Wallace B. Thoreson, Ph.D - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the mechanisms that control neurotransmission from rods and cones. Release of the excitatory neurotransmitter L-glutamate from photoreceptors is regulated by the activity of L-type Ca2+ channels. In the physiological voltage range, there appears to be a linear relationship between the influx of Ca2+ through these channels and the release of glutamate at the photoreceptor synapse. This linearity differs from the non-linear relationship found at most other synapses. One aim of this application is to use electrophysiological (capacitance monitoring and whole cell patch clamp recording) techniques, photolysis of caged Ca2+, and Ca2+ imaging techniques to examine the Ca2+ dependence of release from larval tiger salamander photoreceptors. One way in which a linear relationship between Ca2+ influx and release might arise is if vesicular exocytosis is initiated by the binding of only a single Ca2+ ion. If the binding of multiple Ca2+ ions is required to initiate release, then linearity between ICa and release is likely to reflect the linear summation, accompanying activation of an increasing number of Ca2+ channels, of sparsely distributed release sites with non-overlapping Ca2+ microdomains. These two possibilities will be investigated. The existence of a large number of modulators that can alter the voltage dependence or amplitude of photoreceptor ICa appear to present a challenge for photoreceptors to maintain the stable level of ICa activation necessary for stable synaptic output. The second major aim of this application is to use whole cell patch clamp recording as well as Ca2+ and Cl- imaging techniques to test the relative contribution of three specific intrinsic modulatory mechanisms to stabilizing ICa activation in rod and cone photoreceptors: (1) Ca-dependent inactivation of ICa, (2) depletion of synaptic cleft Ca2+, and (3) activation of Ca2+-activated Cl- channels. In addition to their importance in normal vision, regulation of photoreceptor ICa, intracellular Ca2+ concentration, and glutamate release are also important in pathophysiology of the retina. For example, increased intracellular Ca2+ levels in rods and cones may contribute to photoreceptor degeneration, and increased glutamate release arising from enhanced activation of ICa can have excitotoxic consequences on post-synaptic neurons. Thus, understanding the intrinsic mechanisms in rods and cones that regulate ICa, intracellular Ca2+ levels, and synaptic transmission is important for understanding the physiology of both diseased and normal retina.

DESCRIPTION (provided by applicant): Photoreceptors transmit their light responses across the first synapse in the retina by regulating the continuous release of glutamate-containing vesicles. The mechanisms by which light-evoked changes in membrane potential regulate synaptic transmission from photoreceptors are not well understood. We propose experiments to analyze the biophysical mechanisms of release from photoreceptors. Synaptic release from photoreceptors involves both fast transient and slow sustained components of release. Sustained release is important for shaping post-synaptic responses to slow changes in illumination and transient release contributes more to responses at abrupt light offset. In Aim 1, we test whether sustained and transient components of release are both due to release from the synaptic ribbon or whether non-ribbon synaptic release sites are also involved. In Aim 2, we determine how voltage-dependent changes in release probability, the size of the releasable pool of vesicles, and the rate of vesicle replenishment interact to shape sustained and transient post-synaptic responses to light and dark at the cone synapse. In Aim 3, we test whether quantal synaptic currents evoked by release of individual synaptic vesicles are regulated by changes in cytosolic glutamate levels at the cone synapse. Understanding the mechanisms of synaptic release from photoreceptors is important for understanding basic mechanisms of vision and how vision is disrupted by mutations in synaptic proteins or mis-regulation of glutamate release. Understanding normal retinal physiology is also important for designing therapies to restore normal retinal function to diseased eyes using retinal stem cells or prosthetic devices. PUBLIC HEALTH RELEVANCE: This project studies the mechanisms by which visual signals are transmitted to downstream neurons at the first synapse in the retina. In addition to providing a better understanding of early visual processing by the retina, understanding the mechanisms by which rod and cone photoreceptors release the neurotransmitter glutamate is necessary to understand how mutations in synaptic proteins or mis-regulation of glutamate release lead to eye disease and vision loss. An understanding of normal retinal physiology is also needed for restoring vision to diseased eyes by the use of retinal stem cells, prosthetic devices, or other means.

Light responses of rod and cone photoreceptors are encoded by the release of glutamate-filled vesicles at photoreceptor synapses. Synaptic transmission at the first synapse in the retina thus fundamentally shapes visual perception and damage to photoreceptor synapses by protein mutation or diseases such as macular degeneration and ischemia causes vision loss. To understand the consequences of damage to these synapses and how to restore vision by therapeutic means requires a thorough understanding of their normal operation. Release from photoreceptors involves a plate-like protein structure known as the synaptic ribbon. Unlike most central nervous system (CNS) synapses that release only one or two synaptic vesicles at a time, ribbon synapses in photoreceptors and other sensory neurons are specialized for continuous release. In addition to the ribbon itself, the specialized capabilities of ribbon synapses are also determined by the use of certain proteins that differ from those at more conventional synapses. Rod and cone photoreceptors differ further from both conventional and other ribbon synapses in their use of an exocytotic Ca2+ sensor with unusual Ca2+ dependence. At most synapses, synaptic vesicle release rate rises with the 5th power of [Ca2+]i but release from photoreceptors has a weaker 1-3rd order Ca2+-dependence. The identity of the atypical Ca2+ sensor that regulates vesicle release from photoreceptors is a major unresolved question about the mechanisms of release at the first synapse in the retina. Isoforms of the protein synaptotagmin (Syt) serve as the exocytotic Ca2+ sensors in most neurons. Our first aim is to identify the Ca2+ sensor controlling release from photoreceptors by testing mice in which specific Syt proteins have been selectively deleted from rods or cones. Our second aim is to confirm that the exocytotic Ca2+ sensors in mouse rod and cone synapses retain the unusually low Ca2+ cooperativity seen in lower vertebrates. In Aim 3, we propose to characterize how the Ca2+-dependence of release rate is shaped by different combinations of Syt, Complexin, and SNARE proteins that reproduce components of the rapid release machinery at different conventional and ribbon synapses, using unique in vitro approaches that can probe single fusion pores with sub-ms time resolution. Together, these experiments will reveal the mechanisms responsible for the atypical Ca2+-dependence of neurotransmission at the critical first synapse in vision and allow us to understand how the expression of particular proteins shapes the properties of release to meet specific signaling needs at different CNS synapses.

In sensory cells of the eye and inner ear the neurotransmitter glutamate is released at active zones in a graded and continuous manner. These cells have evolved specialized structures known as synaptic ribbons. These proteinaceous structures tether synaptic vesicles near active zones. Based on their location, abundance of tethered vesicles, and properties of the sensory neurons in which they are found, these organelles have long been thought to be important for maintaining the continuous release of glutamate. Several other functions have also been ascribed to ribbons. However, recent evidence casts doubt on these ideas. The focus of this grant is to understand the role of synaptic ribbons in sensory synaptic transmission using animal models that lack Ribeye, a protein that is both the most abundant within the ribbon and not found anywhere else. Lack of Ribeye leads to loss of membrane associated synaptic ribbons, without loss of other presynaptic proteins. Specific Aim 1 investigates how Ribeye removal and loss of synaptic ribbons affects neurotransmitter release from photoreceptors. Specific Aim 2 looks at the properties of synaptic release in bipolar cells from the same animals. In Specific Aim 3, we will image single synaptic vesicles in cells lacking Ribeye to measure rates of vesicle movement and replenishment to test the role of the ribbon in these processes. Understanding ribbon function may provide clues to help understand diseases that specifically affect vision and hearing. In addition, the fundamental understanding of presynaptic processes in these specialized neurons will have broader implications for neuronal communication in general and thus, may contribute to our understanding of various aspects of mental health and neurological disorders.