Shedding light on balance: Interrogating individual synapses within vestibular epithelia

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Project Summary The present application proposes a developmental research plan to investigate the structure-function relationship of individual presynaptic complexes in mammalian vestibular hair cells. These complexes incorporate synaptic ribbons that can exhibit broad architectural heterogeneity, the functional significance of which is not known. The distribution of presynaptic architectures in the primary hair cell phenotypes (i.e. types I and II) within vestibular epithelia, and the unique dendritic specialization known as the calyx (encapsulating type I hair cells), render traditional methods of investigating synaptic function inappropriate for elucidating the functional characteristics of individual synaptic sites. Recent advances in the development of optical biosensors and viral transduction strategies provide the foundation for novel capabilities to detect and quantify neurotransmitter release at individual synapses. iGluSnFR is a genetically encoded, membrane bound glutamate sensor. When expressed at the postsynaptic membrane, it emits a fluorescent signal proportional to glutamate release. Recent key investigations demonstrated that when packaged with an AAV9 capsid and a synapsin promoter, inner ear afferent neurons are transduced and iGluSnFR is expressed. This strategy directs the sensor to the appropriate postsynaptic targets for measuring glutamate release from individual hair cell synapses. When coupled with strategies for post-recording elucidation of synaptic ribbons an association between glutamate release and synapse structure can be made. The project includes three Aims to: 1) optimize transduction in vestibular afferent neurons; 2) develop and optimize recording strategies to capture the time- resolved glutamate release from individual synaptic sites; and 3) evaluate the methods in a mouse model for which presynaptic function in type I hair cells is drastically attenuated. Immunohistochemical processing following optical recording will enable the direct association of immunolabeled ribbons with the recording sites, for which ribbon volume provides a proxy of its architecture. The development of these methods provides the foundation future studies of pathologic conditions whose etiologies are proposed to involve synaptopathies. This investigation will enable future investigations of synaptic function in normal and pathologic conditions, and provide a platform for the rigorous evaluation of novel treatments of inner ear dysfunction.