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Journal of Histochemistry and Cytochemistry, Vol. 46, 1261-1268, November 1998, Copyright © 1998, The Histochemical Society, Inc.

ARTICLE

Pre-embedding Staining for GAD67 Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

Susan M. Murphya, Paul M. Pilowskya, and Ida J. Llewellyn–Smitha
a Department of Medicine and Centre for Neuroscience, Flinders University, Bedford Park, Australia

Correspondence to: Ida J. Llewellyn–Smith, Dept. of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia..

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) 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. 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 GABA-immunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals. (J Histochem Cytochem 46:1261–1268, 1998)

Key Words: amino acid, immunocytochemistry, motor neuron, spinal cord, ultrastructure


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