Denis A. Baylor

Receptive fields of cones in the retina of the turtle

1. Intracellular recordings have been made of the responses to light of single cones in the retina of the turtle. The shape of the hyperpolarizing response to a flash depends on the pattern of retinal illumination as well as the stimulus intensity.

The photocurrent, noise and spectral sensitivity of rods of the monkey Macaca fascicularis.

Visual transduction in rods of the cynomolgus monkey, Macaca fascicularis, was studied by recording membrane current from single outer segments projecting from small pieces of retina. Light flashes evoked transient outward‐going photocurrents with saturating amplitudes of up to 34 pA. A flash causing twenty to fifty photoisomerizations gave a response of half the saturating amplitude. The response‐stimulus relation was of the form 1‐e‐x where x is flash strength. The response to a dim flash usually had a time to peak of 150‐250 ms and resembled the impulse response of a series of six low‐pass filters. From the average spectral sensitivity of ten rods the rhodopsin was estimated to have a peak absorption near 491 nm. The spectral sensitivity of the rods was in good agreement with the average human scotopic visibility curve determined by Crawford (1949), when the human curve was corrected for lens absorption and self‐screening of rhodopsin. Fluctuations in the photocurrent evoked by dim lights were consistent with a quantal event about 0.7 pA in peak amplitude. A steady light causing about 100 photoisomerizations s‐1 reduced the flash sensitivity to half the dark‐adapted value. At higher background levels the rod rapidly saturated. These results support the idea that dim background light desensitizes human scotopic vision by a mechanism central to the rod outer segments while scotopic saturation may occur within the outer segments. Recovery of the photocurrent after bright flashes was marked by quantized step‐like events. The events had the properties expected if bleached rhodopsin in the disks occasionally caused an abrupt blockage of the dark current over about one‐twentieth of the length of the outer segment. It is suggested that superposition of these events after bleaching may contribute to the threshold elevation measured psychophysically. The current in darkness showed random fluctuations which disappeared in bright light. The continuous component of the noise had a variance of about 0.03 pA2 and a power spectrum that fell to half near 3 Hz. A second component, consisting of discrete events resembling single‐photon responses, was estimated to occur at a rate of 0.006 s‐1. It is suggested that the continuous component of the noise may be removed from scotopic vision by a thresholding operation near the rod output.

Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1.

Timely deactivation of the α-subunit of the rod G-protein transducin (Gαt) is essential for the temporal resolution of rod vision. Regulators of G-protein signalling (RGS) proteins accelerate hydrolysis of GTP by the α-subunits of heterotrimeric G proteins in vitro. Several retinal RGS proteins can act in vitro as GTPase accelerating proteins (GAP) for Gαt. Recent reconstitution experiments indicate that one of these, RGS9-1, may account for much of the Gαt GAP activity in rod outer segments (ROS). Here we report that ROS membranes from mice lacking RGS9-1 hydrolyse GTP more slowly than ROS membranes from control mice. The Gβ5-L protein that forms a complex with RGS9-1 (ref. 10) was absent from RGS9-/- retinas, although Gβ5-L messenger RNA was still present. The flash responses of RGS9-/- rods rose normally, but recovered much more slowly than normal. We conclude that RGS9-1, probably in a complex with Gβ5-L, is essential for acceleration of hydrolysis of GTP by Gαt and for normal recovery of the photoresponse.

The electrical response of turtle cones to flashes and steps of light

1. The linear response of turtle cones to weak flashes or steps of light was usually well fitted by equations based on a chain of six or seven reactions with time constants varying over about a 6‐fold range.

Visual transduction in cones of the monkey Macaca fascicularis.

1. Visual transduction in macaque cones was studied by measuring the membrane current of single outer segments projecting from small pieces of retina. 2. The response to a brief flash of light was diphasic and resembled the output of a bandpass filter with a peak frequency near 5 Hz. After the initial reduction in dark current there was a rebound increase which resulted from an increase in the number of open light‐sensitive channels. The response to a step of light consisted of a prominent initial peak followed by a steady phase of smaller amplitude. 3. Responses to dim light were linear and time‐invariant, suggesting that responses to single photons were linearly additive. From the flash sensitivity and the effective collecting area the peak amplitude of the single photon response was estimated as about 30 fA. 4. With flashes of increasing strength the photocurrent amplitude usually saturated along a curve that was gentler than an exponential but steeper than a Michaelis relation. The response reached the half‐saturating amplitude at roughly 650 photoisomerizations. 5. The response‐intensity relation was flatter in the steady state than shortly after a light step was turned on, indicating that bright light desensitized the transduction with a delay. This desensitization was not due to a reduction in pigment content. In the steady state, a background of intensity I lowered the sensitivity to a weak incremental test flash by a factor 1/(1 + I/IO), where IO was about 2.6 x 10(4) photoisomerizations s‐1, or about 3.3 log trolands for the red‐ and green‐sensitive cones. 6. Bleaching exposures produced permanent reductions in flash sensitivity but had little effect on the kinetics or saturating amplitude of subsequent flash responses. The sensitivity reductions were consistent with the expected reductions in visual pigment content and gave photosensitivities of about 8 x 10(‐9) microns2 (free solution value) for the red‐ and green‐sensitive pigments. During a steady bleaching exposure the final exponential decline of the photocurrent had a rate constant given by the product of the light intensity and the photosensitivity. 7. In some cells it was possible to measure a light‐induced increase in current noise. The power spectrum of the noise resembled the spectrum of the dim flash response and the magnitude of the noise was consistent with a single photon response roughly 20 fA in size. 8. The membrane current recorded in darkness was noisy, with a variance near 0.12 pA2 in the band 0‐20 Hz.(ABSTRACT TRUNCATED AT 400 WORDS)

Two components of electrical dark noise in toad retinal rod outer segments.

1. Physiological noise in the visual transduction mechanism was studied by recording membrane current from single rod outer segments in pieces of isolated toad retina. 2. The inward current in darkness showed spontaneous fluctuations which disappeared during the response to bright light. 3. The dark noise consisted of two components, a continuous fluctuation of rms amplitude about 0.2 pA and occasional discrete events about 1 pA in size. 4. Intervals between discrete events followed the exponential distribution expected of a Poisson process with a mean rate of about one event per 50 sec (20 degrees C). 5. The amplitude and power spectrum of the discrete events resembled those of single photon effects in the same rod, suggesting that discrete events may arise from spontaneous activation of single rhodopsin molecules. 6. The temperature dependence of the mean frequency of occurrence of discrete events gave an activation energy of 22 kcal mole‐1, probably characteristic of thermal isomerization of rhodopsin. 7. The variance of the continuous component of the dark noise rose linearly with the length of the outer segment drawn into the suction electrode, indicating that this component is generated in the outer segment. 8. The power spectrum of a rods continuous noise was usually fitted by the square of a Lorentzian with the same time constant as that of the four first‐order delays in the cells single photon response. The shot effects composing the continuous component thus appear to be shaped by two of four sequential processes in transduction. 9. The variance and spectrum of the continuous noise are interpreted to reflect shot effects about 1/400 the size of a single photon effect occurring at a frequency of 6 x 10(3) sec‐1. 10. The rods flash sensitivity was halved by a steady light to giving about 8 photoisomerizations sec‐1. The much lower mean rate of discrete events indicates that Io in increment sensitivity experiments on individual receptors is not set by thermal activation of rhodopsin. 11. Values of sensitivity and time‐to‐peak flash response collected from many cells in darkness were correlated by the same power law relation obtaining in the presence of backgrounds. The correlation observed would be explained if a single variable controlled both the gain and time scale of several stages of the transduction mechanism in background light and in darkness.

Multi-neuronal signals from the retina: acquisition and analysis

Throughout the central nervous system, information about the outside world is represented collectively by large groups of cells, often arranged in a series of 2-dimensional maps connected by tracts with many fibers. To understand how such a circuit encodes and processes information, one must simultaneously observe the signals carried by many of its cells. This article describes a new method for monitoring the simultaneous electrical activity of many neurons in a functioning piece of retina. Extracellular action potentials are recorded with a planar array of 61 microelectrodes, which provides a natural match to the flat mosaic of retinal ganglion cells. The voltage signals are processed in real time to extract the spike trains from up to 100 neurons. We also present a method of visual stimulation and data analysis that allows a rapid characterization of each neurons visual response properties. A randomly flickering display is used to elicit spike trains from the ganglion cell population. Analysis of the correlations between each spike train and the flicker stimulus results in a simple description of each ganglion cells functional properties. The combination of these tools will allow detailed study of how the population of optic nerve fibers encodes a visual scene.

Concerted Signaling by Retinal Ganglion Cells

To analyze the rules that govern communication between eye and brain, visual responses were recorded from an intact salamander retina. Parallel observation of many retinal ganglion cells with a microelectrode array showed that nearby neurons often fired synchronously, with spike delays of less than 10 milliseconds. The frequency of such synchronous spikes exceeded the correlation expected from a shared visual stimulus up to 20-fold. Synchronous firing persisted under a variety of visual stimuli and accounted for the majority of action potentials recorded. Analysis of receptive fields showed that concerted spikes encoded information not carried by individual cells; they may represent symbols in a multineuronal code for vision.

Rapid and Reproducible Deactivation of Rhodopsin Requires Multiple Phosphorylation Sites

Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.

Reconstruction of the electrical responses of turtle cones to flashes and steps of light

1. Theoretical equations which predict the electrical response of turtle cones to a wide range of light stimuli are developed from the experiments described in previous papers.