Noga Vardi - US grants

Abstract My long-term goal is to understand intracellular signaling cascades and their contribution to image processing in retina. As the eye flicks about a scene, a photoreceptor sees an alternating pattern of light and dark. Correspondingly, the photoreceptor transiently decreases and increases its glutamate release. Each pulse of glutamate has two effects: in OFF bipolar cells, it directly opens an AMPA/kainate cation channel, and in ON bipolar cells, it activates the metabotropic receptor, mGluR6, that indirectly closes a cation channel. The light response signaled by the ON bipolar cell is crucial for night vision, and subserves half the dynamic range in day vision. Though central to retinal processing, the basic molecular mechanisms that underly the the light ON response are still enigmatic, and are therefore the focus of this proposal. We previously showed that the ¿ subunit of the heterotrimeric G-protein, G¿o1, is required for the ON response. However, which G¿ and which G¿ isoforms comprise the other two subunits of this G- protein is unknown. Once Go is activated, either of its activated arms, G¿o1GTP or the free G¿¿ dimer, can lead to channel closure, but which one does so is yet unknown. The next step of the cascade was thought to involve cGMP as a second messenger, but recent evidence suggests cGMP is a modulator. Still, whether cGMP activates a kinase to phosphorylate the receptor or the channel is controversial. Here we propose three Aims to answer these questions. In Aim 1, based on our profiling of ON bipolar cells and published immunocytochemistry, we hypothesize that the G-protein mediating the light ON response is G¿o1¿3¿13. Aim 1 will test this hypothesis by using RNA interference to silence the genes that encode G¿3 and G¿13. Specific shRNA vectors will be injected subretinally to postnatal P0-P2 mice and transfected to bipolar cells by electroporation. At P21-P40, the compound light response of ON bipolar cells will be recorded using electroretinograms, and a single rod bipolar cell's response will be recorded with whole cell configuration. Aim 2 will determine which arm of Go leads to channel closure by uncoupling G¿o1 from the G¿¿ dimer. We will record from a rod bipolar cell and dialyze either G¿¿-activating peptides to activate G¿¿, G¿¿ scavengers to deactivate G¿¿, or active G¿o1 to test its direct effect. During dialysis we will monitor the agents' effects on holding current, input resistance, and light response. In Aim 3, we will first study cGMP's effect on the mGluR6 cascade in oocytes expressing the appropriate proteins. We will then test the hypothesis that cGMP's synthetic enzyme is guanylyl cyclase ¿1¿1 and hydrolyzing enzyme is phosphodiesterase type 9A. Once identified, we will use knockout mice to study how these genes' deletion affect the rod bipolar light responses. Our research will contribute to the fundamental knowledge of the first synapse on the visual pathway and will identify new molecular players whose mutations may lead to night blindness, thus extending the basic foundation for future clinical studies.

DESCRIPTION (provided by applicant): Retinal bipolar cells are the key link between photoreceptors and ganglion cells. One bipolar cell type, the rod bipolar cell, transmits the dim light signal at night, while about 10 types of cone bipolar cells transmit the detailed information of the visual image in daylight. Because the visual image contains information from various features (contrast, spatial, temporal, color, etc.), each cone bipolar type extracts certain features and transmits them optimally. The largest class of bipolar cells, the ON class, conveys positive contrast with responses that are mediated by a transduction cascade. When whole-cell patched, their light responses runs down rapidly. Consequently, information about the physiological properties of different ON cone bipolar cell types is scarce. Recently, a new calcium indicator protein (GCaMP3) was developed, and it can specifically be targeted to ON bipolar cells (under control of mGluR6 promoter) or to the closely connected AII amacrine cells (under control of mGluR1 promoter). We here propose to image this indicator with two-photon microscopy and combined it with electrophysiology to investigate the physiology and visual contribution of these cells. Aim 1 will investigate the rod bipolar cell's adaptation mechanism that critically depends on calcium accumulation to lower the response gain. Retinas will be stimulated with ascending light intensities and calcium signal will be recorded in rod bipolar dendrites and axon terminals. Input-output functions will determine the amount of calcium that causes adaptation. The source of calcium will be determined by either emptying calcium stores, blocking intracellular calcium channels, or blocking TRPM1 transduction channels. Aim 2 will determine the physiological differences among the types of ON cone bipolar cells in two ways. First, the retina will be stimulated with flashing or temporally modulated sinusoidal light with varying intensities, and the calcium responses of different cone bipolar types will be recorded by imaging axon terminals that reside in all ON layers of the inner plexiform layer. Second, an AII cell will be depolarized, and the strength of its coupling to the cone bipolar types will be measured by calcium imaging. In order to reveal the cell type identity of the imaged terminals, at the end of the recording session, dye will be injected into multiple cells with a microelectrode. Aim 3 will measure the dynamics of coupling and noise within the AII network under different light intensities using two complementary methods. First, AII amacrine cells will be infected with channelrhodopsin fused to GFP;an AII cell will be patched with whole cell configuration;channelrhodopsin at various distances from the patched cell will be stimulated;and the resulting voltage in the cell will be recorded. Second, AII amacrine cells will be infected with GCaMP3;current will be injected into a cell that is whole-cell patched;and the resulting calcium response in neighboring AII cells will be measured. These experiments will be repeated after blocking gap junctions and/or Na+ channels. The proposed experiments will greatly facilitate our understanding of retinal circuits and parallel processing and they will help apply this knowledge to efforts in restoring vision. PUBLIC HEALTH RELEVANCE: Our goal of imaging light-evoked calcium responses in the ON bipolar cells and the AII amacrine cells will have a substantial impact on the field because these recordings are still novel and they promise to pave the way for efficient recordings from specific cell compartments in the retina. These will yield important new information relatively fast, and will gain greater understanding of the principle of visual processing in night and day vision. This understanding in turn will help design more optimal approaches for the ever developing tools of genetic therapy.