Spinal neurons in the zebrafish labeled with fluoro-gold and wheat-germ agglutinin

This study concerns the identification and location of interneurons in the spinal cord of the zebrafish by way of retrograde tracing from the body musculature. To distinguish motoneurons from interneurons; two tracers were applied: the fluorochrome Fluoro-Gold to label motoneurons, and the trans-synaptically transported compound wheat-germ agglutinin to label motoneurons and the associated interneurons that are considered to be premotoneurons. Because the tracer accumulated mainly in cell bodies (not in neurites), premotoneurons labeled directly from motoneurons could not be distinguished from those labeled via interjacent cells. Both tracers were unilaterally injected into the myotomal muscle at the anal level of the animal. The number of labeled cell bodies in the spinal cord was examined 6, 10 and 16 days after injection. Several technique-oriented experiments were performed in order to map all pathways along with the tracers were incorporated in the neurons. The following observations were made. (1) All Fluoro-Gold-positive cells contained wheat-germ agglutinin, but yet more cells contained only wheat-germ agglutinin; the number of wheat-germ agglutinin-labeled cells was about tenfold higher than the number of Fluoro-Gold-labeled cells. (2) Fluoro-Gold labeling was restricted to cells within one to two spinal cord segments corresponding to the injection site, whereas wheat-germ agglutinin labeling was more diffuse. (3) The position and size of Fluoro-Gold-labeled cells corresponded to those of motoneurons described in previous horse-radish peroxidase experiments. (4) Statistical analysis of the group of wheat-germ agglutinin-labeled cells showed two subpopulations, one with a mean cell size and position corresponding to motoneurons and one with a smaller mean cell size, also positioned within the motor column. The smaller cells were considered to be premotoneurons. The ratio motoneuron:premotoneuron was lowest in the ventrolateral area of the motor column.