A fixation technique, which allows a “near-to-native” state tissue preservation, such as high-pressure freezing in combination with electron tomography is required for studying synapses systematically at high resolution in 3D. With these techniques we are studying the ultrastructural architecture of synapses in the zebrafish Danio rerio which can be high-pressure frozen as intact animals at early stages. Currently we are focusing at a group of cytoskeletal proteins, the septins, which play important roles in synapse formation and synaptic function. We localize them with various microscopy techniques ranging from light to 3D electron microscopy in the nervous system. A limiting factor in 3D electron microscopy is the annotation of structures. We therefore developed an automatic vesicle annotation macro with Fiji, which helps us to automatically measure the size of synaptic vesicles and their density.  

Figures:

3D model of a zebrafish neuromuscular junction at 4 dpf (Scale bar = 500nm).

Automatic annotated zebrafish neuromuscular junction with Fiji at 4dpf (Scale bar = 200nm).

Expression pattern of sept5a in 1 dpf zebrafish larvae (Scale bar = 300µm).

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EMC Abstracts - https://emc-proceedings.com/abstract/studying-synaptic-architecture-in-the-zebrafish-danio-rerio/