Graduation Date

Summer 8-17-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmacology and Experimental Neuroscience

First Advisor

Wallace Thoreson

Abstract

Photoreceptors are the first neurons in the visual system. They transduce changes in light intensity into graded changes in membrane potential that are then transformed into chemical signals by regulating the release of glutamate-filled synaptic vesicles. Rod and cone photoreceptors release glutamate continuously in darkness and release slows in light. To help track rapid changes in light intensity, photoreceptors are capable of both rapid exocytosis and rapid endocytosis of synaptic vesicles.

Endocytosis is needed for recycling synaptic vesicles but also appears to be important for removing proteins and lipids from active zones to restore release site function after prior vesicle fusion. Synaptic exocytosis from vertebrate photoreceptors involves synaptic ribbons that cluster vesicles near the presynaptic membrane. We hypothesized that such clustering increases the likelihood that exocytosis at one ribbon release site may disrupt release at an adjacent site. Consistent with this, studies described in Chapter 2 showed that endocytosis is needed to rapidly restore release site competence at photoreceptor ribbon synapses. We combined optical and electrophysiological techniques to show that endocytosis is important for restoring late steps in the vesicle fusion process but does not appear to be needed for vesicles to dock successfully at the membrane. Release site clearance by endocytosis is thus essential for continuous release in photoreceptors.

We explore mechanisms that contribute to efficient synaptic vesicle exocytosis and endocytosis in Chapter 3. Exocytosis and endocytosis of synaptic vesicles can be coupled in two general ways. In the full-collapse model, the vesicle membrane merges completely with the plasma membrane and so vesicles must be fully reconstructed before they can be retrieved by endocytosis. In the kiss-and-run model, a vesicle briefly contacts the plasma membrane through a small fusion pore that permits release of small molecules but the vesicle does not flatten into the plasma membrane. The vesicle with its complement of proteins is quickly recycled to the cytoplasm after closure of the fusion pore during kiss-and-run. Using a combination of techniques including total internal reflectance fluorescence microscopy (TIRFM), confocal microscopy, electron microscopy, and membrane capacitance measurements, we found that kiss-and-run exocytosis and endocytosis contributes to more than 50% of the release events in photoreceptors. In addition to speeding endocytosis, kiss-and-run fusion may limit disruption of release site structure during fusion, providing an efficient mechanism to facilitate sustained release.

HCs not only receive excitatory feedforward signals from photoreceptors, but also send inhibitory feedback signals back to photoreceptors. At normal physiological membrane potentials in cones, inhibitory feedback from HCs to cones increases the activity of L-type voltage-gated Ca2+ channels producing inward feedback currents that increase the synaptic release of glutamate. In the final chapter of this thesis, we describe studies using paired whole cell recordings to determine if, in addition to Ca2+ currents, other currents also contribute to these inward feedback currents in cones. We found that feedback currents in cones involve a smaller than expected contribution from Ca2+-activated Cl- currents and a larger than expected contribution from Cl- currents associated with glutamate transporter activity in cones.

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