Versatile, high-resolution anterograde labeling of vagal efferent projections with dextran amines

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None of the anterograde tracers used to label and investigate vagal preganglionic neurons projecting to the viscera has proved optimal for routine and extensive labeling of autonomic terminal fields. To identify an alternative tracer protocol, the present experiment evaluated whether dextran conjugates, which have produced superior results in the CNS, might yield widespread and effective labeling of long, fine-caliber vagal efferents in the peripheral nervous system. The dextran conjugates that were evaluated proved reliable and versatile for labeling the motor neuron pool in its entirety, for single- and multiple-labeling protocols, for both conventional and confocal fluorescence microscopy, and for permanent labeling protocols for brightfield microscopy of the projections to the gastrointestinal (GI) tract. Using a standard ABC kit followed by visualization with DAB as the chromagen, Golgi-like labeling of the vagal efferent terminal fields in the GI wall was achieved with the biotinylated dextrans. The definition of individual terminal varicosities was so sharp and detailed that it was routinely practical to examine the relationship of putative vagal efferent contacts (by the criteria of high magnification light microscopy) with the dendritic and somatic architecture of counterstained neurons in the myenteric plexus. Overall, dextran conjugates provide high-definition labeling of an extensive vagal motor pool in the GI tract, and offer considerable versatility when multiple-staining protocols are needed to elucidate the complexities of the innervation of the gut.