INTRACELLULAR LABELING OF CAT SPINAL NEURONS USING A TETRAMETHYLRHODAMINE-DEXTRAN AMINE CONJUGATE

Tetramethylrhodamine-dextran is a highly fluorescent neuroanatomical t racer that, in its 10,000 MW form, has seen widespread use as a sensit ive anterograde tract-tracing label. We report here the use of a lower molecular weight tetramethylrhodamine-dextran (3000 MW; Molecular Pro bes, OR) as an in vivo intracellular marker of locomotor-related spina l neurons. In the paralyzed, decerebrate cat preparation, fictive loco motion was evoked by electrical stimulation of the mesencephalic locom otor region. Extracellular and intracellular potentials of rhythmicall y active spinal neurons were recorded using microelectrodes filled wit h 2% tetramethylrhodamine-dextran (3000 MW) in 0.9% saline (impedance 5-20 Mohm). Following impalement and electrophysiological characteriza tion, neurons were iontophoretically injected for 2-30 min with 3-10 n A of pulsed positive current. Animals were then perfused 30 min to 7 h postinjection with a variety of paraformaldehyde- and glutaraldehyde- containing fixatives. After tissue sectioning, more than 90% of the in jected neurons were recovered. Choline acetyltransferase-immunoreactiv ity could be demonstrated in a subpopulation of tetramethylrhodamine-d extran-labeled neurons. This technique, in addition to producing high- quality electrodes, has the advantages of rapid yet extensive filling of neuronal processes, no tissue processing prior to visualization, an d compatibility with immunohistochemistry.