Intracellular labeling of cat spinal neurons using a tetramethykhodamine-dextran amine conjugate

P. A. Carr, B. R. Noga, D. M. Nance, L. M. Jordan

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Tetramethylrhodamine-dextran is a highly fluorescent neuroanatomical tracer that, in its 10,000 MW form, has seen widespread use as a sensitive anterograde tract-tracing label. We report here the use of a lower molecular weight tetramethylrhodamine-dextran (3000 MW; Molecular Probes, OR) as an in vivo intracellular marker of locomotor-related spinal neurons. In the paralyzed, decerebrate cat preparation, fictive locomotion was evoked by electrical stimulation of the mesencephalic locomotor region. Extracellular and intracellular potentials of rhythmically active spinal neurons were recorded using microelectrodes filled with 2% tetrarnethybrhodamine-dextran (3000 MW) in 0.9% saline (impedance 5-20 Mohm). Following impalement and electrophysiological characterization, neurons were iontophoretically injected for 2-30 min with 3-10 nA 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 injected neurons were recovered. Choline acetyltransferase-immunoreactivity could be demonstrated in a subpopulation of tetramethylrhodamine-dextran-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, and compatibility with immunohistochemistry.

Original languageEnglish (US)
Pages (from-to)447-451
Number of pages5
JournalBrain Research Bulletin
Volume34
Issue number5
DOIs
StatePublished - 1994
Externally publishedYes

Keywords

  • Dextran amine conjugate
  • Fluorescent tracer
  • Immunohistochemistry
  • Intracellular staining
  • Locomotion
  • Spinal cord

ASJC Scopus subject areas

  • Neuroscience(all)

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