Ronald S. Petralia and Ya-Xian Wang have been running the Advanced Imaging Core facility of NIDCD now since October 2011. This core is dedicated primarily to collaborating with and training others scientists in NIDCD in studies utilizing electron microscopy (EM) either alone or in conjunction with light microscopy. Also, to a limited extent, some work is done with other NIH institutes or outside NIH. Many of the projects reported here were continuations of those described in the FY12 Annual Report. The specific research projects (collaboration and/or training) that the Core has been involved in this year are described below. Also, the Core generally oversees common equipment (microscopes, freezers, biological microwave oven, lockers, culture room, etc.) and space of the NIDCD PIs in building 50. And Ronald Petralia regularly reviews papers for professional journals and reviews many various documents for NIDCD PIs, reviews grants, writes review papers and letters of recommendation, serves on several NIH committees and helps coordinate lab+office safety for NIDCD. Projects (RCDC=Neuro, Aud Sys): In calendar year 2012 so far, the Core has 5 research papers published or in press (including 2 with RSP as first author, 1 in collaboration with 2 NIDCD PIs, and 1 each in J. Neurosci. and J. Cell Biol.), as well as 1 review (plus a 2nd in review). (STARS codes) NIDCD: Matt Kelley/Richard Chadwick: The Core worked with fellow, Kate Szarama in the past 2 years on mechanical properties of developing pillar cells in comparison to outer hair cells, in mouse cochlea;this project involved the Core in active research and extensive training. We published a paper in early 2012, and 2 more papers in late 2012/early 2013. The Core currently is planning more collaboration with Dr. Szarama in the near future. (1F-Dev-Aud Sys) Matt Kelley: Work with fellow, Helen May-Simera, on the ciliary proteins, BBS8 and IFT20, as part of a study on 'ciliary'proteins and their association with the PCP protein Vangl2;The Core has been performing EM immunogold labeling plus EM on both the developing mouse cochlea and ventricular zone of the rat brain. For this study, the Core also has been involved in the extensive cochlea tissue preparation, in collaboration with Kate Szarama. A manuscript was submitted recently to Development. We also are doing preliminary studies on another ciliary protein. (1F-Dev-Aud Sys) Bechara Kachar: The Core has been involved in 4 projects in progress with Dr. Kachars lab during the designated time period of this report. The first is with Taro Fujikawa on the kainate receptors of the outer hair cells and their afferent terminals;it has involved both training and extensive performance of research. Dr. Fujikawa has returned to Japan, but we continue to work on this study and hope to submit it for publication soon. Also, Dr. Petralia presented a poster at the NIDCD retreat on this study. The second project is with Janaina Brusco, on EM ultrastructure of synapses in the posterodorsal medial amygdala. The manuscript is in review currently. The third project is directly with Bechara and involves the examination of specially prepared auditory and vestibular tissue. The Core expects to expand the versatility of the Core by learning new methodologies from Bechara. The fourth project is with Seham Ebrahim and it currently is one of the major active projects of our Core. We have been performing tissue preparation, ultramicrotomy, immunolabeling, staining, and EM of auditory and vestibular tissue, especially looking at the structure of normal stereocilia and comparing this to the structure of stereocilia in espin mutant mice. The Core also gives help with other projects of Becharas lab. (1G&2B-Per Aud/Vest S&F) Tom Friedman/Inna Belyantseva: The Core has been trained Inna and assisted her with tissue prep and interpretation of data involving EM. Currently, Dr. Belyantseva works more on her own on EM, but the Core continues to help her as needed. Stephan Brenowitz: The Core continues to work with postdoctoral fellows from Dr. Brenowitzs laboratory, Shan He and Milos Sedlacek on 2 major projects. Drs. He and Sedlacek presented this work recently at an NIDCD meeting. For the study with Dr. He, the Core prepares tissue from the dorsal cochlear nucleus (DCN) for immunogold analysis. Sections are cut using ultramicrotomy, labeled with primary antibodies related to DCN function (especially cartwheel cells), and photographed with EM. We expect to submit a manuscript soon. The work with Dr. Sedlacek has been along 2 lines. In the first, DCN sections with neurobiotin-injected fusiform cells are prepared and examined with EM, to identify and reconstruct synaptic terminal types on these identified neurons. In the second line of study, we developed a new method called Serial Block Face Imaging for the DCN sections. These sections are sliced and imaged as long serial sections in a scanning electron microscope. From these series of images, we can reconstruct basal dendrites of fusiform cells and trace the various excitatory and inhibitory synaptic terminals that contact them. Ultimately, we hope to identify the types and origins of these synaptic inputs and correlate them with the physiology. This summer, Dr. Petralia mentored a student who made reconstructions from these image series and presented the findings at the 2013 NIH Summer Student Poster Day. (1A-Cen Aud S&F) Lisa Cunningham/Lindsey May: We have begun planning a morphological analysis of utricle exosomes. (2B-Vest S&F) Yong Zeng/Ron Bush NIDCD/NEI: The Core has been preparing tissue and studying sections with EM for a study on changes in immunolocalization of a protein related to a retinal disease. The Core currently is working with Gail Seabold (formerly NIDCD/now NEI), on synaptic adhesion-like molecule 1 (SALM1) trafficking in neurons in collaboration with a lab in France and involves a recent SALM1-associated human pathology. We also have worked together on some associated studies. Other NIH institutes: NIA /Mark Mattson/Pamela Yao lab: Projects include: sonic hedgehog in synapse development and function;another looks at endocytic accessory proteins in neurons. We published 2 papers in 2012 and 2 in 2013. The Core continues to be involved in research studies with this group. In addition, we submitted a review paper on aging synapses. NICHD Chris McBain/Ken Pelkey/Megan Wyeth lab: The Core has been work on a project looking at the role of a kainate receptor auxiliary protein in hippocampal circuitry. This involves work of the Core on preparation of tissue (KO and WT mice), immunogold labeling, and EM, as well as training of the fellow, Megan. A poster on this was presented at the Soc. Neurosci. meeting (late 2012) and we are revising the paper for J. Neurosci. NICHD Andres Buonanno lab: Last year we looked at EM immunolocalization of antibodies to some neuregulin-associated proteins. It is not clear yet if we will do more in 2013. NINDS Katherine Roche lab: We have worked with this lab in previous years and may collaborate in the near future. NHLBI John Hammer lab: We have been studying the localization of the motor protein, myosin My18A in the cerebellum, utilizing postembedding immunogold and preembedding immunoperoxidase methods. They presented a poster at the ASCB meeting in late 2012. NEI: We also continue to work to some extent with Gail Seabold who now works in NEI, as noted above. NIHMS: Currently we have a paper in revision with Song Jiao and Zheng Li;it is on autophagy and synaptic vesicles affected by BAD-BAX-caspase. Outside NIH: We have 2 papers on protein phosphatase-1 (published or in press) in 2013 with Houhui Xia/LSUSM. We also work with Gavin Rumbaugh (Scripps) on SynGAP mutant effects on synapses of somatosensory cortex (associated with schizophrenia).
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