Ultrastructural Analysis of Synaptic Profiles

Ultrastructure was analyzed with two electron microscopes (EM). One EM is a JEOL JEM 100S transmission electron microscope with which areas of interest were photographed at 15,000–40,000 magnification with high-resolution film (Kodak SO-163; Kodak, Rochester, NY, USA). The negatives were scanned at 1200–2000 pixels/inch (ScanMaker 800, Microtek, Santa Fe Springs, CA, USA) to produce digital images. A second EM is a Phillips CM10 equipped with a digital camera for direct capture of digital images. Synapses were identified by the presence of vesicles in the presynaptic profile, a clear synaptic cleft and the presence of a postsynaptic density. Tracer-labeled profiles were readily identified by the presence of DAB precipitate. GABA-positive (GABA+) profiles were easily distinguished from GABA-negative profiles by a distinct difference in the density of overlying gold particles (Nakamoto et al., 2013a (link)). GABA immunostaining was also readily distinguished from the DAB label (Nakamoto et al., 2013b (link)).