Microimplantation of [3H]proline on a single bead of ion-exchange resin.

An advantage of the use of radioautography of axonally transported proteins to trace neuroanatomical pathways is its precision and selectivity. Only cell bodies at the site of injection incorporate the precursor, so that axons of passage are not affected 1,9,11. The selectivity of the method is thus limited only by the size of the injection site. In studies of the goldfish visual system, injection is made into the vitreous humor 10, and the use of [SH]proline has been demonstrated to be a particularly suitable precursor3,11. The ganglion cell bodies synthesize labeled protein, which is then transported to the brain via the optic nerve. Injection of precursor into an intracerebral site is less simple, since it is necessary to disrupt the brain substance with the shaft of a pipet or needle. If the isotope is injected hydrostatically, there may be considerable diffusion at the injection site, depending upon the amount of prior physical trauma, rate of injection and on the volume in-je~ted. To minimize these problems, we examined the feasibility of concentrating the precursor on the surface of a cation ion-exchange resin bead which was implanted by first fixing it to the tip of a fine wire. An immediate application of the method was to map the projections of the olfactory bulb in the teleost fish, Macropodus opercularis. The 0.5 mm wide bulbs protrude from beneath the rostral edge of the telencephalon and are readily visualized by cutting a 2 mm hole in the dorsal midline of the craniumL In the experiment described below, a resin bead containing tritiated proline was implanted in the center of the visible portion of the bulb without touching the telen-cephalon. Preparation of precursor-labeled beads. Ten DOWEX 50W-X8, H + form (200-400 mesh, Bio-Rad) beads, 60-80 #m in diameter, were set out in the center well of a glass depression slide. They had been selected for size under the dissecting microscope after having been air-dried for 5 min. The beads were washed twice with 100/zl of distilled, deionized water and then dried. Twenty #1 of water containing 15-20/~Ci of L[3H-2,3]proline were added and the beads were dried under a flow of air. An additional 20/~1 of water was added prior to final drying. Single beads were mounted with a film of Ficoll (Pharmacia) on the flattened tip