Graduation Date

Summer 8-14-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Integrative Physiology & Molecular Medicine

First Advisor

Wallace B. Thoreson

Abstract

Neurons communicate via Ca2+-dependent release of neurotransmitters packaged into vesicles (quanta). Some CNS neurons, especially sensory synapses, can release multiple vesicles at a time, increasing information transmission and overcoming the unreliability of a stochastic process. Ribbon-bearing neurons, including retinal photoreceptors, face the challenge of encoding sensory receptor potentials into an ever-changing train of vesicle release events. We studied release of glutamate using voltage clamp to measure anion currents activated during glutamate reuptake into presynaptic terminals (IA(glu)) of salamander and mouse rods, finding that each employ distinct mechanisms for multiquantal release. In amphibian rods, we found that 1/3 of the spontaneous IA(glu) fusion events involve synchronous fusion of multiple vesicles. By varying intracellular buffering to localize Ca2+-dependent events, we found that multiquantal release occurs near Ca2+ sources. In photoreceptors, Ca2+ influx occurs just below synaptic ribbons. Vesicles house SNARE machinery so we hypothesized that vesicles on the ribbon undergo homotypic fusion prior to exocytosis. Destruction of ribbons and disruption of the SNARE-protein syntaxin3B prevented spontaneous multiquantal release, suggesting that salamander rods are capable of multivesicular release due to homotypic fusion of vesicles along ribbons. In mouse rods, spontaneous release at −70 mV involved the stochastic fusion of single vesicles. With depolarization, glutamate release increased linearly with voltage-gated Ca2+ currents. As the membrane approached the resting potential in darkness of −40 mV, rods began to release glutamate in multivesicular bursts of 17±7 vesicles every 2801±598 ms. Release evoked by brief depolarizations and bursts both involved the same pool of ribbon-associated vesicles with fusion regulated by the vesicular Ca2+ sensor synaptotagmin-1. A second, slower component of release controlled by synaptotagmin-7 is also present in rods but not cones. We hypothesized a v role for coordinated bursts of release in transmitting single photon signals. The rate of bursting was responsive to small voltage changes of 1.0-3.5 mV and the voltage waveform that triggered bursts most effectively was similar to single photon responses. We propose that multiquantal bursts contribute to mechanisms that filter out small noisy events to improve reliable detection of single photons by the retina.

Available for download on Tuesday, July 13, 2021

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