A Technique to Perform Structure–Function Analysis of Synapses

In this chapter, we will present a detailed methodology for nanoscale structure–function analysis of an ultrafast and high-performing synapse, the auditory ribbon synapse. Ribbon synapses are found in sensory systems and are important to convey sensory information without synaptic fatigue. These synapses are characterized by a highly specialized structure, the so-called synaptic ribbon. This ribbon tethers a large number of synaptic vesicles to facilitate constant neurotransmission. Auditory ribbon synapses, located in the cochlear inner hair cells, furthermore display a specific set of synaptic proteins, that are partially distinct from those found in conventional central synapses or even other ribbon synapses. In order to understand how such specialized structures mediate indefatigable neurotransmission, we perform structure–function analysis of hair cell synapses by combining a simple stimulation method with electron microscopy. We describe in detail how to use chemical solutions in order to depolarize inner hair cell ribbon synapses and then rapidly immobilize the tissue utilizing cryofixation by high-pressure freezing followed by freeze-substitution. In this chapter, we will put emphasis on the sample depolarization as well as further processing steps. Finally, we provide a short description of how to analyze chemically induced changes of morphological vesicle pools and active zone parameters to obtain information about the fine structures of individual synapses.