Simultaneous immunofluorescence and autoradiography: a useful technique for investigating neurotransmitter uptake by neurons and glia in primary central nervous system culture.
Previous studies on the localization of radiolabelled neurotransmitters in cultured cells of neural origin have relied on the comparison of cell morphology, as determined by immunocytochemistry, with the patterns of labelling on autoradiograms. We present here a method combining simultaneously autoradiography, following the uptake of tritium-labelled amino acid transmitters, with indirect immunofluorescence using antibodies against both surface and intracellular antigens. Using a fixative containing only a low concentration of glutaraldehyde (4% paraformaldehyde, 0.1% glutaraldehyde), a similar retention of gamma-[3H]aminobutyric acid (GABA) and D-[3H]aspartate was achieved as with the higher concentrations commonly used, with the advantage that the autofluorescence associated with glutaraldehyde fixed tissue was eliminated, and the immunoreactivity of the antigens to be localized was not destroyed. Using this method GABA and D-aspartate accumulating cells, in dissociated mouse central nervous system (CNS) cultures, could be reliably identified as oligodendrocytes, and some multiprocessed astrocytes, by anti-galactocerebroside (GC) and anti-glial fibrillary acidic protein (GFAP) immunofluorescence respectively. GABA-accumulating neuron-specific enolase (NSE) positive neurons could be clearly identified but no D-aspartate accumulating neurons were found. This technique should have a wide application in the investigation of whether selective transport mechanisms coexist with antigens characteristic of a certain cell type or sub-type.
Reynolds
R
R
Department of Pediatrics, University of Berne, Inselspital, Switzerland.
Herschkowitz
N
N
D000818:Animals; D000831:Animals, Newborn; D001224:Aspartic Acid / Q000493:pharmacokinetics*; D001253:Astrocytes / Q000378:metabolism*; D001345:Autoradiography; D001921:Brain / Q000166:cytology*; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D005455:Fluorescent Antibody Technique; D051379:Mice; D009457:Neuroglia / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D009836:Oligodendroglia / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000493:pharmacokinetics*
D001224:Aspartic Acid; D005680:gamma-Aminobutyric Acid