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 underlie the light ON response are still enigmatic, and are therefore the focus of this proposal. We previously showed that the a subunit of the heterotrimeric G-protein, Gao1, 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"""""""", Gao1GTP 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 Gao1?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 Gao1 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 Gao1 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 a1?1 and hydrolyzing enzyme is phosphodiesterase type 9A. Once identified, we will use knockout mice to study how these genes'deletion affects 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.

Public Health Relevance

The ON bipolar cells comprise 3/4 of the second order neurons in retina, and one type, the rod bipolar cell is essential for night vision. Any mutation in the signaling cascade of this cell type may render people night blind, thus justifying strong efforts to identify the molecules that mediate light signaling in this cell type.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Biology and Diseases of the Posterior Eye Study Section (BDPE)
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Greenwell, Thomas
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University of Pennsylvania
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Tummala, Shanti R; Dhingra, Anuradha; Fina, Marie E et al. (2016) Lack of mGluR6-related cascade elements leads to retrograde trans-synaptic effects on rod photoreceptor synapses via matrix-associated proteins. Eur J Neurosci 43:1509-22
Xu, Ying; Orlandi, Cesare; Cao, Yan et al. (2016) The TRPM1 channel in ON-bipolar cells is gated by both the ? and the ?? subunits of the G-protein Go. Sci Rep 6:20940
Tummala, Shanti R; Neinstein, Adam; Fina, Marie E et al. (2014) Localization of Cacna1s to ON bipolar dendritic tips requires mGluR6-related cascade elements. Invest Ophthalmol Vis Sci 55:1483-92
Sulaiman, Pyroja; Xu, Ying; Fina, Marie E et al. (2013) Kir2.4 surface expression and basal current are affected by heterotrimeric G-proteins. J Biol Chem 288:7420-9
Nikonov, Sergei S; Lyubarsky, Arkady; Fina, Marie E et al. (2013) Cones respond to light in the absence of transducin ? subunit. J Neurosci 33:5182-94
Devi, Sulochana; Markandeya, Yogananda; Maddodi, Nityanand et al. (2013) Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes. Pigment Cell Melanoma Res 26:348-56
Xu, Ying; Dhingra, Anuradha; Fina, Marie E et al. (2012) mGluR6 deletion renders the TRPM1 channel in retina inactive. J Neurophysiol 107:948-57
Dhingra, Anuradha; Vardi, Noga (2012) ""mGlu Receptors in the Retina"" - WIREs Membrane Transport and Signaling. Wiley Interdiscip Rev Membr Transp Signal 1:641-653
Dhingra, Anuradha; Ramakrishnan, Hariharasubramanian; Neinstein, Adam et al. (2012) G?3 is required for normal light ON responses and synaptic maintenance. J Neurosci 32:11343-55
Dhingra, Anuradha; Fina, Marie E; Neinstein, Adam et al. (2011) Autoantibodies in melanoma-associated retinopathy target TRPM1 cation channels of retinal ON bipolar cells. J Neurosci 31:3962-7

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