Rapid anterograde and retrograde tracing from mesenteric nerve trunks to the guinea-pig small intestine in vitro

A novel technique for rapid anterograde labelling of cut axons in vitro was used to visualise the peripheral branches of mesenteric nerve trunks suppl ying the guineapig small intestine. Biotinamide, dissolved in an artificial intracellular solution, was applied to the cut ends of the mesenteric nerv es and the tissue was maintained in organ culture overnight. Labelled nerve fibres were visualised by fluorescein isothiocyanate (FITC)-conjugated str eptavidin. Intense staining of nerve fibres and terminal varicosities in th e ganglia and internodal strands of the myenteric plexus was achieved up to 15 mm from the application site. Filled fibres formed baskets around some myenteric nerve cell bodies, suggesting target-specific neurotransmission. When combined with multiple-labelling immunohistochemistry for tyrosine hyd roxylase (TH), calcitonin gene-related protein (CGRP) or choline acetyltran sferase (ChAT), most anterogradely labelled nerve fibres, and many pericell ular baskets, were found to be TH immunoreactive, indicating their postgang lionic sympathetic origin. Double-labelling immunohistochemistry revealed t hat the postganglionic sympathetic pericellular baskets preferentially surr ounded 5-hydroxytryptamine (5-HT)-handling myenteric neurons. Some biotinam ide-filled fibres were CGRP immunoreactive, and are likely to originate fro m spinal sensory neurons. We describe for the first time many pericellular baskets labelled from the mesenteric nerves which were ChAT immunoreactive. Retrogradely filled intestinofugal nerve cell bodies were also observed, a ll of which had a single ax-on arising from a small nerve cell body with sh ort filamentous or lamellar dendrites. Many of these cells were ChAT immuno reactive. This in vitro technique is effective in identifying the fine arra ngement of nerve terminals arising from nerve trunks in the periphery.