gms | German Medical Science

57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN)

German Society for Neuropathology and Neuroanatomy

12. - 15.09.2012, Erlangen

57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN)

Optical nanoscopy of synaptic architecture in mammalian brain slices using a genetically encoded biotag

Meeting Abstract

  • presenting/speaker Christian Kempf - University Hospital Zurich, Institute for Nuropathology, Zurich, Switzerland; University of Heidelberg, Institute for Anatomy and Cell Biology, Heidelberg, Germany; Max-Planck-Institute for Medical Research, Institute for Anatomy and Cell Biology, Zurich, Germany
  • Kirsten Reuter - Max-Planck-Institute for Medical Research, Heidelberg, Germany
  • Pit Bingen - German Cancer Research Center, Optical Nanoscopy, Heidelberg, Germany; Max-Planck-Institute for Biophysical Chemistry, NanoBiophotonics, Göttingen, Germany
  • Daniel Barzan - Bioquant, Single Molecule Spectroscopy, Heidelberg, Germany
  • Thorsten Staudt - German Cancer Research Center, Optical Nanoscopy, Heidelberg, Germany; Max-Planck-Institute for Biophysical Chemistry, NanoBiophotonics, Göttingen, Germany
  • Heinz Horstmann - University of Heidelberg, Institute for Anatomy and Cell Biology, Heidelberg, Germany; Max-Planck-Institute for Medical Research, Institute for Anatomy and Cell Biology, Heidelberg, Germany
  • Dirk-Peter Herten - Bioquant, Single Molecule Spectroscopy, Heidelberg, Germany
  • Stefan Hell - German Cancer Research Center, Optical Nanoscopy, Heidelberg, Germany; Max-Planck-Institute for Biophysical Chemistry, NanoBiophotonics, Göttingen, Germany
  • Thomas Kuner - University of Heidelberg, Institute for Anatomy and Cell Biology, Heidelberg, Germany; Max-Planck-Institute for Medical Research, Institute for Anatomy and Cell Biology, Heidelberg, Germany

Deutsche Gesellschaft für Neuropathologie und Neuroanatomie. 57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN). Erlangen, 12.-15.09.2012. Düsseldorf: German Medical Science GMS Publishing House; 2012. Doc12dgnnPP4.14

DOI: 10.3205/12dgnn091, URN: urn:nbn:de:0183-12dgnn0912

Published: September 11, 2012

© 2012 Kempf et al.
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Outline

Text

Question: Understanding the principles that govern neural signaling and their aberrations in pathological conditions is one of the foremost challenges in neuroscience. The functional properties of synaptic proteins have been extensively studied; however, spatiotemporal and quantitative properties remain largely uncharacterized. The major obstacle remains the limitations of conventional imaging.

Methods: The calyx of Held, a glutamatergic terminal in the auditory brainstem circuit, is often employed to study presynaptic mechanisms in the central nervous system. Using stimulated emission depletion (STED) nanoscopy, we examined the molecular architecture of this synaptic terminal in rat brain sections by visualizing cellular components with subdiffraction resolution. To optimize labeling specificity, we employed a multidisciplinary approach, which combines the specificity of genetic fusion and versatility of chemically engineered probes. Since fluorophores linked to O6-benzylguanine (BG) can be directly bound to any protein fused with AGT, we applied this technique in brain slices using virus-mediated overexpression of AGT fusion proteins.

Results: Membrane permeable quenched BG-conjugated fluorescent probes can be targeted to AGT-tags via stereotactic virus injections into anaesthetized rats. In our study, we demonstrated that multi-color STED imaging can resolve synaptic proteins to a resolution of 35 nm inside 4 µm thick tissue sections. Further, we showed that STED imaging complemented with AGT fusion proteins is superior to conventional immunohistochemistry to analyze quantitative, topological and dynamic changes in synaptic protein expression in vivo. The signal is more specific, closer to the protein of interest, labeled in a detergent-free environment that preserves tissue structure, covalently bound to the target and labeled in a stoichiometric manner that facilitates quantitative analysis.

Conclusion: Alteration of presynaptic structure and protein topology following genetic perturbations may be observed directly and correlated with changes in synaptic physiology, thus deepening our understanding of the spatial and functional organization of synaptic processes.