Retinal bipolar cells are the first 'projection neurons' of the vertebrate visual system and transmit all of the information needed for vision. Bipolar cells can signal change in contrast while providing an analog read-out of luminance via changing the rate of neurotransmitter release (NTR). To maintain this ability, the bipolar cells must have dynamic control over release rate and the efficient recruitment of release-ready vesicles to fusion sites. However, the spatiotemporal properties of Ca2+ signals that control NTR, and the molecular entities that control the interplay between Ca2+ signal and vesicle dynamics in sustaining kinetically distinct NTR components remain poorly understood. The long-term goal is to unveil the regulation of Ca2+ signaling in retinal ribbon synapses during development, normal adulthood, and disease. Within this goal, the overall objective of this proposal is to determine the spatiotemporal properties of Ca2+ signals that control kinetically distinct pools of NTR and the role of local Ca2+ signals in governing vesicle dynamics that sustain neurotransmission in bipolar cell ribbon synapses. The central hypothesis is that Ca2+ domains governing kinetically distinct components of NTR are different because the ribbon itself adds an additional compartment responsible for spatial segregation of kinetically different synaptic vesicles and the underlying molecular targets that sense Ca2+ concentration and/or alter Ca2+ signals. This hypothesis is based on preliminary data, acquired in applicant?s laboratory using novel techniques developed for evaluating the traffic of single synaptic vesicles at ribbons while simultaneously measuring the underlying changes in [Ca2+], all with millisecond temporal precision. This hypothesis will be tested by pursuing two specific aims using a confluence of state-of-the-art fluorescence imaging, voltage-clamp electrophysiology, computational modeling, electron microscopy of individual physiologically identified cells, and pharmacological tools: 1) Reveal the mechanisms that determine the spatiotemporal properties of calcium signals which control kinetically distinct neurotransmitter release pools; and 2) Determine the interplay between local calcium signaling and vesicle replenishment that is required for sustaining kinetically distinct components of NTR in rod bipolar cell ribbon synapses as a model system. Dysregulation of Ca2+ signaling is a key early?stage process of neurodegeneration in age-related retinal degenerations, glaucoma, diabetic, and optic neuropathies. The knowledge gained from studying Ca2+ dynamics in bipolar cell synaptic transmission will allow us to determine if defects with local Ca2+ homeostasis are a prelude to disease in the future. Data generated from this proposal will have a broad impact that extends beyond our specific investigation of rod bipolar cells and will be applicable to similar ribbon synapses located within and outside the visual system and encoding distinct aspects of sensory information. More widely, our data will be relevant to synapses throughout the central nervous system because the CAZ of ribbon synapses shares many molecular components with conventional synapses.
The proposed research is relevant to public health because developing strategies to directly monitor calcium signaling and neurotransmitter release will significantly advance fundamental knowledge of how neurons communicate with each other. The contribution will be highly significant because dysregulation of Ca2+ signaling is a key early?stage process of neurodegeneration in age-related retinal degenerations, glaucoma, diabetic, and optic neuropathies. Thus, the proposed research is relevant to the part of NIH?s mission that pertains to developing fundamental knowledge that will help to reduce the burdens of human disability.
