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Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow, but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

The elongated cilia of the outer segment of rod and cone photoreceptor cells can contain concentrations of visual pigments of up to 5mM. The rod visual pigments, G protein-coupled receptors called rhodopsins, have a propensity to self-aggregate, a property conserved among many G protein-coupled receptors. However, the effect of rhodopsin oligomerization on G protein signaling in native cells is less clear. Here, we address this gap in knowledge by studying rod phototransduction. As the rod outer segment is known to adjust its size proportionally to overexpression or reduction of rhodopsin expression, genetic perturbation of rhodopsin cannot be used to resolve this question. Therefore, we turned to high-throughput screening of a diverse library of 50,000 small molecules and used a novel assay for the detection of rhodopsin dimerization. This screen identified nine small molecules that either disrupted or enhanced rhodopsin dimer contacts invitro. In a subsequent cell-free binding study, we found that all nine compounds decreased intrinsic fluorescence without affecting the overall UV-visible spectrum of rhodopsin, supporting their actions as allosteric modulators. Furthermore, exvivo electrophysiological recordings revealed that a disruptive, hit compound #7 significantly slowed down the light response kinetics of intact rods, whereas compound #1, an enhancing hit candidate, did not substantially affect the photoresponse kinetics but did cause a significant reduction in light sensitivity. This study provides a monitoring tool for future investigation of the rhodopsin signaling cascade and reports the discovery of new allosteric modulators of rhodopsin dimerization that can also alter rod photoreceptor physiology.

Two-photon vision arises from the perception of pulsed infrared (IR) laser light as color corresponding to approximately half of the laser wavelength. The physical process responsible for two-photon vision in rods has been delineated and verified experimentally only recently. Here, we sought to determine whether IR light can also be perceived by mammalian cone photoreceptors via a similar activation mechanism. To investigate selectively mammalian cone signaling in mice, we used animals with disabled rod signal transduction. We found that, contrary to the expected progressive sensitivity decrease based on the one-photon cone visual pigment spectral template, the sensitivity of mouse cone photoreceptors decreases only up to 800 nm and then increases at 900 nm and 1000 nm. Similarly, in experiments with the parafoveal region of macaque retinas, we found that the spectral sensitivity of primate cones diverged above the predicted one-photon spectral sensitivity template beyond 800 nm. In both cases, efficient detection of IR light was dependent on minimizing the dispersion of the ultrashort light pulses, indicating a non-linear two-photon activation process. Together, our studies demonstrate that mammalian cones can be activated by near IR light by a nonlinear two-photon excitation. Our results pave the way for the creation of a two-photon IR-based ophthalmoscope for the simultaneous imaging and functional testing of human retinas as a novel tool for the diagnosis and treatment of a wide range of visual disorders.

Photoreceptors are specialized neurons that rely on Ca2+ to regulate phototransduction and neurotransmission. Photoreceptor dysfunction and degeneration occur when intracellular Ca2+ homeostasis is disrupted. Ca2+ homeostasis is maintained partly by mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU), which can influence cytosolic Ca2+ signals, stimulate energy production, and trigger apoptosis. Here we discovered that zebrafish cone photoreceptors express unusually low levels of MCU. We expected that this would be important to prevent mitochondrial Ca2+ overload and consequent cone degeneration. To test this hypothesis, we generated a cone-specific model of MCU overexpression. Surprisingly, we found that cones tolerate MCU overexpression, surviving elevated mitochondrial Ca2+ and disruptions to mitochondrial ultrastructure until late adulthood. We exploited the survival of MCU overexpressing cones to additionally demonstrate that mitochondrial Ca2+ uptake alters the distributions of citric acid cycle intermediates and accelerates recovery kinetics of the cone response to light. Cones adapt to mitochondrial Ca2+ stress by decreasing MICU3, an enhancer of MCU-mediated Ca2+ uptake, and selectively transporting damaged mitochondria away from the ellipsoid toward the synapse. Our findings demonstrate how mitochondrial Ca2+ can influence physiological and metabolic processes in cones and highlight the remarkable ability of cone photoreceptors to adapt to mitochondrial stress.

Sensing changes in the environment is crucial for survival. Animals from invertebrates to vertebrates use both visual and olfactory stimuli to direct survival behaviors including identification of food sources, finding mates, and predator avoidance. In primary sensory neurons there are signal transduction mechanisms that convert chemical or light signals into an electrical response through ligand binding or photoactivation of a receptor, that can be propagated to the olfactory and visual centers of the brain to create a perception of the odor and visual landscapes surrounding us. The fundamental principles of olfactory and phototransduction pathways within vertebrates are somewhat analogous. Signal transduction in both systems takes place in the ciliary sub-compartments of the sensory cells and relies upon the activation of G protein-coupled receptors (GPCRs) to close cyclic nucleotide-gated (CNG) cation channels in photoreceptors to produce a hyperpolarization of the cell, or in olfactory sensory neurons open CNG channels to produce a depolarization. However, while invertebrate phototransduction also involves GPCRs, invertebrate photoreceptors can be either ciliary and/or microvillar with hyperpolarizing and depolarizing responses to light, respectively. Moreover, olfactory transduction in invertebrates may be a mixture of metabotropic G protein and ionotropic signaling pathways. This review will highlight differences of the visual and olfactory transduction mechanisms between vertebrates and invertebrates, focusing on the implications to the gain of the transduction processes, and how they are modulated to allow detection of small changes in odor concentration and light intensity over a wide range of background stimulus levels.

Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.

Neuronal plasticity of the inner retina has been observed in response to photoreceptor degeneration. Typically, this phenomenon has been considered maladaptive and may preclude vision restoration in the blind. However, several recent studies utilizing triggered photoreceptor ablation have shown adaptive responses in bipolar cells expected to support normal vision. Whether such homeostatic plasticity occurs during progressive photoreceptor degenerative disease to help maintain normal visual behavior is unknown. We addressed this issue in an established mouse model of Retinitis Pigmentosa caused by the P23H mutation in rhodopsin. We show robust modulation of the retinal transcriptomic network, reminiscent of the neurodevelopmental state, and potentiation of rod – rod bipolar cell signaling following rod photoreceptor degeneration. Additionally, we found highly sensitive night vision in P23H mice even when more than half of the rod photoreceptors were lost. These results suggest retinal adaptation leading to persistent visual function during photoreceptor degenerative disease.

The retina, a highly metabolic tissue in the central nervous system, consumes the most oxygen and energy stores in the body by tissue mass/volume. Consequently, it is not surprising that retinal ischemia leads to a rapid loss of retinal light responses and electrical transmission. In this study, we show that despite a swift decline of retinal light responses after circulatory death (decay time constant τ = ∼1-2 min), we were able to restore mouse rod and cone photoreceptor light signals from enucleated eyes up to 3 h postmortem with significantly better postmortem recovery of cone versus rod light responses. We also demonstrate that both rod and cone phototransduction are more resistant to postmortem enucleation delay than synaptic transmission to second order neurons (bipolar cells). Encouraged by these analyses and the lack of previously successful postmortem retinal light recordings from human foveal/macular photoreceptors, we attempted to restore light responses in the human macula using donor eyes harvested 0.5 – 5 hours postmortem. Here we show successful recordings of human macular cone photoresponses in samples obtained from eyes enucleated up to 5 hours postmortem. Comparing ‘freshly’ enucleated non-human primate eyes with human eyes enucleated and reoxygenated at different times after death, we show the exponential decay of the light response amplitudes has a time constant of 74 min. We find that both cause of death and donor age are useful parameters for predicting the recovery of retinal light responses in postmortem human macular tissue. Moreover, we present evidence that hypoxia and secondary acidosis, two modifiable factors, are primary contributors to the rapid loss of retinal light signaling after death. Finally, we show postmortem hypoxia rather than acidosis is the major cause of irreversible decay of the light response amplitudes. The criteria and methodology that we have established here for reviving electrical photoresponses in the postmortem human eye will serve as a new starting point for studying neurophysiology and disease in the human retina.

Neuronal plasticity of the inner retina has been observed in response to photoreceptor degeneration. Typically, this phenomenon has been considered maladaptive and may preclude vision restoration in the blind. However, several recent studies utilizing triggered photoreceptor ablation have shown adaptive responses in bipolar cell dendrites expected to support normal vision. Whether such homeostatic plasticity occurs during progressive photoreceptor degenerative disease to help maintain normal visual behavior is unknown. We addressed these issues in an established mouse model of Retinitis Pigmentosa caused by the P23H mutation in rhodopsin. We show robust modulation of the retinal transcriptomic network reminiscent of the neurodevelopmental state as well as potentiation of rod – rod bipolar cell signaling following rod photoreceptor degeneration. Additionally, we found highly sensitive night vision in P23H mice even when more than half of the rod photoreceptors were lost. The results implicate retinal adaptation leading to persistent visual function during photoreceptor degenerative disease.

ObjectiveDiabetic retinopathy is a major complication of diabetes recently associated with compromised photoreceptor function. Multiple stressors in diabetes, such as hyperglycemia, oxidative stress and inflammatory factors, have been identified, but systemic effects of diabetes on outer retina function are incompletely understood. We assessed photoreceptor physiology in vivo and in isolated retinas to better understand how alterations in the cellular environment compared to intrinsic cellular/molecular properties of the photoreceptors, affect light signal transduction and transmission in the retina in chronic type 2 diabetes.Research Design and MethodsPhotoreceptor function was assessed in BKS.Cs-Dock7m +/+ Lepr db/J mice, using homozygotes for Leprdb as a model of type 2 diabetes and heterozygotes as non-diabetic controls. In vivo electroretinogram (ERG) was recorded in dark adapted mice at both 3 and 6 months of age. For ex vivo ERG, isolated retinas were superfused with oxygenated Ames’ media supplemented with 30 mM glucose or mannitol as iso-osmotic control and electrical responses to light stimuli were recorded.ResultsWe found that both transduction and transmission of light signals by rod photoreceptors were compromised in 6 month old (n=9-10 eyes from 5 animals,*** pin vivo (n=4-8 eyes from 2-4 animals). In contrast, rod signaling was similar in isolated retinas from 6 month old control and diabetic mice under normoglycemic conditions (n=11). Acutely elevated glucose ex vivo increased light-evoked rod photoreceptor responses in control mice (n=11, *** pConclusionsOur data suggest that long-term diabetes does not irreversibly change the ability of rod photoreceptors to transduce and mediate light signals. However, type 2 diabetes appears to induce adaptational changes in the rods that render them less sensitive to increased availability of glucose.Significance of this studyWhat is already known about this subject?Retinal neuronal dysfunction in diabetic retinopathy frequently precedes microvascular changes. Damage to photoreceptors has been reported, although the literature is not entirely conclusive, and the underlying mechanisms and reversibility of photoreceptor dysfunction in diabetes remain unknown.What are the new findings?We confirm that retinal function is impaired in vivo in diabetic mice, but report normal function of the isolated diabetic retina under optimal physiological conditions. However, elevated glucose concentrations augment photoreceptor function in the isolated non-diabetic retina, whereas this response is impaired by long-term diabetes.How might these results change the focus of research or clinical practice?Our results clarify that photoreceptor function is not irreversibly damaged and that systemic factors may suppress photoreceptor function in diabetic mice in vivo. The consequences of altered glucose handling in long-term diabetic photoreceptors remain to be evaluated.

Based on clinical findings, diabetic retinopathy (DR) has traditionally been defined as a retinal microvasculopathy. Retinal neuronal dysfunction is now recognized as an early event in the diabetic retina before development of overt DR. While detrimental effects of diabetes on the survival and function of inner retinal cells, such as retinal ganglion cells and amacrine cells, are widely recognized, evidence that photoreceptors in the outer retina undergo early alterations in diabetes has emerged more recently. We review data from preclinical and clinical studies demonstrating a conserved reduction of electrophysiological function in diabetic retinas, as well as evidence for photoreceptor loss. Complementing in vivo studies, we discuss the ex vivo electroretinography technique as a useful method to investigate photoreceptor function in isolated retinas from diabetic animal models. Finally, we consider the possibility that early photoreceptor pathology contributes to the progression of DR, and discuss possible mechanisms of photoreceptor damage in the diabetic retina, such as enhanced production of reactive oxygen species and other inflammatory factors whose detrimental effects may be augmented by phototransduction.

Calcium plays important roles in the function and survival of rod and cone photoreceptor cells. Rapid regulation of calcium in the outer segments of photoreceptors is required for the modulation of phototransduction that drives the termination of the flash response as well as light adaptation in rods and cones. On a slower time scale, maintaining proper calcium homeostasis is critical for the health and survival of photoreceptors. Decades of work have established that the level of calcium in the outer segments of rods and cones is regulated by a dynamic equilibrium between influx via the transduction cGMP-gated channels and extrusion via rod- and cone-specific Na+/Ca2+, K+ exchangers (NCKXs). It had been widely accepted that the only mechanism for extrusion of calcium from rod outer segments is via the rod-specific NCKX1, while extrusion from cone outer segments is driven exclusively by the cone-specific NCKX2. However, recent evidence from mice lacking NCKX1 and NCKX2 have challenged that notion and have revealed a more complex picture, including a NCKX-independent mechanism in rods and two separate NCKX-dependent mechanisms in cones. This review will focus on recent findings on the molecular mechanisms of extrusion of calcium from the outer segments of rod and cone photoreceptors, and the functional and structural changes in photoreceptors when normal extrusion is disrupted.

Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods. In addition, cones can function over a wide range of light conditions whereas rods saturate in moderately bright light. Calcium plays an important role in regulating phototransduction and light adaptation of rods and cones. Notably, the two dominant Ca2+-feedbacks in rods and cones are driven by the identical calcium-binding proteins: guanylyl cyclase activating proteins 1 and 2 (GCAPs), which upregulate the production of cGMP; and recoverin, which regulates the inactivation of visual pigment. Thus, the mechanisms producing the difference in adaptation capacity between rods and cones have remained poorly understood. Using GCAPs/recoverin-deficient mice, we show that mammalian cones possess another Ca2+-dependent mechanism promoting light adaptation. Surprisingly, we also find that, unlike in mouse rods, a unique Ca2+-independent mechanism contributes to cone light adaptation. Our findings point to two novel adaptation mechanisms in mouse cones that likely contribute to the great adaptation capacity of cones over rods.

Sensing light without forming images In the rodent retina, intrinsically photosensitive retinal ganglion cells (ipRGCs) entrain circadian rhythms, modulate mood, and signal pupillary accommodation. Such responses are light-driven but not image-based. Working with donated human organ tissues, Mure et al. used electrophysiological approaches to identify ipRGCs in the human retina. The human retina has more cones than the retinas of nocturnal mice and rats. Differences in sensitivity, latency, and duration of responses identified three subtypes of human ipRGCs. Science , this issue p. 1251

Light adaptation of photoreceptor cells is mediated by Ca(2+)-dependent mechanisms. In darkness, Ca(2+) influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca(2+) extrusion via Na(+)/Ca(2+), K(+) exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca(2+) levels in photoreceptor outer segment because of continuing Ca(2+) extrusion by NCKXs. Guanylate cyclase–activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca(2+). This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca(2+)-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca(2+). Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca(2+) when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones.

The classic concept that GPCRs function as monomers has been challenged by the emerging evidence of GPCR dimerization and oligomerization. Rhodopsin (Rh) is the only GPCR whose native oligomeric arrangement was revealed by atomic force microscopy demonstrating that Rh exists as a dimer. However, the role of Rh dimerization in retinal physiology is currently unknown. In this study, we identified econazole and sulconazole, two small molecules that disrupt Rh dimer contacts, by implementing a cell-based high-throughput screening assay. Racemic mixtures of identified lead compounds were separated and tested for their stereospecific binding to Rh using UV-visible spectroscopy and intrinsic fluorescence of tryptophan (Trp) 265 after illumination. By following the changes in UV-visible spectra and Trp265 fluorescence in vitro, we found that binding of R-econazole modulates the formation of Meta III and quenches the intrinsic fluorescence of Trp265. In addition, electrophysiological ex vivo recording revealed that R-econazole slows photoresponse kinetics, whereas S-econazole decreased the sensitivity of rods without effecting the kinetics. Thus, this study contributes new methodology to identify compounds that disrupt the dimerization of GPCRs in general and validates the first active compounds that disrupt the Rh dimer specifically.-Getter, T., Gulati, S., Zimmerman, R., Chen, Y., Vinberg, F., Palczewski, K. Stereospecific modulation of dimeric rhodopsin.

Photoreceptors are specialized neurons that rely on Ca2+ to regulate phototransduction and neurotransmission. Photoreceptor dysfunction and degeneration occur when intracellular Ca2+ homeostasis is disrupted. Ca2+ homeostasis is maintained partly by mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU), which can influence cytosolic Ca2+ signals, stimulate energy production, and trigger apoptosis. Here we discovered that zebrafish cone photoreceptors express unusually low levels of MCU. We expected that this would be important to prevent mitochondrial Ca2+ overload and consequent cone degeneration. To test this hypothesis, we generated a cone-specific model of MCU overexpression. Surprisingly, we found that cones tolerate MCU overexpression, surviving elevated mitochondrial Ca2+ and disruptions to mitochondrial ultrastructure until late adulthood. We exploited the survival of MCU overexpressing cones to additionally demonstrate that mitochondrial Ca2+ uptake alters the distributions of citric acid cycle intermediates and accelerates recovery kinetics of the cone response to light. Cones adapt to mitochondrial Ca2+ stress by decreasing MICU3, an enhancer of MCU-mediated Ca2+ uptake, and selectively transporting damaged mitochondria away from the ellipsoid toward the synapse. Our findings demonstrate how mitochondrial Ca2+ can influence physiological and metabolic processes in cones and highlight the remarkable ability of cone photoreceptors to adapt to mitochondrial stress.

Mitochondrial Ca2+regulates key cellular processes including cytosolic Ca2+signals, energy production, and susceptibility to apoptosis. Photoreceptors are specialized neurons that have extraordinarily high energy demands and rely on cytosolic Ca2+signals for light adaptation and neurotransmission. Here we show that unlike other neurons zebrafish cone photoreceptors express low levels of the mitochondrial Ca2+uniporter (MCU), the channel that allows mitochondrial Ca2+entry. To determine why MCU expression is kept low, we overexpressed MCU specifically in cones. This increases mitochondrial [Ca2+], causes faster cytosolic Ca2+clearance, and accelerates photoresponse recovery. Moreover, flux through the citric acid cycle increases despite dramatic changes in mitochondrial ultrastructure and localization. Remarkably, cones survive this ongoing stress until late adulthood. Our findings demonstrate the importance of tuning mitochondrial Ca2+influx to modulate physiological and metabolic processes and reveal a novel directed movement of abnormal mitochondria in photoreceptors.

Cones comprise only a small portion of the photoreceptors in mammalian retinas. However, cones are vital for color vision and visual perception, and their loss severely diminishes the quality of life for patients with retinal degenerative diseases. Cones function in bright light and have higher demand for energy than rods; yet, the mechanisms that support the energy requirements of cones are poorly understood. One such pathway that potentially could sustain cones under basal and stress conditions is macroautophagy. We addressed the role of macroautophagy in cones by examining how the genetic block of this pathway affects the structural integrity, survival, and function of these neurons. We found that macroautophagy was not detectable in cones under normal conditions but was readily observed following 24 h of fasting. Consistent with this, starvation induced phosphorylation of AMPK specifically in cones indicating cellular starvation. Inhibiting macroautophagy in cones by deleting the essential macroautophagy gene Atg5 led to reduced cone function following starvation suggesting that cones are sensitive to systemic changes in nutrients and activate macroautophagy to maintain their function. ATG5-deficiency rendered cones susceptible to light-induced damage and caused accumulation of damaged mitochondria in the inner segments, shortening of the outer segments, and degeneration of all cone types, revealing the importance of mitophagy in supporting cone metabolic needs. Our results demonstrate that macroautophagy supports the function and long-term survival of cones providing for their unique metabolic requirements and resistance to stress. Targeting macroautophagy has the potential to preserve cone-mediated vision during retinal degenerative diseases.

Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods. In addition, cones can function over a wide range of light conditions whereas rods saturate in moderately bright light. Calcium plays an important role in regulating phototransduction and light adaptation of rods and cones. Notably, the two dominant Ca2+-feedbacks in rods and cones are driven by the identical calcium-binding proteins: guanylyl cyclase activating proteins 1 and 2 (GCAPs), which upregulate the production of cGMP; and recoverin, which regulates the inactivation of visual pigment. Thus, the mechanisms producing the difference in adaptation capacity between rods and cones have remained poorly understood. Using GCAPs/recoverin-deficient mice, we show that mammalian cones possess another Ca2+-dependent mechanism promoting light adaptation. Surprisingly, we also find that, unlike in mouse rods, a unique Ca2+-independent mechanism contributes to cone light adaptation. Our findings point to two novel adaptation mechanisms in mouse cones that likely contribute to the great adaptation capacity of cones over rods.

The Function of the retina and effects of drugs on it can be assessed by recording transretinal voltage across isolated retina that is perfused with physiological medium. However, building ex vivo ERG apparatus requires substantial amount of time, resources and expertise. Here we adapted a commercial in vivo ERG system for transretinal ERG recordings from rod and cone photoreceptors and compared rod and cone signaling between ex vivo and in vivo environments. We found that the rod and cone a- and b-waves recorded with the transretinal ERG adapter and a standard in vivo ERG system are comparable to those obtained from live anesthetized animals. However, ex vivo responses are somewhat slower and their oscillatory potentials are suppressed as compared to those recorded in vivo. We found that rod amplification constant (A) was comparable between ex vivo and in vivo conditions, ∼10–30s−2 depending on the choice of response normalization. We estimate that the A in cones is between 3 and 6s−2 in ex vivo conditions and by assuming equal A in vivo we arrive to light funnelling factor of 3 for cones in the mouse retina. The ex vivo ERG adapter provides a simple and affordable alternative to designing a custom-built transretinal recordings setup for the study of photoreceptors. Our results provide a roadmap to the rigorous quantitative analysis of rod and cone responses made possible with such a system.

Photoreceptor cell death is the proximal cause of blindness in many retinal degenerative disorders; hence, understanding the gene regulatory networks that promote photoreceptor survival is at the forefront of efforts to combat blindness. Down-regulation of the microRNA (miRNA)-processing enzyme DICER1 in the retinal pigmented epithelium has been implicated in geographic atrophy, an advanced form of age-related macular degeneration (AMD). However, little is known about the function of DICER1 in mature rod photoreceptor cells, another retinal cell type that is severely affected in AMD. Using a conditional-knockout (cKO) mouse model, we report that loss of DICER1 in mature postmitotic rods leads to robust retinal degeneration accompanied by loss of visual function. At 14 wk of age, cKO mice exhibit a 90% reduction in photoreceptor nuclei and a 97% reduction in visual chromophore compared with those in control littermates. Before degeneration, cKO mice do not exhibit significant defects in either phototransduction or the visual cycle, suggesting that miRNAs play a primary role in rod photoreceptor survival. Using comparative small RNA sequencing analysis, we identified rod photoreceptor miRNAs of the miR-22, miR-26, miR-30, miR-92, miR-124, and let-7 families as potential factors involved in regulating the survival of rods.—Sundermeier, T. R., Zhang, N., Vinberg, F., Mustafi, D., Kohno, H., Golczak, M., Bai, X., Maeda, A., Kefalov, V. J., Palczewski, K. DICER1 is essential for survival of postmitotic rod photoreceptor cells in mice.

Calcium ions (Ca2+) modulate the phototransduction cascade of vertebrate cone photoreceptors to tune gain, inactivation, and light adaptation. In darkness, the continuous current entering the cone outer segment through cGMP-gated (CNG) channels is carried in part by Ca2+, which is then extruded back to the extracellular space. The mechanism of Ca2+ extrusion from mammalian cones is not understood. The dominant view has been that the cone-specific isoform of the Na+/Ca2+, K+ exchanger, NCKX2, is responsible for removing Ca2+ from their outer segments. However, indirect evaluation of cone function in NCKX2-deficient (Nckx2−/−) mice by electroretinogram recordings revealed normal photopic b-wave responses. This unexpected result suggested that NCKX2 may not be involved in the Ca2+ homeostasis of mammalian cones. To address this controversy, we examined the expression of NCKX2 in mouse cones and performed transretinal recordings from Nckx2−/− mice to determine the effect of NCKX2 deletion on cone function directly. We found that Nckx2−/− cones exhibit compromised phototransduction inactivation, slower response recovery and delayed background adaptation. We conclude that NCKX2 is required for the maintenance of efficient Ca2+ extrusion from mouse cones. However, surprisingly, Nckx2−/− cones adapted normally in steady background light, indicating the existence of additional Ca2+-extruding mechanisms in mammalian cones.

A previously unidentified calcium-dependent mechanism contributes to light adaptation in mammalian rods., Sensory cells adjust their sensitivity to incoming signals, such as odor or light, in response to changes in background stimulation, thereby extending the range over which they operate. For instance, rod photoreceptors are extremely sensitive in darkness, so that they are able to detect individual photons, but remain responsive to visual stimuli under conditions of bright ambient light, which would be expected to saturate their response given the high gain of the rod transduction cascade in darkness. These photoreceptors regulate their sensitivity to light rapidly and reversibly in response to changes in ambient illumination, thereby avoiding saturation. Calcium ions (Ca2+) play a major role in mediating the rapid, subsecond adaptation to light, and the Ca2+-binding proteins GCAP1 and GCAP2 (or guanylyl cyclase–activating proteins [GCAPs]) have been identified as important mediators of the photoreceptor response to changes in intracellular Ca2+. However, mouse rods lacking both GCAP1 and GCAP2 (GCAP−/−) still show substantial light adaptation. Here, we determined the Ca2+ dependency of this residual light adaptation and, by combining pharmacological, genetic, and electrophysiological tools, showed that an unknown Ca2+-dependent mechanism contributes to light adaptation in GCAP−/− mouse rods. We found that mimicking the light-induced decrease in intracellular [Ca2+] accelerated recovery of the response to visual stimuli and caused a fourfold decrease of sensitivity in GCAP−/− rods. About half of this Ca2+-dependent regulation of sensitivity could be attributed to the recoverin-mediated pathway, whereas half of it was caused by the unknown mechanism. Furthermore, our data demonstrate that the feedback mechanisms regulating the sensitivity of mammalian rods on the second and subsecond time scales are all Ca2+ dependent and that, unlike salamander rods, Ca2+-independent background-induced acceleration of flash response kinetics is rather weak in mouse rods.

Mutations that affect calcium homeostasis (Ca(2+)) in rod photoreceptors are linked to retinal degeneration and visual disorders such as retinitis pigmentosa and congenital stationary night blindness (CSNB). It is thought that the concentration of Ca(2+) in rod outer segments is controlled by a dynamic balance between influx via cGMP-gated (CNG) channels and extrusion via Na(+)/Ca(2+), K(+) exchangers (NCKX1). The extrusion-driven lowering of rod [Ca(2+)]i following light exposure controls their light adaptation and response termination. Mutant NCKX1 has been linked to autosomal-recessive stationary night blindness. However, whether NCKX1 contributes to light adaptation has not been directly tested and the mechanisms by which human NCKX1 mutations cause night blindness are not understood. Here, we report that the deletion of NCKX1 in mice results in malformed outer segment disks, suppressed expression and function of rod CNG channels and a subsequent 100-fold reduction in rod responses, while preserving normal cone responses. The compensating loss of CNG channel function in the absence of NCKX1-mediated Ca(2+) extrusion may prevent toxic Ca(2+) buildup and provides an explanation for the stationary nature of the associated disorder in humans. Surprisingly, the lack of NCKX1 did not compromise rod background light adaptation, suggesting additional Ca(2+)-extruding mechanisms exist in these cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Vision relies on photoactivation of visual pigments in rod and cone photoreceptor cells of the retina. The human eye structure and the absorption spectra of pigments limit our visual perception of light. Our visual perception is most responsive to stimulating light in the 400- to 720-nm (visible) range. First, we demonstrate by psychophysical experiments that humans can perceive infrared laser emission as visible light. Moreover, we show that mammalian photoreceptors can be directly activated by near infrared light with a sensitivity that paradoxically increases at wavelengths above 900 nm, and display quadratic dependence on laser power, indicating a nonlinear optical process. Biochemical experiments with rhodopsin, cone visual pigments, and a chromophore model compound 11-cis-retinyl-propylamine Schiff base demonstrate the direct isomerization of visual chromophore by a two-photon chromophore isomerization. Indeed, quantum mechanics modeling indicates the feasibility of this mechanism. Together, these findings clearly show that human visual perception of near infrared light occurs by two-photon isomerization of visual pigments.

The cone-driven flash responses of mouse electroretinogram (ERG) increase as much as twofold over the course of several minutes during adaptation to a rod-compressing background light. The origins of this phenomenon were investigated in the present work by recording preflash-isolated (M-)cone flash responses ex vivo in darkness and during application of various steady background lights. In this protocol, the cone stimulating flash was preceded by a preflash that maintains rods under saturation (hyperpolarized) to allow selective stimulation of the cones at varying background light levels. The light-induced growth was found to represent true enhancement of cone flash responses with respect to their dark-adapted state. It developed within minutes, and its overall magnitude was a graded function of the background light intensity. The threshold intensity of cone response growth was observed with lights in the low mesopic luminance region, at which rod responses are partly compressed. Maximal effect was reached at intensities sufficient to suppress ∼90% of the rod responses. Light-induced enhancement of the cone photoresponses was not sensitive to antagonists and agonists of glutamatergic transmission. However, applying gap junction blockers to the dark-adapted retina produced qualitatively similar changes in the cone flash responses as did background light and prevented further growth during subsequent light-adaptation. These results are consistent with the idea that cone ERG photoresponses are suppressed in the dark-adapted mouse retina by gap junctional coupling between rods and cones. This coupling would then be gradually and reversibly removed by mesopic background lights, allowing larger functional range for the cone light responses.

BACKGROUND: The rate-limiting step that determines the dominant time constant (τ(D)) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca(2+)-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τ(D) is modulated by background light in mouse rods. METHODOLOGY/PRINCIPAL FINDINGS: We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca(2+) shortens the dominant time constant (τ(D)) of saturated photoresponse recovery, i.e., when extracellular free Ca(2+) is decreased from 1 mM to ∼25 nM, the τ(D) is reversibly increased ∼1.5-2-fold. CONCLUSIONS: We conclude that the increase in τ(D) during low Ca(2+) treatment is not due to increased [cGMP], increased [Na(+)] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca(2+)-dependent mechanism controls the life time of active PDE.

The leading edge of the a-wave of the ERG is generally believed to accurately reflect the changes in the circulating current through the cGMP-gated channels in the outer segment plasma membrane of rods and cones. The aspartate-isolated mammalian electroretinogram (ERG) to a rod-saturating flash contains a fast "nose"-like wave temporally overlapping with the a-wave. We characterize the nature of this nose, investigate the membrane current mechanisms involved in the nose mechanism, and propose a model that can explain the generation of the nose component in the rod inner segment. On the basis of pharmacological treatments and perfusate ion composition alterations we rule out the possible role of most of the known rod membrane current mechanisms that might participate in the generation of the ERG nose component and we propose that the nose is generated by the interplay of voltage-dependent K(x) and h channels together with the Na(+)/K(+) ATPase. Our results strengthen the view that the kinetics of the leading edge of the ERG photoresponses should correspond to that of the outer segment light-sensitive current.

Determine the impact of rod photoreceptor-specific expression of Cre recombinase on the kinetics of phototransduction in the mouse eye and identify changes in gene expression that underlie any observed phenotypic differences.Transretinal ERG and single-cell suction electrode recordings were used to measure the kinetics of phototransduction in a mouse line exhibiting rod photoreceptor-specific Cre recombinase expression, and the results were compared with those from control non-Cre-expressing littermates. Gene expression changes were evaluated using RNA sequencing transcriptome analysis. The pattern of expression of Rgs9bp was determined by mapping sequencing reads to the mouse genome and performing 3'-rapid amplification of cDNA ends (3'-RACE).Expression of the rod-specific iCre75 transgene was accompanied by accelerated phototransduction inactivation, likely due to overexpression of the Rgs9bp gene, which encodes the Rgs9 anchor protein (R9AP). R9AP upregulation stabilized the RGS9 GAP complex, altering phototransduction kinetics. 3'-Race identified an abundant, unexpected Rgs9bp-Prm1 fusion mRNA in Cre-expressing mouse retinas, which was determined to be derived from a second transgene present in the iCre75 line.Here we report the presence of a second, R9AP-expressing transgene in the iCre75 mouse line, leading to altered kinetics of phototransduction. These results highlight an important caveat that must be considered when utilizing this mouse line for rod photoreceptor-specific gene loss of function studies.

Purpose To examine the amplification and kinetics of murine rod photoresponses by recording ERG flash responses in vivo and ex vivo from the same retina. We also aimed to evaluate the two available methods for isolating the rod signal from the ERG flash response, that is, pharmacology and paired flash method on the isolated retina. Methods Dark-adapted ERG responses to full-field flashes of green light were recorded from anesthetized (ketamine/xylazine) C57BL/6N mice. ERG flash responses to homogenous light stimuli arriving from the photoreceptor side were then recorded transretinally from the same retinas, isolated and perfused with Ringer's or Ames' solution at 37°C. The responses were analyzed to determine the a-wave kinetics as well as the estimated flash sensitivity and kinetics of the full rod responses derived with the paired flash protocol. The analysis was complemented with pharmacologic blockade of glutamatergic transmission in the isolated retina. Results The a-waves were of comparable size, sensitivity and kinetics in vivo and in the isolated retina, but the onset of the b-wave was delayed in the isolated retina. The Lamb-Pugh activation constants determined for the a-waves were similar in both preparations. The kinetics of the derived photoreceptor responses were similar in both conditions, although the responses were consistently slightly slower ex vivo. This was not explicable as a direct effect of ketamine or xylazine on the photoreceptors or as their indirect effect through hyperglycemia, as tested on the isolated retina. Conclusions Through comparison to the corneal ERG, the transretinal ERG is a valuable tool for assaying the physiologic state of isolated retinal tissue. The rod photoreceptor responses of the intact isolated retina correspond well to those recorded in vivo. The origin of their faster kinetics compared to single cell recordings remains to be determined.