title: Combined intracellular injection of Neurobiotin and pre-embedding immunocytochemistry using silver-intensified gold probes in myenteric neurons.
abstract: We have developed methods to examine the neurochemistry of synaptic inputs to individual myenteric neurons labeled by dye injection through intracellular recording electrodes. Myenteric neurons of the guinea-pig ileum were filled with Neurobiotin, fixed, washed in 50% ethanol, exposed to sodium cyanoborohydride, incubated in avidin-biotin-horseradish peroxidase and incubated in antisera to calretinin or calbindin. The Neurobiotin-filled cells were revealed using the diaminobenzidine (DAB) reaction. The tissue was examined at the light microscope level to determine the morphology and projections of the Neurobiotin-filled neurons, and then incubated in 1 nm gold-labeled secondary antibodies. Following osmication, the gold probes were silver-intensified and the tissue embedded flat in resin. The tissue was re-examined at the light microscope level. Neurons containing a DAB reaction product could be distinguished from neurons containing a silver-intensified gold reaction product using oblique or epipolarized illumination. Ultrathin sections were taken through the injected neurons and examined. At the ultrastructural level, Neurobiotin-filled cell bodies and their processes (labeled with DAB) were easily distinguished from the structures labeled by silver-intensified gold. Gold-labeled terminals of enteric interneurons made synapses and close contacts with Neurobiotin-filled nerve cell bodies and their processes. This technique is valuable for the neurochemical identification of synaptic inputs to morphologically and/or functionally characterized myenteric neurons and could be easily applied to other preparations, such as brain slices.
authors.affiliation: Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: H M
authors.initials: HM
authors.lastname: Young
authors.forename: W A
authors.initials: WA
authors.lastname: Kunze
authors.forename: S
authors.initials: S
authors.lastname: Pompolo
authors.forename: J B
authors.initials: JB
authors.lastname: Furness
authors.forename: J C
authors.initials: JC
authors.lastname: Bornstein
mesh_terms: D000818:Animals; D001710:Biotin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism; D004594:Electrophysiology; D005260:Female; D006046:Gold; D006168:Guinea Pigs; D007150:Immunohistochemistry; D007267:Injections; D008297:Male; D009197:Myenteric Plexus / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D009474:Neurons / Q000378:metabolism*; D012834:Silver; D013569:Synapses / Q000648:ultrastructure; D016610:Tissue Embedding; D004124:p-Dimethylaminoazobenzene
chemical_list: C070614:neurobiotin; D012834:Silver; D001710:Biotin; D006046:Gold; D004124:p-Dimethylaminoazobenzene