Test Flash Research Articles

Cellular mechanisms that underlie bleaching and background adaptation.

Experiments were performed on rod photoreceptors isolated from the eye of the larval tiger salamander to determine if the same or different mechanisms underlie the desensitization produced by dim background light (background adaptation) and that which persists in the steady state in darkness after a significant fraction of the photopigment is bleached (bleaching adaptation). We have examined adaptational effects after light that bleached between approximately 50% and 95% of the photopigment under conditions which preclude pigment regeneration. The steady-state desensitization, far greater than that predicted by quantum-catch loss, is relieved upon regeneration of the visual pigment with 11-cis retinal. We measured the spread of desensitization along the long axis of the rod after a local bright bleach at one end by comparing responses to dim local test flashes elicited in different regions of the outer segment, before and after bleaching. The space constant for this spread was less than 2.5 microns. We have previously measured the space constant for the longitudinal spread of desensitization during a local dim background in Ambystoma rods to be 7 microns. This is similar to a space constant of 6 microns measured under similar conditions in Bufo rods by Lamb et al. (1981. J. Physiol. 319:463-496). If calcium carries the signal for background desensitization, this difference in space constant for background and bleaching adaptation precludes it as the messenger for the steady component of bleaching adaptation. Experiments with isobutylmethyl xanthine (IBMX) also indicate that Ca2+ as well as c-GMP are unlikely regulators of bleaching desensitization, since elevation of cytosolic levels of both of these internal messengers by IBMX has little effect on sensitivity in bleach-adapted cells. All of our findings are consistent with the notion that bleaching adaptation is not mediated by a freely diffusible cytoplasmic messenger.

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)

Rod suppression of cone‐mediated information about colour and form during dark adaptation

Following substantial bleaches, the specific form and hue thresholds were measured during dark adaptation with a test stimulus of 1 x 2 degrees at 40 degrees extrafoveally. The wavelength of the test field was varied between runs. The results show that both thresholds started to rise at about the cone-rod break of the dark-adaptation curve, irrespective of wavelength used in the test. Furthermore, the specific threshold for form was found to rise when a scotopic stimulus was superimposed on a photopic test flash. On the other hand, both thresholds remained at the cone-plateau level when the test flash was confined within the rod-free fovea. In order to explain the rise in the specific thresholds, it is suggested that signals from rods generated directly in response to the test stimulus may suppress both cone-mediated form and colour. It is also suggested that this type of rod-cone interaction represents a general characteristic involved in several kinds of visual information processing.

Microvillar components of light adaptation in blowflies.

The process of light adaptation in blowfly photoreceptors was analyzed using intracellular recording techniques and double and triple flash stimuli. Adapting flashes of increasing intensity caused a progressive reduction in the excitability of the photoreceptors, which became temporarily suppressed when 3 x 10(6) quanta were absorbed by the cell. This suppression was confirmed by subsequently applying an intense test flash that photoactivated a considerable fraction of the 10(8) visual pigment molecules in the cell. The period of temporary desensitization is referred to as the refractory period. The stimulus intensity to render the receptor cell refractory was found to be independent of the extracellular calcium concentration over a range of 10(-4) and 10(-2) M. During the refractory period (30-40 ms after the adapting flash) the cell appears to be "protected" against further light adaptation since light absorption during this period did not affect the recovery of the cell's excitability. Calculations showed that the number of quantum absorptions necessary to induce receptor refractoriness is just sufficient to photoactivate every microvillus of the rhabdomere. This coincidence led to the hypothesis that the refractoriness of the receptor cells is due to the refractoriness of the individual microvilli. The sensitivity of the receptor cells after relatively weak adapting flashes was reduced considerably more than could be accounted for by the microvilli becoming refractory. A quantitative analysis of these results suggests that a photoactivated microvillus induces a local adaptation over a relatively small area of the rhabdomere around it, which includes several tens of microvilli. After light adaptation with an intense flash, photoactivation of every microvillus by the absorption of a few quanta produced only a small receptor response whereas photoactivation of every rhodopsin molecule in every microvillus produced the maximum response. The excitatory efficiency of the microvilli therefore increases with the number of quanta that are absorbed simultaneously.

Evidence for light-induced release of Ca 2+ from intracellular stores in bee photoreceptors

In the drone retina light elicits an increase in the extracellular Ca 2+ concentration ([Ca] o). After withdrawal of extracellular Ca 2+ and addition of 1 mM EGTA (ethyleneglycol-bis (β-aminoethyl ether) N, N, N′, N′-tetraacetic acid) a short light flash still caused an increase in [Ca] o as measured with Ca 2+-sensitive microelectrodes. This increase vanished after a few flashes. When [Ca] o was reduced to 10 −5 M Ca 2+ (no EGTA) the rise in Ca o disappeared after several light flashes. Subsequent stimulation with a dim steady light produced a decrease in [Ca] o and caused a recovery of the increase in [Ca] o elicited by a test flash. The results show that bee photoreceptors have a light-depletable intracellular Ca 2+ store which can be reloaded by dim continuous lights.

The slope of the psychometric function at different wavelengths

Many current models of visual detection predict that the slope of psychometric functions for detection will be independent of the spectral power distribution of the test light once the spatial and temporal characteristics of the light are fixed. This article examines reports that the slope of the psychometric function depends on the wavelength of a spectrally-narrowband test light of fixed size and duration. Three sources of difficulty that any experimental measurement of slope must encounter are addressed: (1) the different spatial distributions of the photoreceptor classes across the retina; (2) possible variations in threshold over the course of the experiment; and (3) the large variability of estimates of slope and the need for a method of assessing this variability. Measurements of slope with 2 and 4 deg test flashes against a bright 510 nm field show no significant trend with the wavelength of the test. A novel statistical test bounds the magnitude of possible variations in slope across the visible spectrum.

Double-flash electroretinography in human eyes.

A tri-color Ganzfeld stimulator with light-emitting diodes as light sources is used to study the suppression of the second electroretinographic response in human eyes to double-flash stimulation. The mechanism suppressing the a- and b-waves of the response to the second (test) flash has a scotopic spectral sensitivity to the first (conditioning) flash. Responses to mild test stimuli are more sensitive to suppression by a conditioning flash than responses to strong test stimuli.

Nonlinearity and facilitation in phosphoinositide signaling studied by the use of caged inositol trisphosphate in Xenopus oocytes

The phosphoinositide signaling pathway, which mediates neurotransmitter responses, was studied in Xenopus oocytes by recording membrane currents evoked using lightflash photolysis of caged inositol trisphosphate (caged IP3) to produce rapid and reproducible transients in intracellular IP3 levels. Photolysis of caged IP3 evoked currents which were carried largely by chloride ions and depended upon intracellular, but not extracellular, calcium. A given light flash evoked larger responses when the amount of caged IP3 loaded into the oocyte was increased, and illumination of the vegetal hemisphere gave larger responses than the animal. Long (10 sec) light exposures produced oscillatory currents, resembling responses to serotonin and other agonists, which became larger, more transient, and of shorter latency as the light intensity was increased. Brief (ca. 100 msec) flashes evoked a single "spike" of current. The caged IP3 response showed a threshold, in that light flashes had to be greater than a certain intensity and duration before currents could be detected. Associated with this, sub- and suprathreshold light flashes caused a long-lasting (seconds or minutes) potentiation of responses to subsequent test flashes. The lightflash response was also potentiated by a preceding intracellular injection of IP3 and by extracellular application of an agonist thought to induce IP3 liberation. However, intracellular injections of calcium depressed the response. We conclude that the liberation of calcium from intracellular stores varies nonlinearly with the intracellular level of IP3. This phenomenon may explain earlier observations, including the long latency of currents evoked by low doses of agonists such as acetylcholine and serotonin, and the nonlinear facilitation seen between these agonists. Further, it suggests a mechanism for "chemical integration," which may be important in the functioning of neurons and other cells which use IP3 as an intracellular messenger.

Cytoplasmic calcium as the messenger for light adaptation in salamander rods.

1. In order to study the role of cytoplasmic calcium concentration (Ca2+i) in rod photoreceptor light adaptation, we have attempted to prevent light-induced changes in Ca2+i by minimizing calcium fluxes across the outer segment plasma membrane. This was achieved by exposing the outer segment to a low-Ca2+, 0-Na+ solution, in which sodium was replaced with either guanidinium or lithium and the external calcium concentration (Ca2+o) was reduced to micromolar levels. 2. With guanidinium and 1-3 microM-Ca2+o, the circulating current in darkness was maintained for a period of at least 15 s, consistent with approximate stability of Ca2+i. With Li+ rather than guanidinium most of the initial current was suppressed, but the residual current was again relatively stable. 3. During prolonged exposures (greater than 30 s) to low-Ca2+, 0-Na+ solution followed by dim illumination, the circulating current did not remain constant but slowly increased. Incorporation of calcium buffer into the cytoplasm greatly reduced the rate of change of current, consistent with the idea that the increase arose from a gradual decrease in Ca2+i. 4. Light responses of rods exposed to low-Ca2+, 0-Na+ solution in darkness were altered in a characteristic manner. Although the initial rising phase of the light response was little changed, the peak amplitude of the response was larger and occurred later, and the response decayed more slowly than in control. The response-intensity relation was steepened and was shifted towards lower intensities both for flashes and for steps of light. The normal sag in the response to steps disappeared, and the waveform of the step response could be predicted to a close approximation from the integral of the dim flash response. 5. Presentation of background illumination in Ringer solution produced a marked acceleration of the response to a subsequent bright flash. No such acceleration was observed if the background was given in low-Ca2+, 0-Na+ solution. 6. The results described in paragraphs 4 and 5 indicate that, under conditions expected to minimize changes in Ca2+i, all manifestations of light adaptation disappear, and the rod simply sums the effects of incident photons with an invariant integration time. 7. Exposure of a light-adapted rod to low-Ca2+, 0-Na+ solution altered the responses to superimposed test flashes in much the same way as for rods in darkness. The initial rising phases in low-Ca2+, 0-Na+ solution were unchanged, but the responses were larger, reached peak later and decayed more slowly.(ABSTRACT TRUNCATED AT 400 WORDS)

Absence of visual sampling in infantile nystagmus.

The possibility that patients with infantile nystagmus achieve spatial constancy by sampling the visual scene only during certain range of velocities or certain phases of their nystagmus cycle was investigated by asking patients to detect a flashed test target that was presented repeatedly during all phases of the nystagmus cycle. After observing a 543 nm fixation spot projected on a diffusely illuminated tangent screen 1 m in front of the eye, patients were asked to detect a 2 msec test flash of the spot, now locked to the retina, that occurred 200 msec after the fixation spot was extinguished. The test target appeared randomly at 3,6,9, or 12 o'clock at, in separate trials, 0.8 deg or 10.0 deg from central vision. To avoid forcing patients from guessing a direction cued by the disappearance of the fixation spot, sometimes the flash did not occur at all. To avoid dark adaptation in our patients, the diffuse background illumination was adjusted to the brightest level allowing easy detection of the test flash. In every condition, all patients reported the fixation spot stationary. The probability of detection was the same across all velocity ranges. Remarkably, patients were just as likely to detect the test flash when the eye was nearly stationary even when it was moving more than 100 deg/sec. In one patient, the background illumination was raised so that he began missing the test flash more often. Here too, the probability of detection, now reduced to about 50%, was the same across velocity ranges.(ABSTRACT TRUNCATED AT 250 WORDS)

Saturation of rod-initiated signals in 2-month-old human infants.

Absolute and increment thresholds were measured for 2-month-old human infants by using long-wavelength adapting fields and short-wavelength test flashes. The test flashes were 500 or 10 msec in duration and varied from 6 degrees to 16 degrees in diameter. The psychometric functions measured in the dark were steep for large, long-duration test flashes and shallow for small, brief flashes. Under conditions of light adaptation, the psychometric functions were consistently steep. The shape of the area-threshold function depended on the criterial percent correct responses; when a criterion of 75% correct responses was used, the slope of the area-threshold function was much steeper than predicted by Ricco's law. These results are interpreted in the context of a model in which rod-initiated signals saturate at a locus proximal to the rods themselves but distal to most of the considerable spatial pooling of visual signals that occurs in infants.

Long-term variability in the spectral loci of unique blue and unique yellow.

The spectral loci of both unique blue and unique yellow were measured over a 16-month period. Using the method of constant stimuli, two neutrally adapted observers made forced-choice green or red responses to monochromatic test flashes. Results showed a consistent difference between observers (about 5 nm), considerable within-subject variability (standard deviation 3 nm), and significant long-term drifts (greater than 5 nm) in spectral loci. These results demonstrate the importance of frequently redetermining unique-hue loci when they are used as baselines in chromatic contrast and adaptation studies.

Melatonin enhances horizontal cell sensitivity in salamander retina

Intracellular electrophysiological recording techniques were utilized to investigate the possible function of retinal melatonin in the larval tiger salamander. Endogenous retinal melatonin was present and appeared to bind to a membrane-enriched fraction of the salamander retina, as determined by radioimmunoassay and receptor binding studies. Melatonin added through the purfusion bath to flat-mounted retinas resulted in a horizontal cell(HC) hyperpolarization of 10–20 mV. Additionally, the amplitude of HC responses to short test flashes increased in the presence of melatonin. Voltage-intensity plots revealed that application of 500 μM of melatonin caused an increase of the HC light sensitivity and this effect was reversible. These results suggest that melatonin synthesized and released during the dark period of the diurnal cycle may alter the sensitivity of second-order neurons at a time of day when photopic input is at its lowest level.

The directional sensitivity of the photoreceptors in the human achromat.

1. The anatomical nature of the retinal photoreceptors in a typical, complete achromat was investigated by measuring their directional sensitivity. 2. A small (0.5 deg), brief (94 ms) test flash was placed at threshold by varying either its intensity (indirect method) or the intensity of a large (5 deg) adapting field (direct method). The dependent variable was the intensity of light required for the detection threshold as a function of the position of entry in the pupil. An infra-red viewing system was used to monitor the achromat's pupil and eye position. 3. At both scotopic and mesopic adapting field luminances, the complete achromat's receptors displayed only a small directional sensitivity effect. The effect was not wavelength dependent and could be attributed solely to the rods. 4. The results are consistent with other psychophysical evidence indicating that the complete achromat's retinae lack the post-receptoral function of the cone photoreceptors. Therefore they do not confirm previous reports that complete achromats have a second, high-intensity type of photoreceptor.

Theoretical interpretation and derivation of flash-on-flash threshold parameters in visual system diseases

Visual increment thresholds measured on backgrounds flashed on simultaneously with the test flash exhibit saturation rather than following Weber's law. These flash-on-flash thresholds have been modeled with saturating nonlinearities similar to those used to describe intensity-response functions of retinal neurons. Recently, the flash-on-flash technique has been used to assess the mechanisms of threshold elevations in visual system diseases. However, the results were interpreted in a qualitative way. This paper extends the clinical application of flash-on-flash threshold measures by providing a theoretically derived algorithm for estimating nonlinear model parameters from flash-on-flash threshold data. The performance of the algorithm is evaluated with Monte Carlo techniques, and the theoretical interpretations of the model parameters are tested experimentally. Published clinical flash-on-flash threshold data are reanalyzed using the algorithm. A previously unappreciated decrease in the half-saturation constant of retinitis pigmentosa patients was revealed by this analysis.

Perceptual latencies are shorter for motion towards the fovea than for motion away

Subjects had to align a brief test flash with a peripheral steady reference target during ocular pursuit, or with a moving reference target during steady eye fixation. The performance was determined by the direction of movement, of the reference target image on the retina—towards or away from fovea. The data are explained by the hypothesis that the passage of a retinal locus by a moving target image is experienced faster when the image moves foveopetally, regardless of whether the passage is caused by real target movement or by ocular pursuit.

Improvement in human vision under bright light: grain or gain?

1. The factor by which increment threshold changes with changing background intensity is less if the test flash is small than if it is large. This is commonly attributed to a reduction of the area over which visual signals are integrated as light adaptation increases. 2. We propose and test an alternative hypothesis that the change in slope is the result of purely local processes: if it is assumed that increasing the background intensity increases the exponent of the local response function, but does not alter the extent of spatial integration, then the threshold of the small test flash will rise more slowly than the threshold of the large test flash simply because the small test flash is of a higher intensity than the large and therefore evokes a correspondingly greater local response. 3. We measured small and large test field increment thresholds and dichoptic brightness matches as a function of background intensity. 4. The log-log slopes of the small and large field increment threshold functions differed by not more than about 20%, suggesting that even under the conventional interpretation of such data, the change of spatial integration is less than is usually supposed. 5. The intensity of a large (2.3 deg) suprathreshold test field matched to a standard in the other eye varies with increasing background intensity with the same shallow slope as the small test (2.6 min) threshold versus intensity function; this is in agreement with the predictions of the local non-linearity hypothesis and suggests that there is no substantial change in spatial integration during light adaptation.

VARIATION OF SACCADIC SUPPRESSION WITH TARGET ECCENTRICITY

Two LEDs separated by 24 degrees from each other were employed as saccadic fixation sources located on a circle about the centre of the saccade path. Either horizontal, vertical or oblique saccade paths were used and a brief test flash was presented at one of five retinal eccentricities during each saccade. The percentage of detected flashes was measured as a function of the location of the test stimulus and of its time delay with respect to the onset of the saccade. The results suggest that saccadic suppression is maximal at the fovea and is significantly decreased as retinal eccentricity increases.

Rod influence on thresholds using different detection criteria during dark adaptation

Following adaptation to intense long wavelength (LW) light, dark adaptation was measured with an extrafoveal 660 nm test flash using three different criteria: reporting the test flash as present (detection threshold), as colored (the chromatic threshold), and as red (the color-specific threshold). The detection threshold followed the typical dark adaptation course. The chromatic threshold first fell to a steady level but then rose to a higher level. The color-specific threshold first dropped, and then rose. These threshold rises are attributed to the action of rods since they do not occur in the fovea and are delayed following adaptation to short wavelength light of equal photopic, and stronger scotopic, intensity to the LW light adaptation.

Field additivity of Stiles's Pi-4 color mechanism.

Stiles's middle-wave color mechanism Pi-4 is field additive for mixtures of 410-, 480-, 530-, 622-, and 670-nm adaptation fields, when studied with a 1-deg, 200-msec, 500-nm foveal test flash at 10 times absolute threshold.