Pre-embedding staining for GAD₆₇ versus postembedding staining for GABA as markers for central GABAergic terminals

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD₆₇) and postembedding staining for γ-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. Nerve terminals with or without GAD₆₇ immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD₆₇-positive and GAD₆₇-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD₆₇-positive terminals. However, some GAD₆₇-negative terminals also showed high densities of gold particles, and some GAD₆₇-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD₆₇-positive and GAD₆₇-negative terminals. These results show that the presence of GAD₆₇ immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABAimmunoreactive terminals lack GAD₆₇, suggesting that both GAD₆₇ and GABA are reliable markers of GABAergic nerve terminals.