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Author ORCID Identifier

https://orcid.org/0000-0003-0477-986X

AccessType

Campus-Only Access for Five (5) Years

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded

2020

Month Degree Awarded

September

First Advisor

Luke R. Remage-Healey

Subject Categories

Molecular and Cellular Neuroscience

Abstract

Hair cell ribbon synapses in vertebrate hearing organs are capable to encode neural signals with extraordinary precision and exhibit multiple hallmark features, including multivesicular release (MVR). Compared to single vesicle releases, MVRs introduce more neurotransmitter to the synaptic cleft, but the impact of extra neurotransmitter on postsynaptic receptor activation is poorly understood. In the first part of the study, we preformed non-stationary noise analysis and examined receptor activation in spontaneous excitatory postsynaptic currents (EPSCs) recorded in hair cell ribbon synapses in the bullfrog amphibian papilla. We found that MVR activates more receptors with higher open probability within the synapse. To account for EPSCs of >300 pA, we hypothesize that MVR of high quantal content could reach and activate distant receptors in neighboring release sites. Given the geometry of these release sites, we demonstrated this is not only feasible but also consistent with multiple observations on EPSC amplitude, kinetics, and charge.

Another hallmark of hair cell ribbon synapses is its remarkable temporal precision, requiring intracellular Ca2+, the trigger for synaptic vesicle releases, to be controlled precisely. In the second part of the study, we examined Ca2+ extrusion in hair cells and its implication in phase-locking. We found that Ca2+ clearance from synaptic ribbons follows a double-exponential function, and the weight of the fast component, but not the two time constants, is significantly reduced during inhibition of Ca2+ extrusion. Consistently, we found synaptic phase-locking was also disrupted in the same treatments, suggesting involvement of active Ca2+ extrusion in phase-locking.

The third hallmark of hair cell ribbon synapses is the tight coupling between Ca2+ channels and docked synaptic vesicles. In the third and last part of the study, we investigated synaptic vesicle priming and fusion by manipulating Ca2+ diffusion in hair cells with dialysis of exogenous Ca2+ buffer. We found that with strong Ca2+ buffer introduced into hair cells, the reduction in priming occurred earlier than the reduction in fusion. This result, while still in its preliminary form, suggests that synaptic vesicle priming and fusion are mediated by two distinct Ca2+ sensors, possibly two different Ca2+ binding domains in otoferlin.

DOI

https://doi.org/10.7275/18286036

Available for download on Wednesday, September 01, 2021

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