Visualization of enteric and gallbladder ganglia with a vital fluorescent dye

A method is described here for the vital staining of the nerve plexuses of the gastrointestinal tract with the fluorescent dye 4-(4-diethylaminostyryl)-methylpyridinium iodide (4-Di-2-ASP). This dye stained nerve fibers in the myenteric and submucosal plexuses and in the intramural ganglia of the gallbladder. In addition to staining the ganglia, the dye clearly revealed the secondary and the fine tertiary fibers of the myenteric plexus. In the submucosal plexus thin fibers innervating the blood vessels were also stained. This method proved to be effective in tissues that are relatively thick and in which the visualization of the nerves is quite difficult without staining--the myenteric plexus of rat small intestine, guinea-pig gallbladder and human large intestine. The staining did not fade within at least 6-8 h, or after long exposure to bright light. Intracellular recordings from myenteric neurons in the guinea pig ileum showed that cellular properties were not significantly altered after incubation in the dye solution. 4-Di-2-ASP thus appears to be suitable for rapid vital staining of nerves in whole mount from many parts of the gastrointestinal tract.