Combined intracellular injection of Neurobiotin and pre-embedding immunocytochemistry using silver-intensified gold probes in myenteric neurons

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.