TY  - JOUR
A1  - Heckmann, Manfred
A1  - Pauli, Martin
T1  - Visualizing presynaptic active zones and synaptic vesicles
JF  - Frontiers in Synaptic Neuroscience
N2  - The presynaptic active zone (AZ) of chemical synapses is a highly dynamic compartment where synaptic vesicle fusion and neurotransmitter release take place. During evolution the AZ was optimized for speed, accuracy, and reliability of chemical synaptic transmission in combination with miniaturization and plasticity. Single-molecule localization microscopy (SMLM) offers nanometer spatial resolution as well as information about copy number, localization, and orientation of proteins of interest in AZs. This type of imaging allows quantifications of activity dependent AZ reorganizations, e.g., in the context of presynaptic homeostatic potentiation. In combination with high-pressure freezing and optogenetic or electrical stimulation AZs can be imaged with millisecond temporal resolution during synaptic activity. Therefore SMLM allows the determination of key parameters in the complex spatial environment of AZs, necessary for next generation simulations of chemical synapses with realistic protein arrangements.
KW  - active zone
KW  - depression
KW  - facilitation
KW  - plasticity
KW  - potentiation
KW  - synapse
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-274687
SN  - 1663-3563
VL  - 14
ER  - 
TY  - JOUR
A1  - Capetian, Philipp
A1  - Müller, Lorenz
A1  - Volkmann, Jens
A1  - Heckmann, Manfred
A1  - Ergün, Süleyman
A1  - Wagner, Nicole
T1  - Visualizing the synaptic and cellular ultrastructure in neurons differentiated from human induced neural stem cells - an optimized protocol
JF  - International Journal of Molecular Sciences
N2  - The size of the synaptic subcomponents falls below the limits of visible light microscopy. Despite new developments in advanced microscopy techniques, the resolution of transmission electron microscopy (TEM) remains unsurpassed. The requirements of tissue preservation are very high, and human post mortem material often does not offer adequate quality. However, new reprogramming techniques that generate human neurons in vitro provide samples that can easily fulfill these requirements. The objective of this study was to identify the culture technique with the best ultrastructural preservation in combination with the best embedding and contrasting technique for visualizing neuronal elements. Two induced neural stem cell lines derived from healthy control subjects underwent differentiation either adherent on glass coverslips, embedded in a droplet of highly concentrated Matrigel, or as a compact neurosphere. Afterward, they were fixed using a combination of glutaraldehyde (GA) and paraformaldehyde (PFA) followed by three approaches (standard stain, Ruthenium red stain, high contrast en-bloc stain) using different combinations of membrane enhancing and contrasting steps before ultrathin sectioning and imaging by TEM. The compact free-floating neurospheres exhibited the best ultrastructural preservation. High-contrast en-bloc stain offered particularly sharp staining of membrane structures and the highest quality visualization of neuronal structures. In conclusion, compact neurospheres growing under free-floating conditions in combination with a high contrast en-bloc staining protocol, offer the optimal preservation and contrast with a particular focus on visualizing membrane structures as required for analyzing synaptic structures.
KW  - transmission electron microscopy
KW  - human neurons
KW  - induced neural stem cells
KW  - synapse
KW  - synaptic vesicles
KW  - high contrast
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236053
SN  - 1422-0067
VL  - 21
IS  - 5
ER  -