A nonhuman primate model of blue light-induced progressive outer retina degeneration showing brimonidine drug delivery system-mediated cyto- and neuroprotection.

Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD) characterized by atrophy of the retinal pigment epithelium (RPE), loss of photoreceptors, and disruption of choriocapillaris. Excessive light exposure is toxic to the retina and is a known risk factor for AMD. We first investigated the effects of blue light-induced phototoxicity on RPE and photoreceptors in nonhuman primates (NHPs, a model of progressive retinal degeneration) and then evaluated the potential cyto- and neuroprotective effects of the brimonidine drug delivery system (Brimo DDS). In the first set of experiments related to model development, parafoveal lesions of varying severity were induced using blue light irradiation of the retina of cynomolgus monkeys to evaluate the level of phototoxicity in the RPE and photoreceptors. RPE damage was assessed using fundus autofluorescence imaging to quantify areas of hypofluorescence, while thinning of the outer nuclear layer (ONL, photoreceptor nuclei) was quantified using optical coherence tomography (OCT). Photoreceptor function was assessed using multifocal electroretinography (mfERG). RPE damage progressively increased across all lesion severities from 2 to 12 weeks, as did the extent of ONL thinning. Lesions of high severity continued to show reduction in mfERG amplitude, reaching a statistically significant maximum reduction at 12 weeks. Collectively, the first set of experiments showed that blue light irradiation of the NHP eye resulted in progressive retinal degeneration identified by damage to RPE, ONL thinning, and disrupted photoreceptor function - hallmarks of GA in humans. We then used the model to evaluate the cyto- and neuroprotective effects of Brimo DDS, administered as a therapeutic after allowing the lesions to develop for 5 weeks. Placebo DDS or Brimo DDS were administered intravitreally and a set of untreated animals were used as an additional control. In the placebo DDS group, hypofluorescence area continued to increase from baseline, indicating progressive RPE damage, while progression was significantly slowed in eyes receiving Brimo DDS. Likewise, ONL thinning continued to progress over time in eyes that received the placebo DDS, but was reduced in Brimo DDS-treated eyes. Pharmacologically relevant brimonidine concentrations were sustained in the retina for up to 26 weeks following Brimo DDS administration. In summary, Brimo DDS demonstrated cyto- and neuroprotective effects in a novel NHP GA model of progressive retinal degeneration.

Solar Retinopathy Presenting with Outer Retinal Defects Among Habitants of High Altitude

Solar radiation causes acute foveal injury resulting in outer retinal defects. Symptoms often follow an event of unprotected gazing at a solar eclipse or directly viewing the sun. We encountered a series of cases during winter among habitants of high altitudes who complained of visual field scotomas. All of them had a typical history of prolonged sunbathing but denied gazing at the sun directly. Optical coherence tomography showed outer retinal defects involving the ellipsoid zone characteristic of solar retinopathy in all patients. In this case series, we would like to emphasize the role of geographical factors in the causation of solar retinopathy.

Light-induced modifications of the outer retinal hyperreflective layers on spectral-domain optical coherence tomography in humans: an experimental study.

PURPOSE: Numerous small hyperreflective dots (HRDs) can be seen within the hyporeflective layer between the ellipsoid zone (EZ) and the interdigitation zone (IZ) on C-scan spectral-domain optical coherence tomography (SD-OCT) with a yet unknown variation under light conditions. The aim of this study was to explore light-induced SD-OCT changes in these HRDs. METHODS: The study subjects were randomly assigned to two groups: Group 1 experienced a dark adaptation protocol followed by intense retinal photobleaching, while Group 2, serving as the control group, was exposed to constant ambient light without any variation. The number of HRDs was automatically counted. RESULTS: Twenty healthy volunteers were prospectively included. The number of HRDs differed significantly over time (p = 0.0013). They decreased in Group 1 after dark adaptation and retinal photobleaching before returning to baseline levels 30 min later; conversely, they remained relatively constant in Group 2 throughout the study (p < 0.001). Light-skinned subjects had less HRD than dark-skinned subjects. CONCLUSION: We observed light-induced modifications in the space between the EZ and the IZ. We hypothesize that the HRDs visible in this zone correspond to melanosomes that are mobilized during the light stimulation protocol. Larger studies are recommended to further evaluate and confirm light-induced SD-OCT changes under physiological and pathological conditions.

Photopigment Bleaching Phenomenon With Scanning Laser Ophthalmoscopy and Fluorescein Angiography.

Photopigment bleaching occurs with saturation of photoreceptor pigment by short-wavelength fundus autofluorescence imaging. This phenomenon is seen as characteristic hyperautofluorescence with subsequent imaging acquisition. Herein, a patient with multiple sclerosis was found to exhibit increased choroidal hyperfluorescence during fluorescein angiography (FA) that corresponded with a circumscribed area of intense blue light exposure during initial scanning laser ophthalmoscopy. To the authors' knowledge, this case is the first description of photobleaching phenomenon during FA and should be recognized as nonpathologic by the clinician. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:590-592.].

In vitro phototoxicity of rhodopsin photobleaching products in the retinal pigment epithelium (RPE).

Although the primary biological function of retinal photoreceptors is to absorb light and provide visual information, extensive exposure to intense light could increase the risk of phototoxic reactions mediated by products of rhodopsin bleaching that might accumulate in photoreceptor outer segments (POS). The phototoxicity of POS, isolated from bovine retinas, was examined in cultured retinal pigment epithelium cells (ARPE-19) containing phagocytised POS and in selected model systems by determining POS ability to photogenerate singlet oxygen, and photoinduce oxidation of cholesterol and serum albumin. Bleaching of rhodopsin-rich POS with green light resulted in the formation of retinoid products exhibiting distinct absorption spectra in the near-UV. Irradiation of POS-fed ARPE-19 cells with blue light reduced their survival in a dose-dependent manner with the effect being stronger for cells containing prebleached POS. The specific and non-specific phagocytic activity of ARPE-19 cells was inhibited by sub-lethal photic stress mediated by phagocytised POS. The oxidising ability of POS photobleaching products was demonstrated both in a model system consisting of serum albumin and in ARPE-19 cells. Distinct photooxidation of proteins, mediated by POS, was observed using coumarin boronic acid as a sensitive probe of protein hydroperoxides. Irradiation of POS with blue light also induced oxidation of liposomal cholesterol as determined by HPLC-EC(Hg). Time-resolved singlet oxygen phosphorescence demonstrated the efficiency of retinoids, extracted from POS by chloroform-methanol treatment, to photogenerate singlet oxygen. The results indicate that photic stress mediated by POS photobleaching products could inhibit phagocytic efficiency of RPE cells and, ultimately, compromise their important biological functions.

Protective effects of autophagy against blue light-induced retinal degeneration in aged mice.

The aim of this study was to explore the role of autophagy in response to blue light damage in aged mice and in human retinal pigmented epithelium (hRPE) cells. Blue light damage to the retina was induced in 10-month-old (10 mo) C57 mice and hRPE cells. Flash electroretinography was used to assess retinal function. Retinal structure changes were observed by electron microscopy. Western blot was conducted to determine the expression levels of the following proteins: cleaved caspase-3, p38 mitogen-activated protein kinases, protein kinase R-like endoplasmic reticulum kinase (PERK), autophagy marker light chain 3 (LC3), P62, and Beclin-1. On day 1 after light damage to the 10 mo mice, retinal function was changed. The latent periods of a-wave and b-wave were delayed, and amplitude was reduced. The electron microscopy results revealed mitochondria damage in the retinal pigmented epithelium and a disorganized photoreceptor outer segment (OS). PERK, LC3, and Beclin-1 were upregulated, whereas P62 was not. On day 5 after the blue light damage, restoration of electroretinography and OS was observed. PERK, LC3, and Beclin-1 were downregulated, whereas P62 was not. Protein changes in vitro were consistent with in vivo. The present study provided structural and functional evidence that autophagy plays an important role in the response to blue lightinduced retinal damage.

Light-Induced Thickening of Photoreceptor Outer Segment Layer Detected by Ultra-High Resolution OCT Imaging.

PURPOSE: We examined if light induces changes in the retinal structure that can be observed using optical coherence tomography (OCT). METHODS: Normal C57BL/6J mice (age 3-6 months) adapted to either room light (15 minutes to ∼5 hours, 50-500 lux) or darkness (overnight) were imaged using a Bioptigen UHR-OCT system. Confocal histologic images were obtained from mice killed under light- or dark-adapted conditions. RESULTS: The OCT image of eyes adapted to room light exhibited significant increases (6.1 ± 0.8 µm, n = 13) in total retina thickness compared to the same eyes after overnight dark adaptation. These light-adapted retinal thickness changes occurred mainly in the outer retina, with the development of a hyporeflective band between the RPE and photoreceptor-tip layers. Histologic analysis revealed a light-evoked elongation between the outer limiting membrane and Bruch's membrane from 45.8 ± 1.7 µm in the dark (n = 5) to 52.1 ± 3.7 µm (n = 5) in the light. Light-adapted retinas showed an increase of actin staining in RPE apical microvilli at the same location as the hyporeflective band observed in OCT images. Elongation of the outer retina could be detected even with brief light exposures, increasing 2.1 ± 0.3 µm after 15 minutes (n = 9), and 4.1 ± 1.0 µm after 2 hours (n = 6). Conversely, dark-adaptation caused outer retinal shortening of 1.4 ± 0.4 µm (n = 7) and 3.0 ± 0.5 µm (n = 8) after 15 minutes and 2 hours, respectively. CONCLUSIONS: Light-adaption induces an increase in the thickness of the outer retina and the appearance of a hyporeflective band in the OCT image. This is consistent with previous reports of light-induced fluid accumulation in the subretinal space.

Photopigment self-screening and the determination of macular pigment absorbance using heterochromatic flicker photometry.

PURPOSE: Heterochromatic flicker photometry (HFP) is commonly used to determine macular pigment optical density (MPOD). Since HFP in this application is a locus comparison method, an identical relative spectral response at each locus is required for a perfect measure. We know this requirement cannot be strictly true since the optical density of photopigments increases as the foveal center is approached. Thus, the self-screening effect would result in an underestimate of MPOD. An earlier study concluded that the underestimate is on the order of 30%. We examined this issue by manipulating photopigment optical density, and consequently the degree of selfscreening. METHODS: A continuously exposed, 470 nm, background bleached cone photopigments over a range from 0 to 80%. MPOD was determined 10' and 30' from the foveal center. Two subjects were used in the main experiment. Five additional subjects were studied with just the 0% and 80% bleach levels. Spectral measures were obtained at 0% and 70% bleach levels for the two primary subjects. RESULTS: Subjects in the main experiment showed MPOD estimates that increased with increasing bleaching. The effect, however, was small: one observer's MPOD increased 0.08 and 0.02 for the 10' and 30' loci, respectively; the other observer's values were 0.04 and 0.01 for the same loci. Comparable values were obtained for the other five subjects using the 0% and 80% bleach conditions. Spectral measures were consistent with the findings of the main experiment. CONCLUSIONS: When self-screening is nearly abolished (80% bleach), a relatively small underestimation is revealed for the unbleached state. For the 1° target we show about 2-3% underestimation. Our 20' target reveals a larger underestimate (8-9%), consistent with longer photoreceptor outer-segments nearer the foveal center. We conclude that HFP yields values essentially independent of self-screening for targets of 1° diameter or greater. Smaller targets are less than 10% underestimated for near-zero bleach conditions.

Functional optical coherence tomography reveals transient phototropic change of photoreceptor outer segments.

Dynamic near infrared microscopy has revealed transient retinal phototropism (TRP) correlated with oblique light stimulation. Here, by developing a hybrid confocal microscopy and optical coherence tomography (OCT), we tested sub-cellular source of the TRP in living frog retina. Dynamic confocal microscopy and OCT consistently revealed photoreceptor outer segments as the anatomic source of the TRP. Further investigation of the TRP can provide insights in better understanding of Stiles-Crawford effect (SCE) on rod and cone systems, and may also promise an intrinsic biomarker for early detection of eye diseases that can produce photoreceptor dysfunction.

Cone-like rectification properties of cGMP-gated channels in transmutated retinal photoreceptors of nocturnal geckoes.

Photoreceptors of nocturnal geckoes are scotopic, with rod-shaped outer segments, and sensitivities to light similar to the one of retinal rods from other species of lower vertebrates. However, these cells are not rods, but they originated from cones of ancestral diurnal geckoes with pure-cone retinas, after being forced to adapt to a nocturnal behavior. Several interesting adaptations of these rod-like cones have been studied to date; molecular biology and functional studies confirmed that several proteins of the phototransductive cascade display structural and functional properties that indicate their origin from cones rather than from rods. In this paper, we investigate, with whole cell voltage clamp in the photoreceptor detached outer segment preparation, the voltage rectification properties of cGMP-gated channels in three species, Gekko gecko, Tarentola mauritanica, and Hemidactylus frenatus. We show that the current-voltage properties in the physiological voltage range are reminiscent of the ones of cGMP-gated channels from cones rather than from rods of other cold-blooded vertebrates. The origin and the relevance of the mechanisms investigated are discussed.

Angiotensin II type 1 receptor blockade suppresses light-induced neural damage in the mouse retina.

Exposure to light contributes to the development and progression of retinal degenerative diseases. However, the mechanisms underlying light-induced tissue damage are not fully understood. Here, we examined the role of angiotensin II type 1 receptor (AT1R) signaling, which is part of the renin-angiotensin system, in light-induced retinal damage. Light-exposed Balb/c mice that were treated with the AT1R blockers (angiotensin II receptor blockers; ARBs) valsartan, losartan, and candesartan before and after the light exposure exhibited attenuated visual function impairment, compared to vehicle-treated mice. This effect was dose-dependent and observed across the ARB class of inhibitors. Further evaluation of valsartan showed that it suppressed a number of light-induced retinal effects, including thinning of the photoreceptor cell layer caused by apoptosis, shortening of the photoreceptor cell outer segment, and increased levels of reactive oxygen species (ROS). The role of ROS in retinal pathogenesis was investigated further using the antioxidant N-acetyl-l-cysteine (NAC). Treatment of light-exposed mice with NAC before the light exposure suppressed the visual function impairment and photoreceptor cell histological changes due to apoptosis. Moreover, treatment with valsartan or NAC suppressed the induction of c-fos (a component of the AP-1 transcription factor) and the upregulation of fasl (a proapoptotic molecule whose transcript is regulated downstream of AP-1). Our results suggest that AT1R signaling mediates light-induced apoptosis, by increasing the levels of ROS and proapoptotic molecules in the retina. Thus, AT1R blockade may represent a new therapeutic approach for preventing light-induced retinal neural tissue damage.

Stress reaction in outer segments of photoreceptors after blue light irradiation.

The retina is prone to oxidative stress from many factors which are also involved in the pathogenesis of degenerative diseases. In this study, we used the application of blue light as a physiological stress factor. The aim of this study was to identify the major source of intracellular ROS that mediates blue light-induced detrimental effects on cells which may lead to cytotoxicity. We hypothesized that outer segments are the major source of blue light induced ROS generation. In photoreceptors, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes and the recently found respiratory chain complexes may represent a major source for reactive oxygen species (ROS), beside mitochondria and chromophores. Therefore, we investigated this hypothesis and analysed the exact localization of the ROS source in photoreceptors in an organotypic culture system for mouse retinas. Whole eyeball cultures were irradiated with visible blue light (405 nm) with an output power of 1 mW/cm². Blue light impingement lead to an increase of ROS production (detected by H2DCFDA in live retinal explants), which was particularly strong in the photoreceptor outer segments. Nox-2 and Nox-4 proteins are sources of ROS in blue light irradiated photoreceptors; the Nox inhibitor apocynin decreased ROS stimulated by blue light. Concomitantly, enzyme SOD-1, a member of the antioxidant defense system, indicator molecules of protein oxidation (CML) and lipid oxidation (MDA and 4-HNE) were also increased in the outer segments. Interestingly, outer segments showed a mitochondrial-like membrane potential which was demonstrated using two dyes (JC-1 and TMRE) normally exclusively associated with mitochondria. As in mitochondria, these dyes indicated a decrease of the membrane potential in hypoxic states or cell stress situations. The present study demonstrates that ROS generation and oxidative stress occurs directly in the outer segments of photoreceptors after blue light irradiation.

Effects of chronic exposure to 0.5 Hz and 5 Hz flickering illumination on the eye growth of guinea pigs.

AIMS: To investigate the effect of prolonged flickering illumination exposure on the growth of the guinea pig eye. METHODS: Thirty-six 2-week-old guinea pigs were randomized to one of the three treatment groups (n = 12 for each). Two strobe-reared groups were raised with a duty diurnal cycle of 50 % at a flash rate of 0.5 Hz and 5 Hz respectively. Illumination intensity varied between the minimum-maximum light levels of 0-600 lux during each cycle. The control group was exposed to steady 300 lux illumination. All animals underwent refraction and biometric measurements prior to and after 2, 4, 6, 8, 10 and 12 weeks of treatment. Finally, flash electroretinograms were compared, and retinal microstructures were examined. RESULTS: There was a significant correlation between refractive errors and axial eye elongation, with myopia increasing between 1.5 and 3.4 D per mm eye elongation. After 12 weeks of treatment, the animals raised in 0.5 Hz flickering light were 5.5 ± 0.4 D more myopic than the group raised in continuous illumination, followed by the group raised at 5 Hz flicker light which was about 2.2 ± 1.3 D more myopic. In animals raised in flickering light of 5 or 0.5 Hz for 12 weeks, the implicit time of the a-wave was delayed by 4 and 8.5 ms, respectively. At this time, the outer segment disc membranes were found deformed and detached. CONCLUSION: Chronic exposure to 0.5 and 5 Hz temporally modulated illumination induces electrophysiological and histological changes in retinal activities that alter the emmetropization of the guinea pig eye.

Intense physiological light upregulates vascular endothelial growth factor and enhances choroidal neovascularization via peroxisome proliferator-activated receptor γ coactivator-1α in mice.

OBJECTIVE: Toxicity of intense light to facilitate the development of neovascular age-related macular degeneration has been a health concern although the mechanism remains unclear. METHODS AND RESULTS: Effects of intense, but within physiological range, light on retinal pigment epithelium, a major pathogenic origin of age-related macular degeneration were studied in mice. Intense physiological light upregulated vascular endothelial growth factor (VEGF) expression in retinal pigment epithelium, independent of circadian rhythm, which resulted in enhancement of choroidal neovascularization. In rd1/rd1 mice or Crx(-/-) mice that do not possess outer segment structure, light exposure did not induce VEGF, indicating that VEGF upregulation by light depended on increased outer segment phagocytosis by retinal pigment epithelium. In retinal pigment epithelium cells phagocytosing increased amount of outer segment, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) not hypoxia-inducible factor-1α was induced, leading to VEGF upregulation. The VEGF upregulation and choroidal neovascularization enhancement were abrogated in PGC-1α(-/-) mice and estrogen-related receptor-α(-/-) mice, indicating the involvement of PGC-1α/estrogen-related receptor-α pathway. CONCLUSIONS: Intense physiological light is involved in choroidal neovascularization through excess outer segment phagocytosis and VEGF upregulation mediated by PGC-1α in vivo.

Complement and UV-irradiated photoreceptor outer segments increase the cytokine secretion by retinal pigment epithelial cells.

PURPOSE: Age-related macular degeneration (AMD) is accompanied by increased complement activation, and by lipofuscin accumulation in retinal pigment epithelial (RPE) cells due to incomplete degradation of photoreceptor outer segments (POS). The influence of POS, ultraviolet (UV)-irradiated POS and human complement sera (HCS) on cytokine secretion from RPE cells was therefore examined. METHODS: RPE cells were incubated with POS or UV-POS every other day for 1 week. The autofluorescence (AF) was measured photometrically and by flow cytometry. Senescence-associated genes were analyzed by RT-PCR. Internalization and degradation of POS were determined using phagocytosis and degradation assays, and lysosomal function by neutral red uptake. RPE cells in polycarbonate cell culture inserts were incubated apically with POS or UV-POS and afterward basally with HCS. C7-deficient HCS was used as control. The integrity of the cell monolayer was assessed by measuring the transepithelial electrical resistance (TER) and the permeability. Interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and vascular endothelial growth factor were quantified by ELISA. RESULTS: POS treatment led to an increased AF and senescence marker expression, which were further elevated in response to UV-POS. UV-POS were preferentially accumulated over POS and the lysosomal function was impaired due to UV-POS. HCS intensified the cytokine production compared with controls. POS had no effect, though UV-POS combined with HCS induced a significant increase in all cytokines. CONCLUSIONS: RPE cultivation with UV-POS might serve as a model to investigate the accumulation of lipofuscin-like structures. The enhanced cytokine secretion due to UV-POS with HCS may account for an increased susceptibility for lipofuscin-loaded cells to complement, inducing a proinflammatory environment as observed in AMD.

Flow of energy in the outer retina in darkness and in light.

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.

Chromatic pupil responses: preferential activation of the melanopsin-mediated versus outer photoreceptor-mediated pupil light reflex.

OBJECTIVE: To weight the rod-, cone-, and melanopsin-mediated activation of the retinal ganglion cells, which drive the pupil light reflex by varying the light stimulus wavelength, intensity, and duration. DESIGN: Experimental study. PARTICIPANTS: Forty-three subjects with normal eyes and 3 patients with neuroretinal visual loss. METHODS: A novel stimulus paradigm was developed using either a long wavelength (red) or short wavelength (blue) light given as a continuous Ganzfeld stimulus with stepwise increases over a 2 log-unit range. The pupillary movement before, during, and after the light stimulus was recorded in real time with an infrared illuminated video camera. MAIN OUTCOME MEASURES: The percent pupil contraction of the transient and sustained pupil response to a low- (1 cd/m(2)), medium- (10 cd/m(2)), and high-intensity (100 cd/m(2)) red- and blue-light stimulus was calculated for 1 eye of each subject. From the 43 normal eyes, median and 25th, 75th, 5th, and 95th percentile values were obtained for each stimulus condition. RESULTS: In normal eyes at lower intensities, blue light evoked much greater pupil responses compared with red light when matched for photopic luminance. The transient pupil contraction was generally greater than the sustained contraction, and this disparity was greatest at the lowest light intensity and least apparent with bright (100 cd/m(2)) blue light. A patient with primarily rod dysfunction (nonrecordable scotopic electroretinogram) showed significantly reduced pupil responses to blue light at lower intensities. A patient with achromatopsia and an almost normal visual field showed selective reduction of the pupil response to red-light stimulation. A patient with ganglion cell dysfunction owing to anterior ischemic optic neuropathy demonstrated global loss of pupil responses to red and blue light in the affected eye. CONCLUSIONS: Pupil responses that differ as a function of light intensity and wavelength support the hypothesis that selected stimulus conditions can produce pupil responses that reflect phototransduction primarily mediated by rods, cones, or melanopsin. Use of chromatic pupil responses may be a novel way to diagnose and monitor diseases affecting either the outer or inner retina.

Stimulus-evoked intrinsic optical signals in the retina: pharmacologic dissection reveals outer retinal origins.

PURPOSE: To elucidate the anatomic origins of stimulus-evoked intrinsic optical signals in the mammalian retina by using selective pharmacologic blockade of specific retinal layers. METHODS: Four adult cats were used to investigate the stimulus-evoked intrinsic signals. The retinas were visually stimulated with a liquid crystal display (LCD) integrated into a modified fundus camera. The evoked signals in the near infrared (NIR) were recorded with a digital camera to image the changes in the optical reflectance of the retinas. Variants of the electroretinogram (pattern ERG and long-pulse ERG) were also recorded as additional measures of retinal function. Specific retinal layers were inactivated via intravitreal injections of the voltage-gated sodium channel blocker, tetrodotoxin (TTX), the metabotropic glutamate receptor (mGluR6) agonist, 2-amino-4-phosphonobutyric acid (APB), and/or the ionotropic glutamate receptor antagonist cis-2,3 piperidinedicarboxylic acid (PDA). The stimulus-evoked intrinsic signals were imaged before and after drug injection. RESULTS: ERG recordings and tests of the consensual pupillary response confirmed the effectiveness of each drug. Yet despite the pharmacologic blockade of the inner retina (TTX) and postreceptoral retinal circuitry (APB and PDA), the stimulus-evoked intrinsic signals remained essentially unaltered from preinjection conditions. Similarly, the time course of the signal did not appreciably shift in time or shape. CONCLUSIONS: The findings demonstrate that stimulus-evoked intrinsic signals persist after injection of APB, PDA, and TTX, drugs that work to suppress inner and postreceptoral retinal circuitry. The persistence of the intrinsic signals after administration of these drugs indicates that the dominant intrinsic signals are likely to arise from the outer retina.