This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our new collaboration with NCMIR allowed us to use electron microscope tomography to investigate mitochondria-associated adherens complexes (MACs) in MNTB neurons. Serial section EM is rather tedious when reconstruction volumes are large. Such is the case when investigating contacts surrounding entire MNTB neurons. Through collaboration with Dr. Perkins at the National Center for Microscopy and Imaging Research (NCMIR), we have applied the technique of electron tomography to reconstruct neurons at ultrastructural resolution. Utilizing intermediate voltage electron microscopes at their facility, we collected 5 tilt series through thick tissues, 0.5 ? 2.0 m in thickness that permitted reconstruction and rendering of calyceal elements. Tomography enabled us to visualize linkages among synaptic vesicles leading away from the active zone, and ultimately tethered to nearby mitochondria. Software for aligning, segmenting and reconstructing the tissue was developed at NCMIR and imported to my facility at WVU School of Medicine. To further this effort, we will collaborate with not only Dr. Guy Perkins, but also with Drs. Maryann Martone and Mark Ellisman.
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