MAR 09, 2022 10:00 AM PST

Time-resolved electron microscopy: synaptic ultrastructure millisecond by millisecond with high-pressure freezing

Sponsored by: Leica Microsystems

Event Date & Time
Date:  March 09, 2022
Time: 10:00am (PST),  1:00pm (EST), 7:00pm (CEST)
Transmission electron microscopy reveals the architecture of cells at nanometer resolution, but chemical fixation severely warps their native structure. The true ultrastructure of cells can be captured by fast high-pressure freezing before processing by electron microscopy. High-pressure freezing has been an enabling technology in many fields, notable among them the cell biology of synapses. Many questions in synaptic physiology remain unresolved because the millisecond-scale speed of neurotransmission and nanometer-scale structure of the synapse has made it difficult to visualize ultrastructural changes during neurotransmission. High-pressure freezing can be combined with optogenetics (‘flash-and-freeze’) or electrical field stimulation (‘zap-and-freeze’) to close this gap. In these methods, neurons are stimulated then frozen at defined time points down to the millisecond by high-pressure freezing, and processed and imaged by transmission electron microscopy. This system can be used for single action potentials or more complex stimulation protocols to capture structural changes during synaptic plasticity, and in dissociated neurons or specific circuits in intact tissue. This approach already been used to discover and interrogate the molecular mechanisms of novel synaptic phenomena, such as ultrafast endocytosis and ‘transient docking’, a movement of synaptic vesicles that may underly short-term plasticity. Further use of high-pressure freezing and time-resolved electron microscopy will lead to more such discoveries.
Learning Objectives
  • Recognize how high-pressure freezing preserves synapses in their near-native state for electron microscopy
  • Identify problems in cellular neurobiology that can be addressed by combining high-pressure freezing with electrical stimulation (‘zap-and-freeze’)
  • Explain what features, such as docking and exocytosis, can be directly quantified using zap-and-freeze
  • Discuss a new synaptic process, transient docking, that was discovered using zap-and-freeze
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