter mouse for ChAT and nNOS proteins is a powerful tool to study of the effects of various diseases on the ENS without the need for immunostaining.
Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, California.
Yanfen
Y
Jiang
Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, California.
Hui
H
Dong
Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, California.
Lars
L
Eckmann
Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, California.
Elaine M
EM
Hanson
Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, California.
Katherine C
KC
Ihn
Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, California.|San Diego VA Health Care Systemm, San Diego, California.
Ravinder K
RK
Mittal
D000818:Animals; D002795:Choline O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D059329:Cholinergic Neurons / Q000378:metabolism; D017615:Enteric Nervous System / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D041981:Gastrointestinal Tract / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D007150:Immunohistochemistry; D051379:Mice; D008822:Mice, Transgenic; D057054:Molecular Imaging; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021381:Protein Transport
D052248:Nitric Oxide Synthase Type I; D002795:Choline O-Acetyltransferase