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 loss or damaged after hearing loss. This report summarizes the 8th year for the Section on Sensory Cell Development and Function. Our main focus has been publishing high quality research projects initiated at the NIH and exploring ways to expand upon and initiate new research. We use 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. Understanding how hair cell activity impacts hair cell health Our functional studies show that although all pLL hair cells mechanotransduce, the majority of cells are synaptically silent. We propose that synaptic silencing may be in place to alleviate the metabolic burden and prevent the unnecessary cellular stress associated with presynaptic activity. This cellular stress may be part of what renders hair cells susceptible to excitotoxic noise or ototoxins. In support of activity impacting ototoxin susceptibility, previous work in zebrafish indicates that over time, an accumulated history of activity renders hair cells more susceptible to the ototoxin neomycin. Currently it is not clear whether presynaptic activity specifically impacts this susceptibility. Because of the presynaptic silencing in the pLL system, we hypothesize that presynaptic activity may be a driving factor underlying cellular stress and ototoxin susceptibility. Overall, understanding why hair cells are susceptible to excitotoxic noise or ototoxins is important to develop protective therapies against these insults. Determine how spontaneous activity modulates hair-cell synapse assembly Numerous studies in mammals have shown that spontaneous, presynaptic calcium influx is a feature of hair cell development. Work in zebrafish has demonstrated that loss of presynaptic CaV1.3-channel function during development dramatically increases the size of ribbons. But mechanistically how presynaptic calcium controls ribbon size during development is unknown. Our recent work indicates that presynaptic calcium loads calcium into synaptic mitochondria. Mitochondrial calcium loading can alter the metabolic redox state of the hair cell by altering NAD+/NADH levels. Ribeye, the main component of ribbon synapses contains a NAD(H) binding domain that can impact Ribeye-Ribeye self-assembly and ribbon formation. NAD+ and NADH, act directly to increase and decrease ribbon formation during hair-cell development. This work provides mechanistic insight into how activity in hair cells regulate presynapse assembly during development and represents important knowledge required to reform synapses under pathological conditions. But how exactly this activity impacts the dynamics of ribbon formation is unclear. Our gap in knowledge is exacerbated by the fact that the dynamics underlying ribbon formation are not fully understood. To study ribbon assembly in vivo, we are using established transgenic lines that reliably label the main component of ribbons and ribbon precursors, the protein Ribeye (Ribeye b-mCherry). For our analyses we are using Airyscan confocal microscopy and light sheet fluorescent microscopy to uncover the in vivo dynamics of ribbon assembly. Overall this work will reveal novel insights into the dynamics underlying ribbon formation. Understanding ribbon formation is important to understand how to reform or repair these structures after they are lost or damaged.