Cone Photoreceptor Loss in Light-Damaged Albino Rats.

We investigated the etiology of decreased cone-driven vision in a light damage (LD) model of retinal degeneration. To induce slow, moderate degeneration, albino rats underwent low-intensity light exposure for 10 days. Electroretinography was utilized to assess physiologic function of the rod- and cone-driven retinal function in LD and control rats. Immunohistochemistry targeting cone arrestin allowed for quantification of cone density and for comparison of the decline in function. Photoreceptor loss was quantified by outer nuclear layer thickness decreases, as observed by optical coherence tomography and histology. The LD rats showed decreased rod- and cone-driven function with partial recovery 30 days after cessation of light exposure. In addition, LD rats showed decreased cone photoreceptor densities in the central retinal region compared to control rats. Our results demonstrate that the loss of cone-driven visual function induced by light damage is at least partially due to the death of cone photoreceptors.

Life Cycle and Lensing of a Macular Microcyst.

INTRODUCTION: Microscopic details about retinal conditions can provide insight into pathological mechanisms, but these are ordinarily difficult to obtain in situ. We demonstrate how high-resolution imaging and optical modeling can be combined to reveal morphological features of a macular microcyst, offering insight into microcyst formation. OBJECTIVE: To use adaptive optics scanning laser ophthalmoscopic (AOSLO) images to track a transient retinal microcyst and derive its 3-dimensional shape. METHODS: A series of AOSLO images were gathered before, during, and after a transient retinal microcyst developed in an otherwise normal healthy 26-year-old male subject. Optical coherence tomography (OCT) independently confirmed the location of the microcyst. Optical modeling was conducted to quantify the lensing effect of the optically uniform microcyst and to determine its 3-dimensional shape. Increment threshold sensitivity, targeted within and around the microcyst, was tested to see if cone photoreceptor function was affected. RESULTS: A transient microcyst appeared as a 50 µm diameter circle in AOSLO images, localized to the inner nuclear layer. Based on image distortion of the photoreceptor mosaic, optical modeling suggests that the microcyst had the shape of an aspherical lens, distinguishable from a spherical, cylindrical, or elliptical shape, indicative of an edematous expansion of laminar tissue. The microcyst spontaneously resolved about 30 days after first discovery. No changes to the photoreceptor mosaic ensued from the presence of the microcyst, and functional testing of the photoreceptors below the microcyst indicated no loss of light sensitivity. CONCLUSIONS: Microcysts have been associated with numerous subtypes of optic nerve degeneration, including multiple sclerosis and various inherited neuropathies. This microcyst appeared in a healthy individual and resolved without intervention. Lensing effects can be used to determine microcyst shape, which cannot be resolved by OCT imaging, and to help infer etiology.

Glycogen synthase kinase 3 regulates photic signaling in the suprachiasmatic nucleus.

Glycogen synthase kinase 3 (GSK3) is a serine-threonine kinase that regulates mammalian circadian rhythms at the behavioral, molecular and neurophysiological levels. In the central circadian pacemaker, the suprachiasmatic nucleus (SCN), inhibitory phosphorylation of GSK3 exhibits a rhythm across the 24 h day. We have recently shown that GSK3 is capable of influencing both the molecular clock and SCN neuronal activity rhythms. However, it is not known whether GSK3 regulates the response to environmental cues such as light. The goal of this study was to test the hypothesis that GSK3 activation mediates light-induced SCN excitability and photic entrainment. Immunofluorescence staining in the SCN of mice showed that late-night light exposure significantly increased GSK3 activity (decreased pGSK3ß levels) 30-60 min after the light-pulse. In addition, pharmacological inhibition of GSK3 blocked the expected light-induced excitability in SCN neurons; however, this effect was not associated with changes in resting membrane potential or input resistance. Behaviorally, mice with constitutively active GSK3 (GSK3-KI) re-entrained to a 6-h phase advance in the light-dark cycle in significantly fewer days than WT control animals. Furthermore, the behavioral and SCN neuronal activity of GSK3-KI mice was phase-advanced compared to WT, in both normal and light-exposed conditions. Finally, GSK3-KI mice exhibited normal negative-masking behavior and electroretinographic responses to light, suggesting that the enhanced photic entrainment is not due to an overall increased sensitivity to light in these animals. Taken together, these results provide strong evidence that GSK3 activation contributes to light-induced phase-resetting at both the neurophysiological and behavioral levels.

Signalling beyond photon absorption: extracellular retinoids and growth factors modulate rod photoreceptor sensitivity.

KEY POINTS: We propose that the end product of chromophore bleaching in rod photoreceptors, all-trans retinol, is part of a feedback loop that increases the sensitivity of the phototransduction cascade in rods. A previously described light-induced hypersensitivity in rods, termed adaptive potentiation, is reduced by exogenously applied all-trans retinol but not all-trans retinal. This potentiation is produced by insulin-like growth factor-1, whose binding proteins are located in the extracellular matrix, even in our isolated retina preparation after removal of the retinal pigmented epithelium. Simple modelling suggests that the light stimuli used in the present study will produce sufficient all-trans retinol within the interphotoreceptor matrix to explain the potentiation effect. ABSTRACT: Photoreceptors translate the absorption of photons into electrical signals for propagation through the visual system. Mammalian photoreceptor signalling has largely been studied in isolated cells, and such studies have necessarily avoided the complex environment of supportive proteins that surround the photoreceptors. The interphotoreceptor matrix (IPM) contains an array of proteins that aid in both structural maintenance and cellular homeostasis, including chromophore turnover. In signalling photon absorption, the chromophore 11-cis retinal is first isomerized to all-trans retinal, followed by conversion to all-trans retinol (ROL) for removal from the photoreceptor. Interphotoreceptor retinoid-binding protein (IRBP) is the most abundant protein in the IPM, and it promotes the removal of bleached chromophores and recycling in the nearby retinal pigment epithelium. By studying the light responses of isolated mouse retinas, we demonstrate that ROL can act as a feedback signal onto photoreceptors that influences the sensitivity of phototransduction. In addition to IRBP, the IPM also contains insulin-like growth factor-1 (IGF-1) and its associated binding proteins, although their functions have not yet been described. We demonstrate that extracellular application of physiological concentrations of IGF-1 can increase rod photoreceptor sensitivity in mammalian retinas. We also determine that chromophores and growth factors can limit the range of a newly described form of photoreceptor light adaptation. Finally, fluorescent antibodies demonstrate the presence of IRBP and IGFBP-3 in isolated retinas. A simple model of the formation and release of ROL into the extracellular space quantitatively describes this novel feedback loop.

Increased visual sensitivity following periods of dim illumination.

PURPOSE: We measured changes in the sensitivity of the human rod pathway by testing visual reaction times before and after light adaptation. We targeted a specific range of conditioning light intensities to see if a physiological adaptation recently discovered in mouse rods is observable at the perceptual level in humans. We also measured the noise spectrum of single mouse rods due to the importance of the signal-to-noise ratio in rod to rod bipolar cell signal transfer. METHODS: Using the well-defined relationship between stimulus intensity and reaction time (Piéron's law), we measured the reaction times of eight human subjects (ages 24-66) to scotopic test flashes of a single intensity before and after the presentation of a 3-minute background. We also made recordings from single mouse rods and processed the cellular noise spectrum before and after similar conditioning exposures. RESULTS: Subject reaction times to a fixed-strength stimulus were fastest 5 seconds after conditioning background exposure (79% ± 1% of the preconditioning mean, in darkness) and were significantly faster for the first 12 seconds after background exposure (P < 0.01). During the period of increased rod sensitivity, the continuous noise spectrum of individual mouse rods was not significantly increased. CONCLUSIONS: A decrease in human reaction times to a dim flash after conditioning background exposure may originate in rod photoreceptors through a transient increase in the sensitivity of the phototransduction cascade. There is no accompanying increase in rod cellular noise, allowing for reliable transmission of larger rod signals after conditioning exposures and the observed increase in perceptual sensitivity.

Overexpression of rod photoreceptor glutamic acid rich protein 2 (GARP2) increases gain and slows recovery in mouse retina.

BACKGROUND: The rod photoreceptor cGMP-gated cation channel, consisting of three α- and one ß subunit, controls ion flow into the rod outer segment (ROS). In addition to the ß-subunit, the Cngb1 locus encodes an abundant soluble protein, GARP2 that binds stoichiometrically to rod photoreceptor cGMP phosphodiesterase type 6 (PDE6). To examine the in vivo functional role of GARP2 we generated opsin promoter-driven transgenic mice overexpressing GARP2 three-fold specifically in rod photoreceptors. RESULTS: In the GARP2 overexpressing transgenic mice (tg), the endogenous channel ß-subunit, cGMP phosphodiesterase α-subunit, peripherin2/RDS and guanylate cyclase I were present at WT levels and were properly localized within the ROS. While localized properly within ROS, two proteins cGMP phosphodiesterase α-subunit (1.4-fold) and cGMP-gated cation channel α-subunit (1.2-fold) were moderately, but significantly elevated. Normal stratification of all retinal layers was observed, and ROS were stable in numbers but were 19% shorter than WT. Analysis of the photoresponse using electroretinography (ERG) showed that tg mice exhibit no change in sensitivity indicating overall normal rod function, however two parameters of the photoresponse significantly differed from WT responses. Fitting of the rising phase of the ERG a-wave to an accepted model of phototransduction showed a two-fold increase in phototransduction gain in the tg mice. The increase in gain was confirmed in isolated retinal tissue and by suction electrode recordings of individual rod photoreceptor cells. A measure of response recovery, the dominant time constant (τD) was elevated 69% in isolated retina compared to WT, indicating slower shutoff of the photoresponse. CONCLUSIONS: GARP2 may participate in regulating visual signal transduction through a previously unappreciated role in regulating phototransduction gain and recovery.

Adaptive potentiation in rod photoreceptors after light exposure.

Photoreceptors adapt to changes in illumination by altering transduction kinetics and sensitivity, thereby extending their working range. We describe a previously unknown form of rod photoreceptor adaptation in wild-type (WT) mice that manifests as a potentiation of the light response after periods of conditioning light exposure. We characterize the stimulus conditions that evoke this graded hypersensitivity and examine the molecular mechanisms of adaptation underlying the phenomenon. After exposure to periods of saturating illumination, rods show a 10-35% increase in circulating dark current, an adaptive potentiation (AP) to light exposure. This potentiation grows as exposure to light is extended up to 3 min and decreases with longer exposures. Cells return to their initial dark-adapted sensitivity with a time constant of recovery of ∼7 s. Halving the extracellular Mg concentration prolongs the adaptation, increasing the time constant of recovery to 13.3 s, but does not affect the magnitude of potentiation. In rods lacking guanylate cyclase activating proteins 1 and 2 (GCAP(-/-)), AP is more than doubled compared with WT rods, and halving the extracellular Mg concentration does not affect the recovery time constant. Rods from a mouse expressing cyclic nucleotide-gated channels incapable of binding calmodulin also showed a marked increase in the amplitude of AP. Application of an insulin-like growth factor-1 receptor (IGF-1R) kinase inhibitor (Tyrphostin AG1024) blocked AP, whereas application of an insulin receptor kinase inhibitor (HNMPA(AM)3) failed to do so. A broad-acting tyrosine phosphatase inhibitor (orthovanadate) also blocked AP. Our findings identify a unique form of adaptation in photoreceptors, so that they show transient hypersensitivity to light, and are consistent with a model in which light history, acting via the IGF-1R, can increase the sensitivity of rod photoreceptors, whereas the photocurrent overshoot is regulated by Ca-calmodulin and Ca(2+)/Mg(2+)-sensitive GCAPs.

The age-regulating protein klotho is vital to sustain retinal function.

PURPOSE: To determine whether the age-regulating protein klotho was expressed in the retina and determine whether the absence of klotho affected retinal function. METHODS: Immunohistochemistry and qPCR of klotho knockout and wild-type mice were used to detect klotho expression in retina. Immunohistochemistry was used to probe for differences in expression of proteins important in synaptic function, retinal structure, and ionic flux. Electroretinography (ERG) was conducted on animals across lifespan to determine whether decreased klotho expression affects retinal function. RESULTS: Klotho mRNA and protein were detected in the wild-type mouse retina, with protein present in all nuclear layers. Over the short lifespan of the knockout mouse (∼8 weeks), no overt photoreceptor cell loss was observed, however, function was progressively impaired. At 3 weeks of age neither protein expression levels (synaptophysin and glutamic acid decarboxylase [GAD67]) nor retinal function were distinguishable from wild-type controls. However, by 7 weeks of age expression of synaptophysin, glial fibrillary acidic protein (GFAP), and transient receptor potential cation channel subfamily member 1 (TRPM1) decreased while GAD67, post synaptic density 95 (PSD95), and wheat germ agglutinin staining, representative of glycoprotein sialic acid residues, were increased relative to wild-type mice. Accompanying these changes, profound functional deficits were observed as both ERG a-wave and b-wave amplitudes compared with wild-type controls. CONCLUSIONS: Klotho is expressed in the retina and is important for healthy retinal function. Although the mechanisms for the observed abnormalities are not known, they are consistent with the accelerating aging phenotype seen in other tissues.