Abstract
The neurotransmitter gamma-amino butyric acid (GABA) is synthesized in presynaptic terminals by glutamic acid decarboxylase (GAD). Antibodies to GAD have been used to localize presynaptic terminals in the cerebellar cortex that use GABA as a neurotransmitter. In the cerebellum, the Purkinje cell makes synaptic contacts on dendrites and somata of neurons in the cerebellar nuclei. We were interested in developing a technique that combined light microscopic analysis of GAD distribution in Purkinje cell terminals with high quality electron microscopic localization of GAD.The approach taken in this study was to combine high glutaraldehyde/metabisulfite fixation technique with the preembedding N-propyl gallate (NPG) silver enhanced gold procedure. Mice were perfused with buffered 3% glutaraldehyde and 0.4% sodium metabisulfite. Vibratome sections (60 μm) were permeabilized with 0.3% Triton, and incubated with a monoclonal anti-GAD antibody. The sections were incubated in goat-anti-mouse 1 nm Nanogold, and the gold was enhanced with the NPG silver enhancement solution for 10 minutes.
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