PREEMBEDDING STAINING FOR GAD(67) VERSUS POSTEMBEDDING STAINING FOR GABA AS MARKERS FOR CENTRAL GABAERGIC TERMINALS

Pre-embedding immunocytochemistry for the active form of glutamate dec arboxylase (GAD(67)) and postembedding staining for gamma-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerv e terminals with or without GAD(67) immunoreactivity were identified i n one ultrathin section. GABA was localized with immunogold in an adja cent section after etching and bleaching. GABA labeling density was as sessed over 519 GAD(67)-positive and GAD(67)-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statist ically higher densities of gold particles occurred over most GAD(67)-p ositive terminals. However, some GAD(67)-negative terminals also showe d high densities of gold particles, and some GAD(67)-positive terminal s showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjuga tes, significantly reduced gold labeling over both GAD(67)-positive an d GAD(67)-negative terminals. These results show that the presence of GAD(67) immunoreactivity correlates strongly with high densities of im munogold labeling for GABA in nerve terminals in the phrenic motor nuc leus. Preabsorption controls indicate that authentic GABA was localize d in the postembedding labeling procedure. Only a small proportion of intensely GABA-immunoreactive terminals lack GAD(67), suggesting that both GAD(67) and GABA are reliable markers of GABAergic nerve terminal s.