In vivo investigation of sensory hair cell function and development

National Institute On Deafness And Other Communication Disorders

Investigators

Linked publications, trials & patents

Abstract

Summary and Background: Sensory hair cells are required to reliably transmit auditory and vestibular information to the brain. While the majority of hearing loss results from the loss of hair cells, there is accumulating evidence that in cases of noise-induced and age-related hearing loss, the pathology may be due to damage or loss of hair cell synapses rather than hair cells. In these latter cases, effective clinical treatment requires the restoration of synaptic connections. In order to restore these connections, it is critical to understand how they function and how they are assembled in vivo. Our studies combine genetic, molecular, and imaging-based approaches to identify the structural and functional processes underlying synapse formation and function in hair cells. For our studies, we examine neuromast hair cells in the zebrafish posterior lateral-line system (pLL) in order to study hair-cell system development and function, in a live, transparent preparation. We have an extremely powerful collection of transgenic zebrafish that label synaptic structures to either assess synaptic morphology or function using genetically encoded fluorescent proteins. We are combining these microscopy-based approaches with CRISPR technology to create mutant zebrafish in order to identify genes required for synapse formation, function, and regeneration. With this knowledge, we aim to apply our understanding of these processes in order to understand how to properly reform hair cells and synaptic structures when they are lost or damaged after hearing loss. This report summarizes the 11th year for the Section on Sensory Cell Development and Function. Our main focus has been publishing high-quality research projects and exploring ways to expand upon and initiate new research. We apply numerous advanced microscopy approaches to explore new avenues of live functional and developmental analyses. Projects in the lab: In vivo investigation of sensory system function Based on our published work in the neuromasts of the zebrafish pLL, the majority of hair cells and synapses are silent, but the silent cells can be recruited (unsilenced) after damage. This data indicates that silent hair cells may function as failsafe to ensure sensory information is transmitted even after damage or loss of active cells. In the pLL, multiple afferent neurons (~4) innervate each neuromast. Currently, it is unclear if all anatomically connected afferent neurons are activated when the majority of hair cells and synapses are silent. Due to the abundance of silent hair cells, we hypothesize that not all anatomically connected afferent neurons respond to neuromast stimulation. We are currently investigating the functional circuitry between neuromasts and afferent neurons using light sheet fluorescent microscopy. Constructing this circuitry is essential for our understanding of in vivo sensory circuit function. Determine what molecules are required for hair cell synapse formation Hair cell ribbon synapses are critical for hearing and balance and loss of these synapses is linked to age- and noise-related hearing loss. Currently, what molecules are required to assemble these synapses are not fully understood. We have used mouse and zebrafish models to investigate the role of presynaptic Neurexin3 in hair cell synapse assembly. For our work, we acquired high-resolution of synapses in neurexin3 mutants and found fewer complete synapses in both animal models. In addition, we used functional imaging to demonstrate that pre- and post-synaptic responses are reduced in neurexin3 mutants. Overall, this work identified Neurexin3 as a critical molecule required to assemble ribbon synapses. We are currently using live imaging to visualize how ribbon synapse assembly is disrupted when Neurexin3 is absent. Understanding what molecules are important for ribbon synapse formation is important to understanding how to reform or repair these synapses after they are lost or damaged. Determine how hair cell synapses form in vivo Hair cell ribbon synapses are defined by a presynaptic density called a ribbon. How this unique and highly specialized synapse is formed is unclear. Using Airyscan confocal microscope in live zebrafish we have investigated how ribbons form. We have found that developing ribbon precursors move and fuse along microtubule networks before reaching the presynaptic active zone. Currently, we are using similar approaches to visualize how presynaptic ribbons assemble alongside postsynaptic structures. Overall, this work will outline a dynamic series of events that underlies the formation of a critical synapse required for sensory function. Determine how synaptic vesicles reach the hair cell synapses Ribbon synapses have high rates of spontaneous vesicle release and function without fatigue. To sustain this release level, a continuous supply of synaptic vesicles must be trafficked to the presynapse. In neurons, the kinesin motor protein Kif1a has been shown to transport synaptic vesicles along microtubules to the presynapse. We have found that both microtubules and Kif1a transport and ultimately enrich synaptic vesicles at hair cell presynapses. Loss of synaptic vesicle replenishment at ribbon synapses impairs postsynaptic responses. These synaptic defects affect rheotaxis–the ability to station hold in water flow–a behavior mediated by the lateral line in zebrafish. Currently, we are investigating other players that work with Kif1a to enrich synaptic vesicles at the hair cell synapse. Overall, this work has revealed how synaptic vesicles are transported to and maintained at the hair cell synapses and highlights the significance of synaptic vesicle pools for proper hearing and balance.