COMBINED INTRACELLULAR INJECTION OF NEUROBIOTIN AND PREEMBEDDING IMMUNOCYTOCHEMISTRY USING SILVER-INTENSIFIED GOLD PROBES IN MYENTERIC NEURONS

We have developed methods to examine the neurochemistry of synaptic in puts to individual myenteric neurons labeled by dye injection through intracellular recording electrodes. Myenteric neurons of the guinea-pi g ileum were filled with Neurobiotin, fixed, washed in 50% ethanol, ex posed to sodium cyanoborohydride, incubated in avidin-biotin-horseradi sh peroxidase and incubated in antisera to calretinin or calbindin. Th e Neurobiotin-filled cells were revealed using the diaminobenzidine (D AB) reaction. The tissue was examined at the light microscope level to determine the morphology and projections of the Neurobiotin-filled ne urons, and then incubated in 1 nm gold-labeled secondary antibodies. F ollowing osmication, the gold probes were silver-intensified and the t issue embedded flat in resin. The tissue was re-examined at the light microscope level. Neurons containing a DAB reaction product could be d istinguished from neurons containing a silver-intensified gold reactio n product using oblique or epipolarized illumination. Ultrathin sectio ns were taken through the injected neurons and examined. At the ultras tructural level, Neurobiotin-filled cell bodies and their processes (l abeled with DAB) were easily distinguished from the structures labeled by silver-intensified gold. Gold-labeled terminals of enteric interne urons made synapses and close contacts with Neurobiotin-filled nerve c ell bodies and their processes. This technique is valuable for the neu rochemical identification of synaptic inputs to morphologically and/or functionally characterized myenteric neurons and could be easily appl ied to other preparations, such as brain slices.