title: Immunostaining to visualize murine enteric nervous system development.
abstract: The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.
authors.affiliation: Department of Surgery, University of Wisconsin School of Medicine and Public Health.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Barlow-Anacker
authors.affiliation: Department of Surgery, University of Wisconsin School of Medicine and Public Health.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Erickson
authors.affiliation: Department of Neurosciences, University of Wisconsin School of Medicine and Public Health.
authors.forename: Miles L
authors.initials: ML
authors.lastname: Epstein
authors.affiliation: Department of Surgery, University of Wisconsin School of Medicine and Public Health; Department of Neurosciences, University of Wisconsin School of Medicine and Public Health; Department of Pediatrics, University of Wisconsin School of Medicine and Public Health; gosain@surgery.wisc.edu.
authors.forename: Ankush
authors.initials: A
authors.lastname: Gosain
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002795:Choline O-Acetyltransferase / Q000032:analysis / Q000378:metabolism; D004210:Dissection / Q000379:methods; D047108:Embryonic Development; D017615:Enteric Nervous System / Q000166:cytology / Q000196:embryology* / Q000378:metabolism / Q000601:surgery; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D041981:Gastrointestinal Tract / Q000196:embryology* / Q000294:innervation* / Q000601:surgery; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009432:Neural Crest / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D002795:Choline O-Acetyltransferase; D000109:Acetylcholine