Flash and freeze: combining high-pressure freezing and optogenetics to evaluate synaptic transmission at hippocampal mossy fibers synapses
Creators: name: Kim, Olena
name: Borges-Merjane, Carolina
name: Kaufmann, Walter A.
name: Gütl, Daniel
name: Jonas, Peter
Publisher: name: F1000 Research Limited
Year: 2018

description: The visualization of the ultrastructure of synapses by electron microscopy with high pressure freezing and freeze-substitution (HPF-FS) allowed a better understanding of their morphological properties and fine structures. However, synaptic transmission is a dynamic process, and HPF-FS alone only captures static images. The recently developed “Flash and Freeze” technique (Watanabe <i>et al.</i>, 2013, eLife e00723; Watanabe <i>et al.</i>, 2013, Nature 504:242-247) combines optogenetics with HPF-FS allowing for the visualization of action potential-evoked membrane dynamics at synapses. With this powerful technique, a light pulse activates genetically expressed channelrhodopsin in targeted cells, leading to action potential initiation and, subsequently, vesicle fusion at the active zone membrane. After light stimulation, tissue is frozen by HPF with a pre-set time delay after stimulus onset, thus allowing for capturing of events at defined time points after synaptic transmission onset, from exo- to endocytosis. We implemented “Flash and Freeze” to assess presynaptic mechanisms underlying changes in the ultrastructure after induction of short-term plasticity at the mossy fiber-to-CA3 pyramidal cell synapse in mouse hippocampus. We used acute brain slices from 21- to 28-day-old mice and organotypic slice culture, prepared from 5- to 7-day-old mice and kept maximally three weeks in culture. To selectively express channelrhodopsin in granule cells, we crossed transgenic mice with cre-recombinaseER expressed under the control of the <i>prox1</i> gene promoter (Bazigou <i>et al.</i>, 2011, J Clin Invest 121:2984-2992) to the channelrhodopsin reporter line Ai32. For acute slices, Tamoxifen was injected once at 0.1 mg/g of body weight for close to maximum expression in granule cells based on dose-dependent labeling data. For organotypic cultures, 5 μM of 4-hydroxytamoxifen was added to the culture medium. As a proof of principle, we recorded direct light-evoked currents from granule cells and EPSCs from CA3 pyramidal neurons with up to 40 Hz light stimulation with 1-ms individual pulses. Finally, we implemented a fast freeze-substitution protocol that allowed us to obtain preservation of ultrastructure of acute slices and organotypic cultures while minimizing the time for sample processing (Goldman <i>et al.</i>, 2016, Protist 164:369-376). Preliminary analysis of light-stimulated organotypic cultures showed that the average area of active zones (AZs) in hippocampal mossy fiber boutons is 0.10±0.01 μm<sup>2</sup> and mean number of docked vesicles is 143±18 per μm<sup>2</sup>, corresponding to ~14 docked vesicles per active zone. Our upcoming experiments aim to combine the light stimulation paradigms determined by the electrophysiology experiments with the protocols established by the electron microscopy experiments to capture vesicle fusion events and to determine how the number of docked vesicles changes after stimulation.