Double- and triple-labeling of functionally characterized central neurons projecting to peripheral targets studied in vitro.

The use of in vitro preparations such as brain slices poses difficulties in determining the correct identity of cells under study. To circumvent this problem, we first used a fluorescence pre-labeling technique (rhodamine-dextran-lysine) to identify cranial motoneurons projecting to the tongue (hypoglossal motoneurons) in the guinea-pig. Following preparation of slices, cells were recorded intracellularly and their electrophysiological properties determined. The cells were then intracellularly stained with both a fluorescence label (Lucifer Yellow) and with the stable, non-fading label biocytin. Under fluorescent illumination, the great majority of recorded cells within the hypoglossal nucleus were double-labeled (rhodamine and Lucifer Yellow) suggesting that most are indeed motoneurons. Biocytin injected into the same motoneurons provided permanent and detailed images of their morphology. Intracellularly stained cells surrounding the hypoglossal nucleus were not labeled with rhodamine and had distinct electro-physiological properties. The use of the retrogradely transported marker rhodamine-dextran-lysine allows the unambiguous identification of motoneurons in a brainstem slice. The combined intracellular injection of Lucifer Yellow and biocytin provides a simple means of melding the advantages of a fluorescent label (compatible with other fluorescence labels and with immunocytochemistry) with the benefits of a stable, non-fading, electron-dense marker. Application of this technique should prove useful in establishing morphological and functional correlates in other areas of the CNS.