Photopigment Bleaching Research Articles

Contrast threshold recovery after bleaching during cone‐mediated mesopic adaptation: The effect of test conditions

Aims/Purpose: To determine the optimal luminance conditions and the staircase procedures for evaluating the time course of macular contrast sensitivity recovery after photobleaching in order to decide the best protocol to assess cone‐mediated mesopic adaptation.Methods: The dynamics of contrast threshold (CT) recovery following photopigment bleach were measured psychophysically for five minutes in twenty‐four healthy adults (23.7 ± 3.7 years old). The stimuli used were sine‐wave gratings of low‐spatial frequency (1 cycle‐per‐degree). In each subject, six tests were performed, combining three luminance levels (0.1, 1.0, and 10 cd/m2) and two modified staircase procedures (3‐down/1‐up and 2‐down/1‐up) to estimate CTs. The test order was randomized, conducted in two visits, with a 15‐min washout period between tests. Outlier detection techniques were applied to verify the integrity of the sample. Non‐linear exponential‐based optimization was adopted to fit CT recovery functions to estimate the time constant (τ, seconds) of cone sensitivity recovery and the final CT (CTf, log units). Classical and heteroscedastic ANOVA analyses were performed to assess the effect of test conditions.Results: The 2‐down/1‐up staircase allowed for denser point sampling of CTs measurements than the 3‐down/1‐up. Under the lowest luminance test condition, the CT recovery was modelled by bumpier functions, for both staircases procedures, than the other luminance levels. Mean cone τ increased (21.7, 42.5, and 72.0 seconds (p < 0.001)) and CTf worsened (−1.99, −1.85, and − 1.47 log units (p < 0.001)) as luminance level decreased. The best fit (median R2 = 0.87) was achieved under 1 cd/m2 mesopic luminance conditions. There were no statistically significant differences in mean cone τ and CTf between the two staircases procedures.Conclusions: An intermediate mesopic light level and a 2‐down/1‐up staircase provided the best protocol for assessing cone‐mediated mesopic adaptation.

Development of a cone‐mediated dark adaptation test on a smartphone

Aims/Purpose: Dark adaptometry is considered a useful tool for investigating retinal neurodegenerative diseases. While it has been demonstrated that the loss of rod photoreceptors occurs before the loss of cones, foveal cones are also impacted during the initial stages of age‐related macular degeneration. Our aim was to develop a test to assess cone‐mediated dark adaptation (DA) based on the MOBILE‐DA smartphone app.Methods: The MOBILE‐DA smartphone app for testing rod‐mediated DA was modified to assess cone‐mediated DA. Using an Android smartphone, and a 3° diameter achromatic flashing stimulus centered on the fovea, the measurements were made using an adaptive staircase of 0.007 cd/m2 level up – 0.009 cd/m2 level down. In a dark room, the smartphone was placed 40 cm from the subject, and the luminance thresholds were measured monocularly for 5 min following almost complete photopigment bleaching using a long‐duration light exposure (60 seconds). Luminance Threshold (LT) recovery functions were fitted to an exponential decay model to determine the time constant (τ, seconds) of cone sensitivity recovery and final cone LT.Results: The cone‐mediated DA measured by the smartphone app resulted in τ of 91 ± 15 seconds; final cone LT of −2.29 ± 0.10 log cd/m2, and R2 of 0.96 ± 0.02. The time constant and final threshold values obtained through this app were comparable to those obtained through other established techniques.Conclusions: The smartphone app for evaluating cone‐mediated dark adaptation proved effective in tracking the rapid changes in luminance threshold that take place during this process. These findings suggest that the MOBILE‐DA app is well‐suited for measuring cone‐mediated dark adaptation.

Diagnosis and management of transient smartphone blindness: A literature review

The development of smartphones worldwide has rapidly increased the number of smartphone users. Although smartphones have many benefits, prolonged use causes various visual disorders, of which transient smartphone blindness (TSB) is one of the most important concerns. This study aims to analyse the phenomenon of TSB and explore its etiology, pathophysiology, symptoms, and examination to determine the diagnosis and management. Transient smartphone blindness is a new physiologic phenomenon caused by exposure to a certain level of smartphone bright light and the body posture of the smartphone user. It leads to the use of monocular vision to stare at the smartphone. By exploring recent literature, this study found that patients with TSB experienced retinal photopigment bleaching, which decreased light sensitivity. Typically, the sensitivity returned physiologically after several minutes. Patients with this blindness showed several symptoms. For example, they perceived shapes, outlines, or figures during the blackout. The diagnosis of TSB should be primarily based on a detailed history of smartphone use, with the differential diagnosis based on circumstance or occurrence. This diagnosis is useful to distinguish it from transient ischemic attack (TIA). Transient smartphone blindness is a basic monocular transient loss that does not require therapy or drugs. However, it was found that the reassurance of patients’ condition and suggestions on smartphone use were sufficient for the intervention. With a limited scientific study of TSB, this review is the first to raise the discussion topic. It is expected to help physicians to diagnose TSB significantly. In addition, this study will enrich the literature, particularly on the scientific and clinical mechanisms of recognising TSB for clinical diagnosis and management.

Contributed Session I: Human cone response models for optoretinography with FF-SS-OCT and adaptive optics.

Recent work has shown that human rod and cone outer segments (ROS and COS, respectively) deform in response to visual stimuli. In phase-based optoretinography (ORG) the phases of backscattered light from the inner/outer segment junction (IS/OS) and the COS/ROS tips (COST/ROST), is measured, which allows observation of stimulus-evoked, nanometer-scale changes in the OS length. In this work, we used a full-field swept-source OCT with AO that allowed up to kHz volume rates. ORG responses were recorded in two healthy volunteers, with photopigment bleaching levels in the range of 1-60 %, and modeled using an exponential sum. The proposed harmonic oscillator-based response model allowed us to describe the shape of the cone's ORG responses by amplitudes of deflection and relaxation times. Our preliminary results show that responses to complex stimuli were consistent with photopigment availability, which in the context of the consensus theory that adaptation in cones is mediated by photopigment suggests that the ORG may be a useful way to probe light adaptation in cones. The development of simple quantitative parameters describing the ORG response should benefit future clinical applications and help to track the progress of blinding diseases.

Fundus autofluorescence imaging using red excitation light

Retinal disease accounts significantly for visual impairment and blindness. An important role in the pathophysiology of retinal disease and aging is attributed to lipofuscin, a complex of fluorescent metabolites. Fundus autofluorescence (AF) imaging allows non-invasive mapping of lipofuscin and is a key technology to diagnose and monitor retinal disease. However, currently used short-wavelength (SW) excitation light has several limitations, including glare and discomfort during image acquisition, reduced image quality in case of lens opacities, limited visualization of the central retina, and potential retinal light toxicity. Here, we establish a novel imaging modality which uses red excitation light (R-AF) and overcomes these drawbacks. R-AF images are high-quality, high-contrast fundus images and image interpretation may build on clinical experience due to similar appearance of pathology as on SW-AF images. Additionally, R-AF images may uncover disease features that previously remained undetected. The R-AF signal increases with higher abundance of lipofuscin and does not depend on photopigment bleaching or on the amount of macular pigment. Improved patient comfort, limited effect of cataract on image quality, and lack of safety concerns qualify R-AF for routine clinical monitoring, e.g. for patients with age-related macular degeneration, Stargardt disease, or for quantitative analysis of AF signal intensity.

IMPAIRMENTS IN CONE PIGMENT REGENERATION AND ABSOLUTE THRESHOLD IN MACULAR TELANGIECTASIA TYPE 2.

To test the hypothesis that Müller cell dysfunction in macular telangiectasia type 2 (MacTel) results in delayed cone adaptation kinetics and to assess absolute cone and rod thresholds in this condition. Adaptation after an approximate 63.5% full-field cone photopigment bleach was assessed for Goldmann size V (1.7° diameter) 640 nm (red) and 480 nm (blue) targets presented at a retinal locus corresponding to 2° temporal to fixation. The cone time constant of adaptation and absolute cone and rod thresholds were calculated from exponential functions fitted to the resultant dark adaptation curves. Eighteen eyes with MacTel (from 11 patients) were compared with 19 control eyes (from 16 normal subjects). Cone adaptation kinetics were significantly impaired in MacTel, as was the absolute cone threshold. Final thresholds for blue targets were also significantly elevated in MacTel, consistent with impaired rod absolute threshold. Losses in sensitivity observed in MacTel were consistent with a so-called d1/2 mechanism (i.e., receptoral) site of sensitivity loss. In addition to previously documented impairments in rod dark adaptation, MacTel results in a significant elevation in cone thresholds because of pathology at the level of the photoreceptors. The delays in cone adaptation that we found in eyes with MacTel may reflect impairment of the Müller cell-mediated cone-specific visual cycle.

Light adaptation characteristics of melanopsin

Following photopigment bleaching, the rhodopsin and cone-opsins show a characteristic exponential regeneration in the dark with a photocycle dependent on the retinal pigment epithelium. Melanopsin pigment regeneration in animal models requires different pathways to rods and cones. To quantify melanopsin-mediated light adaptation in humans, we first estimated its photopigment regeneration kinetics through the photo-bleach recovery of the intrinsic melanopsin pupil light response (PLR). An intense broadband light (~120,000 Td) bleached 43% of melanopsin compared to 86% of the cone-opsins. Recovery from a 43% bleach was 3.4X slower for the melanopsin than cone-opsin. Post-bleach melanopsin regeneration followed an exponential growth with a 2.5 min time-constant (τ) that required 11.2 min for complete recovery; the half-bleaching level (Ip) was ~ 4.47 log melanopic Td (16.10 log melanopsin effective photons.cm−2.s−1; 8.25 log photoisomerisations.photoreceptor−1.s−1). The effect on the cone-directed PLR of the level of the melanopsin excitation during continuous light adaptation was then determined. We observed that cone-directed pupil constriction amplitudes increased by ~ 10% when adapting lights had a higher melanopic excitation but the same mean photometric luminance. Our findings suggest that melanopsin light adaptation enhances cone signalling along the non-visual retina-brain axis. Parameters τ and Ip will allow estimation of the level of melanopsin bleaching in any light units; the data have implications for quantifying the relative contributions of putative melanopsin pathways to regulate the post-bleach photopigment regeneration and adaptation.

The acute effect of cocoa and red-berries on visual acuity and cone-mediated dark adaptation in healthy eyes

• This study examines the acute effect of cocoa and red-berries polyphenols on retinal functions such as visual acuity (VA) and cone-mediated dark adaptation in healthy eyes. • Photopic visual acuity improved significantly after cocoa ingestion. • Mesopic or low luminance VA and dark adaptation was not affected by any of the two polyphenol diets. • The improvement in photopic visual acuity could be attributed to flavanols and Theobromine. The retina is a highly vascularized tissue with a high metabolic and oxygen demand responsible for human vision. Considering that the polyphenolic flavanols and anthocyanins have been shown to be beneficial for endothelial function and cerebral blood-flow, an acute randomized and controlled crossover trial with two different sources of polyphenols, anthocyanins from red-berries and flavanols from cocoa, was designed to better understand the effect of polyphenols on visual acuity (VA) and cone-mediated dark adaptation (DA). Thirty-seven healthy subjects (22.1 ± 2.0 years old) participated in the acute intervention for three times (red-berries, cocoa or vehicle-control) with a washout period of two weeks in-between. VA under photopic and low luminance (mesopic) conditions, DA or dynamic of recovery of contrast threshold (CT) following near-total photopigment bleach for 5 min, urine total polyphenols, theobromine and antioxidant power were measured in the three study-arms after 2-hours ingestion of the study-food. 3-hours postprandial urine showed higher levels of total polyphenols after ingestion of cocoa flavanols or red-berries anthocyanins in comparison with the vehicle-control and higher levels of theobromine only for the cocoa group. There was an increase in photopic VA with cocoa flavanols that with red-berries anthocyanins did not reach statistical significance. Both, cocoa and red berries, failed to improve mesopic VA and the cone time constant for contrast recovery and final CT of DA.

Functional Imaging of the Outer Retinal Complex using High Fidelity Imaging Retinal Densitometry

We describe a new technique, high fidelity Imaging Retinal Densitometry (IRD), which probes the functional integrity of the outer retinal complex. We demonstrate the ability of the technique to map visual pigment optical density and synthesis rates in eyes with and without macular disease. A multispectral retinal imaging device obtained precise measurements of retinal reflectance over space and time. Data obtained from healthy controls and 5 patients with intermediate AMD, before and after photopigment bleaching, were used to quantify visual pigment metrics. Heat maps were plotted to summarise the topography of rod and cone pigment kinetics and descriptive statistics conducted to highlight differences between those with and without AMD. Rod and cone visual pigment synthesis rates in those with AMD (v = 0.043 SD 0.019 min−1 and v = 0.119 SD 0.046 min−1, respectively) were approximately half those observed in healthy controls (v = 0.079 SD 0.024 min−1 for rods and v = 0.206 SD 0.069 min−1 for cones). By mapping visual pigment kinetics across the central retina, high fidelity IRD provides a unique insight into outer retinal complex function. This new technique will improve the phenotypic characterisation, diagnosis and treatment monitoring of various ocular pathologies, including AMD.

Bleaching effects and fluorescence lifetime imaging ophthalmoscopy.

This study investigates the influence of photopigment bleaching on autofluorescence lifetimes in the fundus in 21 young healthy volunteers. Three measurements of 30° retinal fields in two spectral channels (SSC: 498-560 nm, LSC: 560-720 nm) were obtained for each volunteer using fluorescence lifetime imaging ophthalmoscopy (FLIO). After dark-adaptation by wearing a custom-made lightproof mask for 30 minutes, the first FLIO-measurement was recorded (dark-adapted state). Subsequently, the eye was bleached for 1 minute (luminance: 3200 cd/m2), followed by a second FLIO-measurement (bleached state). Following an additional 10 minute dark adaptation using the mask, a final FLIO-measurement was recorded (recovered state). Average values of the fluorescence lifetimes were calculated from within different areas of a standardized early treatment diabetic retinopathy study (ETDRS) grid (central area, inner and outer rings). The acquisition time in the bleached state was significantly shortened by approximately 20%. The SSC did not show any significant changes in fluorescence lifetimes with photopigment bleaching, only the LSC showed small but significant bleaching-related changes in the fluorescence lifetimes τ1 and τ2 from all regions, as well as the mean fluorescence lifetime in the central area. The fluorescence lifetime differences caused by bleaching were by far less significant than pathological changes caused by eye diseases. The magnitudes of fluorescence lifetime changes are <10% and do not interfere with healthy or disease related FLIO patterns. Thus, we conclude that bleaching is not a relevant confounder in current clinical applications of FLIO.

Abdominal obesity linked to a longer cone-mediated dark-adaptation recovery time in healthy eyes

Obesity has been associated with abnormal lipid metabolism and with tissue hypoxia. Human Bruch's membrane (BrM) lipid deposits have been proposed to create a diffusion barrier to metabolic exchange between the choroid and photoreceptors, delaying the regeneration of photopigments. The speed of retinal dark adaptation (DA) is dependent on the regeneration of these photopigments. While the retina is extremely sensitive to hypoxia, the inner retina, which encodes visual contrast, is more affected by hypoxia than the outer retina. This study examines the association between adiposity measures and the time course of DA measured psychophysically through contrast detection to test the functionality of both the outer and inner retina. Cone-mediated DA recovery of contrast threshold (CT) was measured following near-total photopigment bleach for 6 min in 52 healthy eyes of 52 individuals (42.6 ± 18.3 years). Stimuli were sine-wave gratings of low-spatial frequency (1 cycle-per-degree (cpd)) and low luminance (1 cd/m2) generated at the centre of a CRT monitor. CT recovery functions were fitted to an exponential decay model to determine the time constant (τ, seconds) of cone sensitivity recovery, final cone CT (CTf) and CT elevation (CT0). Weight, height and waist circumference (WC) were measured and body mass index (BMI) and waist-to-height ratio (WHtR) calculated. Relationships were examined through Spearman correlation and through multiple linear regression using age, optical and adiposity measures as independent variables. The repeatability of cone time constant measurements was estimated by the Bland-Altman method and reported as the coefficient of repeatability (CoR). Mean ± SD of time constant and CTf were 57.3 ± 27.7 s and −1.78 ± 0.20 log10 units respectively. Cone time constant showed positive Spearman correlation with WC (p = 0.008) and WHtR (p = 0.023) but not with BMI (p = 0.058). Only WHtR emerged as an independent predictor of time constant (p = 0.001). CTf was not correlated with any adiposity measures. Mean cone time constant was 41 s slower in subjects (25%, n = 13) with abdominal obesity (WHtR≥0.5). Mean CTf was not significantly different in subjects with or without abdominal obesity. CoR for cone time constant was ±16 s. In adult subjects, greater abdominal obesity (WHtR) was related to a longer contrast recovery time for cone-mediated DA (time to dark-adapt) suggesting outer retinal dysfunction. Final contrast threshold, preferentially processed by inner retinal cells, was unaffected by abdominal obesity.

New wrinkles in retinal densitometry.

Retinal densitometry provides objective information about retinal function. But, a number of factors, including retinal reflectance changes that are not directly related to photopigment depletion, complicate its interpretation. We explore these factors and suggest a method to minimize their impact. An adaptive optics scanning light ophthalmoscope (AOSLO) was used to measure changes in photoreceptor reflectance in monkeys before and after photopigment bleaching with 514-nm light. Reflectance measurements at 514 nm and 794 nm were recorded simultaneously. Several methods of normalization to extract the apparent optical density of the photopigment were compared. We identified stimulus-related fluctuations in 794-nm reflectance that are not associated with photopigment absorptance and occur in both rods and cones. These changes had a magnitude approaching those associated directly with pigment depletion, precluding the use of infrared reflectance for normalization. We used a spatial normalization method instead, which avoided the fluctuations in the near infrared, as well as a confocal AOSLO designed to minimize light from layers other than the receptors. However, these methods produced a surprisingly low estimate of the apparent rhodopsin density (animal 1: 0.073 ± 0.006, animal 2: 0.032 ± 0.003). These results confirm earlier observations that changes in photopigment absorption are not the only source of retinal reflectance change during dark adaptation. It appears that the stray light that has historically reduced the apparent density of cone photopigment in retinal densitometry arises predominantly from layers near the photoreceptors themselves. Despite these complications, this method provides a valuable, objective measure of retinal function.

Remote Ischemia Influences the Responsiveness of the Retina: Observations in the Rat

Remote ischemic preconditioning (RIP) has been found to be protective of heart and brain against ischemic injury. We have tested the effects of RIP on retinal function using the electroretinogram. Ischemia remote from the retina was induced in one hindlimb, using a pressure cuff applied for between 5 and 10 minutes. A temperature probe on the footpad confirmed blockage of the circulation. To test the impact of RIP on retinal function, we recorded the dark-adapted flash electroretinogram (ERG) in four groups (n = 5 per group) of Sprague-Dawley rats (sham, 5-minute, 10-minute, and 2 × 5-minute ischemia). Heart rate, breath rate, and peripheral oxygen saturation were monitored using infrared pulse oximetry. RIP increased both the a- and b-waves by up to 14%, more markedly after the longer periods (10 minutes or 2 × 5 minutes) of ischemia. The effect was tested up to 30 minutes after ischemia and retested at 1 week and 1 month. RIP did not appear to accelerate the initial stages of recovery from photopigment bleach. Systemic oxygen saturation, heart rate, and respiration did not vary consistently during or after remote ischemia. The effect of RIP on the ERG is a novel finding. Possible mechanisms of this effect are discussed and related to the idea of neuroprotection and to fundamentals of the electroretinogram.

Optimization of In Vivo Confocal Autofluorescence Imaging of the Ocular Fundus in Mice and Its Application to Models of Human Retinal Degeneration

To investigate the feasibility and to identify sources of experimental variability of quantitative and qualitative fundus autofluorescence (AF) assessment in mice. Blue (488 nm) and near-infrared (790 nm) fundus AF imaging was performed in various mouse strains and disease models (129S2, C57Bl/6, Abca4(-/-), C3H-Pde6b(rd1/rd1), Rho(-/-), and BALB/c mice) using a commercially available scanning laser ophthalmoscope. Gray-level analysis was used to explore factors influencing fundus AF measurements. A contact lens avoided cataract development and resulted in consistent fundus AF recordings. Fundus illumination and magnification were sensitive to changes of the camera position. Standardized adjustment of the recorded confocal plane and consideration of the pupil area allowed reproducible recording of fundus AF from the retinal pigment epithelium with an intersession coefficient of repeatability of ±22%. Photopigment bleaching occurred during the first 1.5 seconds of exposure to 488 nm blue light (∼10 mW/cm(2)), resulting in an increase of fundus AF. In addition, there was a slight decrease in fundus AF during prolonged blue light exposure. Fundus AF at 488 nm was low in animals with an absence of a normal visual cycle, and high in BALB/c and Abca4(-/-) mice. Degenerative alterations in Pde6b(rd1/rd1) and Rho(-/-) were reminiscent of findings in human retinal disease. Investigation of retinal phenotypes in mice is possible in vivo using standardized fundus AF imaging. Correlation with postmortem analysis is likely to lead to further understanding of human disease phenotypes and of retinal degenerations in general. Fundus AF imaging may be useful as an outcome measure in preclinical trials, such as for monitoring effects aimed at lowering lipofuscin accumulation in the retinal pigment epithelium.

Electrophysiological characterization of GFP-expressing cell populations in the intact retina.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments6.In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event. Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments6. In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

Topography of Cone Dark Adaptation Deficits in Age-Related Maculopathy

Despite widespread agreement that dark adaptation is abnormal in age-related maculopathy (ARM), the optimal retinal location for detection of this deficit is unclear. We quantified the diagnostic potential of cone dark adaptation as a function of retinal eccentricity and compared this with the diagnostic potential of the time to the rod-cone-break (RCB). Cone dark adaptation was monitored after an 80% cone photopigment bleach in 10 subjects with ARM and 10 age-matched controls, using four achromatic annuli (0.5, 2, 7, and 12° radius) centered on the fovea. Threshold recovery data were modeled and the time constant of cone recovery (τ), final cone threshold, and time to RCB were determined. Diagnostic potential was evaluated by constructing receiver operating characteristic curves for these parameters. Cone τ was significantly longer for the ARM group at 2, 7, and 12°. The greatest difference between groups was observed at 12° from fixation. At this location, the mean τ was 3.49 (±2.02) min and 0.64 (±0.38) min for ARM and control subjects, respectively (p = 0.002), and time to RCB was 17.68 (±5.37) min and 9.05 (±2.11) min for ARM and control subjects, respectively (p = 0.001). Correspondingly, receiver operating characteristic curves showed that the diagnostic potential of dark adaptometry is greatest for stimuli presented 12° from fixation; for cone τ, the area under the curve = 0.99 ± 0.02 and for time to RCB, area under the curve = 0.96 ± 0.04. This study has shown cone-mediated dark adaptation to be significantly impaired in ARM. Our results provide compelling evidence in support of the diagnostic potential of cone dark adaptation and the use of annular stimuli at 12°. The observation that cone τ is highly diagnostic at this eccentricity is significant clinically because this parameter may be quantified within a few minutes.

Origins of retinal intrinsic signals: A series of experiments on retinas of macaque monkeys

Diffuse flash stimuli applied to the ocular fundus evoke light reflectance decreases of the fundus illuminated with infrared observation light. This phenomenon, which is independent of the photopigment bleaching observed as an increase in the reflectance of visible light, is called intrinsic signals. Intrinsic signals, in general, are stimulus-evoked light reflectance changes of neural tissues due to metabolic changes, and they have been extensively investigated in the cerebral cortex. This noninvasive objective technique of functional imaging has good potential as a tool for the early detection of retinal dysfunction. Once the signal properties were studied in detail, however, it became apparent that the intrinsic signals observed in the retina have uniquely interesting properties of their own due to the characteristic layered structure of the retina. Experiments on anesthetized macaque monkeys are reviewed, and the possible origins of the intrinsic signals of the retina are discussed.

Control Of Sensitivity Following Pigment Bleaching By NADPH In Salamander Rods

The recovery of sensitivity following photopigment bleaching requires the quenching of phototransduction, and the reduction of all-trans retinal is key. Retinol fluorescence increases after bleaching as a base to tip gradient in the rod outer segment and broadly matches the recovery of sensitivity. This gradient must result from a key component in retinal reduction, and we sought to determine how NADPH limits this process. Rod outer segment currents were recorded with suction electrodes, and responses were evoked by brief full-field flashes or by a narrow slit to stimulate selectively the base or tip of the outer segment. Simultaneous whole-cell recordings were made prior to bleaching to dialyze the cell with NADPH and track the recovery of sensitivity. After a 50% bleach rods remained in saturation for ∼ 12 minutes. The base recovered sensitivity with tau ∼ 160 s, but the tip recovered with tau ∼ 450 s resulting in a tip-base tau ratio of ∼ 3. Dialysis of 5 mM NADPH accelerated the recovery time by ∼ 2 min, and eliminated the tip-base difference. Dialysis with 1.66 mM NADPH didn't influence recovery and failed to eliminate the tip-base difference. After a 90% bleach rods remained saturated for ∼ 20 minutes with tip-base ratio ∼ 10 (base tau ∼ 250 s and tip tau ∼ 2700 s). Thus 5 mM NADPH eliminates the gradient along the outer segment, while 1.66 mM fails to influence the recovery of sensitivity; suggesting intrinsic NADPH exceeds 1.66 mM. In addition, tau at the base following 50% or 90% bleach are remarkably equivalent, suggesting that NADPH availability is sufficient to reduce all-trans retinal. The slower tip tau following 90% bleach suggests NADPH originates predominantly near the base, which is adjacent to mitochondria.

Fast Recursive Filters for Simulating Nonlinear Dynamic Systems

A fast and accurate computational scheme for simulating nonlinear dynamic systems is presented. The scheme assumes that the system can be represented by a combination of components of only two different types: first-order low-pass filters and static nonlinearities. The parameters of these filters and nonlinearities may depend on system variables, and the topology of the system may be complex, including feedback. Several examples taken from neuroscience are given: phototransduction, photopigment bleaching, and spike generation according to the Hodgkin-Huxley equations. The scheme uses two slightly different forms of autoregressive filters, with an implicit delay of zero for feedforward control and an implicit delay of half a sample distance for feedback control. On a fairly complex model of the macaque retinal horizontal cell, it computes, for a given level of accuracy, one to two orders of magnitude faster than the fourth-order Runge-Kutta. The computational scheme has minimal memory requirements and is also suited for computation on a stream processor, such as a graphical processing unit.