TY  - JOUR
A1  - Lichter, Katharina
A1  - Paul, Mila Marie
A1  - Pauli, Martin
A1  - Schoch, Susanne
A1  - Kollmannsberger, Philip
A1  - Stigloher, Christian
A1  - Heckmann, Manfred
A1  - Sirén, Anna-Leena
T1  - Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse
JF  - Cell Reports
N2  - Rab3A-interacting molecule (RIM) is crucial for fast Ca\(^{2+}\)-triggered synaptic vesicle (SV) release in presynaptic active zones (AZs). We investigated hippocampal giant mossy fiber bouton (MFB) AZ architecture in 3D using electron tomography of rapid cryo-immobilized acute brain slices in RIM1α\(^{−/−}\) and wild-type mice. In RIM1α\(^{−/−}\), AZs are larger with increased synaptic cleft widths and a 3-fold reduced number of tightly docked SVs (0–2 nm). The distance of tightly docked SVs to the AZ center is increased from 110 to 195 nm, and the width of their electron-dense material between outer SV membrane and AZ membrane is reduced. Furthermore, the SV pool in RIM1α\(^{−/−}\) is more heterogeneous. Thus, RIM1α, besides its role in tight SV docking, is crucial for synaptic architecture and vesicle pool organization in MFBs.
KW  - active zone
KW  - acute brain slices
KW  - CA3
KW  - electron tomography
KW  - high-pressure freezing
KW  - hippocampal mossy fiber bouton
KW  - RIM1α
KW  - SV pool
KW  - synaptic ultrastructure
KW  - presynaptic
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300913
VL  - 40
IS  - 12
ER  - 
TY  - JOUR
A1  - Markert, Sebastian Matthias
A1  - Britz, Sebastian
A1  - Proppert, Sven
A1  - Lang, Marietta
A1  - Witvliet, Daniel
A1  - Mulcahy, Ben
A1  - Sauer, Markus
A1  - Zhen, Mei
A1  - Bessereau, Jean-Louis
A1  - Stigloher, Christian
T1  - Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome
JF  - Neurophotonics
N2  - Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.
KW  - caenorhabditis elegans
KW  - localization micoscopy
KW  - fluorescent-probes
KW  - junction proteins
KW  - resolution limit
KW  - direct stochasticoptical reconstruction microscopy
KW  - structured illumination microscopy
KW  - correlative light and electron microscopy
KW  - gap junction
KW  - neural circuits
KW  - nervous-system
KW  - image data
KW  - reconstruction
KW  - innexins
KW  - super-resolution microscopy
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187292
VL  - 3
IS  - 4
ER  -