Graduation Date

Spring 5-6-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Interdisciplinary Graduate Program in Biomedical Sciences

First Advisor

Wallace Thoreson

MeSH Headings

Synaptic Transmission

Abstract

Synaptic transmission relies on the Ca2+-dependent release of neurotransmitters packaged into synaptic vesicles. Ribbon synapses have the capability to release multiple vesicles at a time in a highly coordinated and synchronized manner. Glutamate release from rod and cone photoreceptor cells involves presynaptic ribbons composed largely of the protein RIBEYE. To examine roles of ribbons in rods and cones, we studied mice in which GCamP3 replaced the B-domain of RIBEYE. We discovered that ribbons were absent from rods and cones of both knock-in mice possessing GCamP3 and conditional RIBEYE knockout mice. Mice lacking ribbons showed reduced temporal resolution and contrast sensitivity assessed with optomotor reflexes. ERG recordings showed 50% reduction in scotopic and photopic b-waves. The readily releasable pool (RRP) of vesicles in rods and cones was also halved. We also studied release from cones by stimulating them optogenetically with ChannelRhodopsin2 (ChR2) while recording postsynaptic currents in horizontal cells. Recovery of release from paired pulse depression was twofold slower in rods and cones lacking ribbons. Release from rods at −40 mV in darkness involves regularly spaced multivesicular fusion events. While the regular pattern of release remained in rods lacking ribbons, the number of vesicles comprising each multivesicular event was halved. Our results support conclusions that synaptic ribbons in rods and cones expand the RRP, speed vesicle replenishment, and augment some forms of multivesicular release. Slower replenishment and a smaller RRP in photoreceptors lacking ribbons may contribute to diminished temporal frequency responses and weaker contrast sensitivity.

Synaptotagmins are the primary Ca2+ sensors for synaptic exocytosis.

Previous work suggested synaptotagmin-1 (Syt1) mediates evoked vesicle release from cone photoreceptor cells in the vertebrate retina whereas release from rods may involve another sensor in addition to Syt1. We found immunohistochemical evidence for syntaptotagmin-7 (Syt7) in mouse rod terminals and so performed electroretinograms (ERG) and single-cell recordings using mice in which Syt1 and/or Syt7 were conditionally removed from rods and/or cones. Deleting Syt1 from rods reduced glutamate release evoked by short depolarizing steps but not long steps, whereas deleting Syt7 from rods reduced release evoked by long but not short steps. Deleting both sensors completely abolished depolarization-evoked release from rods. Effects of various intracellular Ca2+ buffers showed that Syt1-mediated release from rods involves vesicles close to ribbon-associated Ca2+ channels whereas Syt7-mediated release evoked by longer steps involves more distant release sites. Spontaneous release from rods was unaffected by eliminating Syt7. While whole animal knockout of Syt7 slightly reduced ERG b-waves and oscillatory potentials, selective elimination of Syt7 from rods had no effect on ERGs. Furthermore, eliminating Syt1 from rods and cones abolished ERG b-waves and additional elimination of Syt7 had no further effect. These results show that while Syt7 contributes to slow non-ribbon release from rods, Syt1 is the principal sensor shaping rod and cone inputs to bipolar cells in response to light flashes.

Synaptotagmin-9 (Syt9) has been shown to be a Ca2+ sensor for fast synaptic release, and most studies investigating Syt9 have been performed in the brain using calyx of Held slice preparations and cultured neurons. Though the role Syt9 plays in retina is unknown, we found evidence for Syt9 in the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner. We crossed these Syt9fl/fl mice with Rhoi-Cre, HRGP-Cre, and CMV-cre mice to generate mice in which Syt9 was eliminated from rods (rodSyt9CKO), cones (coneSyt9CKO), or whole animals (CMVSyt9). CMVSyt9 mice showed an increase in scotopic electroretinogram (ERG) b-waves evoked by bright flashes with no change in a-waves. Oscillatory potentials were also enhanced at the highest flash intensity. Enhanced responses to bright flashes suggested cone involvement, but cone-driven photopic ERG b-waves were not significantly different in CMVSyt9 knockout mice. Selective elimination of Syt9 from rods decreased b-waves and oscillatory potentials. Selective elimination of Syt9 from cones had no significant effect on ERGs. These results suggest that Syt9 performs both an inhibitory “clamping” function and a facilitatory function in exocytosis in the retina.

Comments

2023 Copyright, the authors

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