Eduardo Solessio - US grants

Specific Aims Broadly, there are two major specific aims for the coming project period. One is the identification of the excitatory molecule for the TRP/TRPL channels and the other is the detailed analysis of three mutants that remain uncharacterized. a. Search for the excitatory molecule for TRP/TRPL channels. This project is a direct continuation of the inaE project completed in the previous project period and represents an attempt to finally close the chapter on the long-standing search for arguably the most sought-after molecule in the field. There will be two basic strategies: i) to identify the excitatory molecule by exogenous application of candidate molecules in whole-cell recordings, and ii) to attempt to obtain genetic evidence as to whether or not monoacylglycerol lipase (MAGL) is involved in the channel activation process by identifying the MAGL gene involved and isolating mutations in the gene. The third part of this aim is to explore whether or not diacylglycerol (DAG) has a synergistic enhancing role in channel excitation even though it has no direct excitatory role itself. b. Analysis of three mutants selected from those that remain uncharacterized. Two of these, P226 and ninaF, were chosen in part because we have made striking progress in the preliminary identification of the genes harboring the lesions responsible for the phenotypes. The third mutant, US2985, was chosen solely on the basis of its interesting phenotype. In the case of P226, we will explore the preliminary hypothesis that its dark adaptation-dependent PDA phenotype is related to metarhodopsin deactivation by extensive, multiple phosphorylation. Because the P226 gene has been tentatively identified to be an ortholog of Usher syndrome genes, the project may shed light on the mechanism of retinal degeneration in Usher syndrome. In the ninaF project, we will explore the hypothesis that the protein encoded by this gene performs a function related to those of NINAC myosin III. In the US2985 project, we will explore the hypothesis that this mutant is impaired in Ca2+-mediated adaptation, but not in Ca2+-mediated activation of response. The project has the potential for identifying a protein specifically involved in Ca2+-mediated adaptation.

? DESCRIPTION (provided by applicant): The human visual system has a remarkable capacity to detect differences in contrast (i.e., differences in luminance across space or time) as small as 1:500. This ability underlies the performance of everyday visual tasks such as reading, driving, and face recognition. Indeed, deficits in contrast sensitivity are used as diagnostic signs for the assessment of retinal disease. Despite its importance, the underlying cellular and network mechanisms that encode and limit contrast sensitivity remain elusive. Past work has assigned the photoreceptor frequency response as one mechanism that limits temporal contrast sensitivity (TCS); however, this has not been demonstrated empirically. The goal of this proposal is to determine how rod photoresponse kinetics contributes to visual TCS in mesopic conditions, when rod photoreceptors integrate the response of multiple photoisomerizations. The underlying hypothesis is that rod inactivation kinetics constrains TCS by limiting the speed of the responses of rods to light decrements. To test the hypothesis, a novel operant behavioral assay was developed which establishes in mouse a model of human temporal vision that matches fundamental properties of human visual psychophysics. In the operant assay, mice are trained to detect and respond to a flickering visual stimulus, an action that requires cortical inpt and decision-making. The preliminary data collected with the operant behavior assay suggest that transgenic mice with fast rod inactivation kinetics have higher temporal contrast sensitivity than control mice. Combined with standard electrophysiological tools and transgenic models, the behavioral assay allows dissection of the contributions of rod kinetics to vision. Aim 1 test how rods respond to dynamic stimuli and what impact adaptation mechanisms have on the sensitivity to different frequencies of stimulation in mesopic light levels. Aim 2 tests the contribution of rod photoresponses kinetics to visual (behavioral) contrast sensitivity. Aim 3 test retinal and visual temporal contrast sensitivity in a rhodopsin P23H mouse model of retinitis pigmentosa to test the hypothesis that mutant mice harboring this mutation will exhibit higher TCS than control mice. We predict this because patients and mouse models harboring the P23H rhodopsin mutation exhibit faster rod recovery responses than control subjects. Results of this aim may well provide proof-of-principle for an early and practical visual function test that i diagnostic of certain forms of autosomal dominant retinitis pigmentosa. This project will be significant because it will help understand 1) the contributions of rod kinetics to visual TCS in normal and diseased retinas, and 2) provide insights into the dynamic retinal interactions between rod and cone signals that determine the temporal, spatial, and spectral sensitivities of mesopic vision. The proposed research is innovative, because it examines visual properties common to mouse and humans using a novel operant behavioral assay developed by our research group.