PgmNr Z6210: The Role of Non-Neuronal SNAREs on Synaptic Transmission in Zebrafish Hair Cells.

Authors:
Mike Waltman 1 ; Suna Li 2 ; Katie Kindt 1


Institutes
1) NIH/NIDCD, Bethesda, MD; 2) University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA.


Abstract:

Mechanosensitive hair cells and their associated ribbon synapses are required for hearing and balance. Hair cells have a unique presynaptic structure called a ribbon body which functions to tether glutamatergic vesicles near adjacent presynaptic L-type calcium channels. The ribbon body is believed to be important for encoding vestibular and auditory information by maintaining fast, sustained exocytosis. Despite their similarity to neuronal synapses, previous work suggests that hair cells do not require the classical neuronal soluble NSF attachment protein receptors (SNAREs) VAMP1 and VAMP2 for vesicle release (Nouvian et al., 2011). Therefore, the SNARE machinery functioning in hair cells is not known.

 To determine the SNARE machinery required in hair cells we utilize the genetically tractable zebrafish model. The zebrafish has hair cells in their inner ear as well as in rosettes of hair cells that compose the lateral-line system. The zebrafish is an excellent model to investigate the molecular basis of ribbon synapse function as previous work has shown that ribbon synapses are structurally conserved amongst vertebrates (reviewed in Nicolson., 2015). Due to this level of conservation, combined with CRISPR-Cas9 advances in zebrafish, have enabled us to use the zebrafish to investigate what VAMP family member or members are required for hair-cell exocytosis.

We are currently examining CRISPR-Cas9 generated knockouts of the v-SNAREs (vesicle SNAREs/VAMPs). We are currently screening all VAMP family members. As of yet, no single VAMP has been directly linked to a complete loss of exocytosis or endocytosis in the hair cell. This suggests that no single VAMP subfamily plays a role in hair-cell synaptic transmission, but may require redundant VAMPS or an interaction with the complimentary subfamily of SNARES: t-SNARES (target SNAREs/SNAPs). We are exploring both possibilities now and continue to examine our current mutant vamp strains.

Lastly, in order to quantify alterations to synaptic vesicle release between wildtype and mutant strains, we are utilizing a transgenic line that expresses synaptophluorin in hair cells to measure vesicle release using swept-field confocal microscopy. In the future we plan on expanding our analysis to include electrophysiological studies in order to further examine alterations to neuronal function based on deficiencies in hair-cell release. An update of this research will be presented.