The parallel neural circuits in mammals that connect photoreceptors to several specific types of ganglion cell have been identified in nearly complete detail. The major missing information now concerns 'chemical architecture', that is, which specific ion channels, receptors and second messengers are expressed by each neuron in a circuit and in what region of each cell. The proteins at issue belong to multigene families whose isoforms differ functionally (e.g. binding affinity, conductance, temporal gating, and desensitization), and the ensemble of such properties sets the behavior of each circuit. The applicant proposes to identify for several multigene families the particular isoforms expressed in specific types of neuron. This will be accomplished by isolating an identified neuron, amplifying its mRNA, and probing the amplified message with known nucleotide sequences for the various isoforms. The isoforms so identified will then be localized immunocytochemically at the subcellular level by electron microscopy. The neurons to be studied are: rods, cones, horizontal cells (types A and B) and AII amacrine cells. These cells are chosen because their circuits are known and characterized computationally. The proteins to be identified and localized include the connexins (aim 1) and a subset of their known modulatory proteins (D1 dopamine receptor, Golf, and adenylyl cyclase III) (aim 2), plus the ligand-gated ion channels, GABA alpha and GABA rho (aim 3). The connexins are important because they mediate electrical coupling (homotypic and/or heterotypic) that affects signal processing (filtering, averaging, switching). The modulators are important because they govern the degree of coupling in various circuits (which is tuned to match environmental luminance). The role of G-proteins and their activators in modulating degree of coupling will be determined by dye coupling in superfused whole mount retinas. The GABA A and GABA rho subunits are important because they mediate feedback and feedforward inhibition and are undoubtedly critical to gain control. The new information regarding chemical architecture will be incorporated into computational models of the rod bipolar and cone bipolar circuits to assess (by simulation) how modulation of coupling and inhibition (feedback and feedforward) affect bipolar cell gain control (aim 4). By characterizing the chemical architecture of known circuits, and then simulating the results, the proposed project would provide a basis for understanding the principles of circuit design in retina. This will help assess the molecular basis of human visual performance and identify losses due to mutations of specific isoforms.
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