Vestibular Ganglion Neurons:Heterogeneity and Molecular Regulation of Afferent Terminal Morphology

Project Abstract The vestibular system detects head motion and modulates central nervous system pathways. Disorders of the vestibular system affect about 69 million Americans and can cause disorientation, imbalance, dizziness, and increased rates of falls. Some vestibulopathies are sensorineural in nature, involving injury to or degeneration of vestibular sensory receptors (hair cells or HCs) or primary sensory neurons (vestibular ganglion neurons or VGNs). Two types of vestibular HCs have been defined: HCI and HCII. VGNs innervate HCI with a cup-like calyx synaptic terminal and HCIIs with bouton-only endings, and they can terminate in either the central/striolar zone or the peripheral/extrastriolar zone of the sensory epithelia. One obstacle for developing new treatments for vestibulopathies is our limited knowledge of how VGNs establish and maintain their region-specific and HC-type specific synaptic contacts and how many distinct subtypes of HCs and VGNs exist. Investigations into neuronal heterogeneity in other sensory systems (e.g., spiral ganglion neurons of the auditory system) have begun to unravel the molecular heterogeneity that drives the anatomic and functional classifications of those neurons. Indeed, studies in SGNs have given us new insights into how the auditory system functions and how sensorineural hearing disorders may be treated. Such characterizations have yet to be undertaken in VGNs. I propose two aims that employ single cell RNA sequencing (scRNAseq) in adult mice. In Aim I, I will test whether VGNs can be classified by distinct transcriptional profiles and correlate their profiles to known anatomic VGN subtypes using hybridization chain reaction, immunocytochemistry, and histological analysis. I seek to determine if molecularly distinct VGN subtypes differ with respect to their cell size, location in the ganglion, zonal projections to the utricular epithelium, and/or terminal morphology on HCs. In Aim II, I hypothesize that, by comparing scRNAseq data from VGNs and from utricular HCI and HCII, I can identify potential cell-to-cell signaling pathways that maintain the distinct morphologies of afferent synaptic terminals in the utricle. I will use the cell-to-cell inference program CellChat, to generate a list of candidate ligand-receptor pairs that may mediate HC-VGN communication. To narrow down signaling pairs that are required for proper afferent terminal morphology, I will take advantage of the finding that conditional knockout (cKO) of Sox2 in adult mouse HCIIs triggers a change in VGN terminals from bouton-type to calyx-type. Genes on my candidate list whose expression is altered in HCIIs after Sox2 cKO are more likely to be necessary to maintain boutons and/or repel calyx formation. This project will inform our understanding of VGN structure and function and the regulatory pathways that may maintain the HC type-specific morphology of VGN terminals. I will conduct this project under the mentorship of Jennifer Stone and David Raible as part of a structured training program that includes education in grantsmanship, statistics, responsible conduct of research, and biology of the inner ear.