In situ cryo-electron tomography of actively firing neuronal synapses

• Applicant: Dr. Jana Kroll
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2021 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 458275811

Communication between neurons has been studied for more than 60 years. Today, we know that synapses release neurotransmitters via fusion of vesicles with the cell membrane (exocytosis), and that a set of key proteins, including calcium sensors and SNARE proteins, induce and regulate vesicle fusion. Our current idea of the synaptic architecture, including an active zone, vesicles and endocytic organelles, is mainly based on electron microscopy (EM) experiments. While initial studies described the synaptic morphology more generally, recent studies make use of morphometric analyses to identify subtle changes e.g. in mouse mutants. The establishment of cryofixation for EM, for instance via high-pressure freezing, has opened the door to examine cellular structures in a near-to-native state. With cryo-EM, it is possible to examine frozen cells or tissue directly, meaning without any staining or fixation. Therefore, cryo-EM allows for the best-possible structural resolution. With the project proposed here, I plan to go even one step further: I aim to combine cryo-electron tomography (a form of high-resolution 3D-EM) with preceding physiological stimulation and with a genetic marker to fluorescently label those synapses that are actively firing. The stimulation of neurons will be performed with a high-pressure freezer that allows me to cryofix neurons less than 5 ms after an electrical stimulus, and thus to preserve neurons in an active state. The frozen samples will subsequently be examined with a cryo-confocal microscope to detect active synapses. These preselected synapses will then be used to perform cryo-eletron tomography. My workflow provides three key advantages compared to other currently available methods: (1) I can combine best-possible structural resolution with best-possible temporal resolution, (2) I can examine finest cellular structures in situ, and (3) I can compare synapses that were releasing neurotransmitters with synapses that were stimulated but did not perform exocytosis. Consequently, my workflow will allow me to identify morphological hallmarks of actively firing synapses. These features will lead to new insights into the variability of synaptic responses, and thus into the regulation of synaptic plasticity. Beyond that, I will re-evaluate the tethering and docking processes preceding vesicle fusion with superb structural resolution. This way, I may identify structural differences between docked vesicles in resting and stimulated synapses. Ideally, I will be able to define the fusion-competence of a docked SV based on its morphology. I am sure that my workflow will help not only me but also other researchers to extend our knowledge of neuronal communication.

• DFG Programme: WBP Position