Denis A. Baylor - US grants

The electrical signals of vertebrate photoreceptors are generated by modulation of an inward current of sodium ions at the outer segment. The mechanism by which Na enters the outer segment is not known, and there is little solid evidence about how light controls the conductance of the pores or carrier molecules that mediate the Na influx. We propose to examine the properties of the light-sensitive conductance and the mechaism of its control, using patch clamp and suction electrode recording, optical measurements, and injection of substances into isolated rods. We intend to approach the following questions: 1) What is the electrical conductance, temperature dependence and kinetic behaviour of the light-sensitive "channel" in the surface membrane of the rod outer segment? 2) How does the channel respond to possible internal transmitters such as CA ions and cyclic GMP? 3) How does the internal concentration of free Ca change during a rod's response to light? 4) What is the origin of the continuous component of the electrical dark noise of rods? 5) What is the magnitude and power spectrum of the dark noise of cones, and how is the noise generated? 6) In the outer segment of a dark-adapted rod is there a longitudinal gradient of internal Na concentration? Is a Na gradient responsible for the different kinetics of transduction at the tip and base of the outer segment?

We propose to examine visual transduction in rods and cones of humans and Macaca fascicularis, a monkey with photoreceptors similar to those of man. We will record the photocurrents of single cells to determine their response properties, electrical noise, and spectral sensitivities. This experimental information will be used to develop a quantitative picture of the way in which primate photoreceptors encode visual stimuli. We intend to approach the following questions: 1) What are the kinetics of the photocurrents of rods and the three kinds of cone? 2) How is intensity information represented? 3) How much, and over what range of intensities do background lights desensitize? 4) What electrical noise is present in darkness and after exposure to bright (bleaching) light? 5) What is the spectral sensitivity of the rods and the three kinds of cone throughout the visible region of the spectrum? 6) Do the functional parameters of the transduction mechanism vary systematically with longitudinal position in the outer segments of the rods and cones? 7) Are primate photoreceptors electrically coupled?

We propose to study visual signalling in the mammalian retina. One group of experiments will focus on visual transduction, while a second will focus on the neural circuits over which transduced signals flow to the retinal ganglion cells. The experiments on visual transduction will examine the still poorly- understood processes that terminate the response to light. By making electrical recordings from transgenic mouse rods as well as normal primate photoreceptors we will ask: 1. Must rhodopsin be phosphorylated in the C terminal region for its catalytic activity to shut off normally in vivo? When does phosphorylation occur? Is phosphorylation at a single site sufficient to elicit complete shutoff? 2. Must arrestin bind to phosphorylated rhodopsin to complete shut-off? When does arrestin bind? How much does phosphorylation alone reduce catalytic activity? 3. Does the Ca-binding protein recoverin, which regulates rhodopsin shutoff, make the single photon response reproducible, mediate the gain reduction that occurs in background light, or both? 4. What molecular defect in rhodopsin is responsible for the anomalously- prolonged single photon responses that occur in normal primate rods? Is older rhodopsin more likely to be defective? 5. What are the dynamics of intracellular Ca in primate cones, and how do they relate to the diphasic waveform of the cone flash response? The experiments on neural circuitry will use a multielectrode array to record from ganglion cells in isolated primate retinas. We will ask: 6. How do the receptive fields of populations of ganglion cells sample visual images? 7. Do ganglion cells in the primate retina undergo concerted firing or do they only signal independently? If concerted firing is present, what cells are involved, over what length scale are signals correlated, and how does concerted firing contribute to the detection of contrast and color? 8. What is the contribution to ganglion cell receptive fields of electrical coupling between photoreceptors and horizontal cell feedback on photoreceptors? 9. Are the cones themselves the dominant site of chromatic adaptation in the retina, or are downstream cells involved? Can adaptation of one cone class change the gain of signals from another cone class?