We continued to expand the use of photo-oxidation as a method for selective staining of subcellular structures using both immunological and non-immunogenic probes. A summary of our progress with phalloidin-eosin as a marker for dendritic spines is provided under the highlights' section. We have also experienced a tremendous expansion of the use of tomography and selective stains to reconstruct organelles and other subcellular features. Guy Perkins, a post-doctoral researcher in the laboratory, produced spectacular reconstructions of mitochondrial cristae in collaboration with Dr. Terry Frey. Several of these projects deal with aspects of synaptic structure, because of the superiority of tomography for these types of three-dimensional analyses. A project with Drs. David Lenzi and William Roberts of the University of Oregon on the three-dimensional structure of the frog vestibular hair cell synapse has been completed, and a manuscript is in preparation. Drs. Thomas Schikorski and Charles Stevens of UCSD have produced several reconstructions of synapses onto dendritic spines in the rat hippocampus. Using software developed at NCMIR, they analyzed the distributions of synaptic vesicles and their relationships to the active zone. Finally, in collaboration wit h Drs. Bing Ren Hu and Justin Zivin of UCSD we have employed selective staining with phosphotungstic acid to stain post-synaptic densities in normal brain and in brains from animals subjected to transient ischemia followed by reperfusion. We observed a dramatic alteration in the size and appearance of synapses from post-ischemic hippocampus and cerebral cortex. published in the Journal of Neuroscience (Hu et al., J. Neurosci., 18: 625-633, 1998). Using thick sections and IVEM, we are able to visualize the entire 3-D structure of pre- and post-synaptic specializations. We performed tomographic reconstructions of synapses in the hippocampus of ischemic and control rats at different time points of reperfusion. These reconstructions clearly showed that synapses in area CA1 were more loosely configured in the ischemic brain than in control brains. The 3D images suggest that synapses in CA1 are undergin degenerative changes prior to obvious cell death. This work was presented at the Society for Neurosciences meeting in 1997 and a manuscript has been submitted to the Journal of Neuroscience.
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