Wheat germ agglutinin conjugated to TRITC: A novel approach for labeling primary projection neurons of peripheral afferent nerves

Abstract

Wheat germ agglutinin conjugated to tetramethylrhodamine isothiocyanate-dextran (WGA–TRITC) was studied as a novel tracer of primary projection neurons of pharyngeal (PhN) and superior laryngeal (SLN) branches of the vagus nerve. The SLN and PhN were dissected from rat cervical tissues and the proximal end of the nerves were bathed in tracer for 60–90 min. The animals were sacrificed 42–72 h later. The tissue was fixed, sliced, mounted on slides and viewed under epifluorescence. The clarity of the fluorescent label in projection neurons was confounded in some regions of the brainstem by autofluorescence. A computer image analysis method was developed to quantify fluorescence intensity for definitive identification of labeled neurons. Brainstem neurons labeled by afferent projections of the SLN and PhN were localized to the nucleus tractus solitarius. Efferents were identified in the nucleus ambiguus. WGA–TRITC labeled cells were observed in the ipsilateral brainstem at intensities significantly different from the fluorescence observed in controls ( P<0.01). The distribution and density of labeling is in agreement with results of previous investigations, suggesting that WGA–TRITC is a useful alternative for tracing SLN and PhN projections to brainstem nuclei.