A modified silent substitution electroretinography protocol to separate photoreceptor subclass function in lightly sedated dogs.

OBJECTIVE: A previously published study successfully isolated photoreceptor responses from canine rods, long/medium-wavelength (L/M) cones, and short-wavelength (S) cones using silent substitution electroretinography (ERG) performed under general anesthesia. We hypothesized that responses would be similar in dogs under sedation and that a curtailed protocol suitable for use in clinical patients could effectively isolate responses from all three photoreceptor subtypes. ANIMALS STUDIED: Three normal adult purpose-bred beagles (2 females and 1 male). METHODS: Dogs were dark-adapted for 1 hour. Sine wave color stimuli were delivered using LEDs in a Ganzfeld dome. The ERG protocol under anesthesia was performed as previously published; sedated ERG protocols were performed after a 3-day washout period. Intravenous sedation (dexmedetomidine 1.25 mcg/kg, butorphanol 0.1 mg/kg) was administered for sedation. Statistical analysis was performed using two-way repeated-measures ANOVA and linear regression. RESULTS: In both anesthetized and sedated animals, rod-derived responses peaked at low frequency (4-12 Hz), L/M-cone responses peaked at high frequency (32-38 Hz), and S-cone responses peaked at low frequency (4-12 Hz). The frequencies eliciting maximal responses were similar in sedated and anesthetized protocols, although rod amplitudes were significantly higher in the sedated protocols compared with anesthetized (P < .001). CONCLUSION: We present a clinically applicable method to consistently isolate rod and cone subclass function in sedated dogs. This may allow detailed evaluation of photoreceptor function in clinical patients with rod or cone subclass deficits without the need for general anesthesia or protracted adaptation times.

Specific connectivity between photoreceptors and horizontal cells in the zebrafish retina.

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

Beyond the Eye: Molecular Evolution of Extraocular Photoreception.

The molecular mechanisms used by biological systems to detect light are diverse, with at least 10 classes of photosensor proteins and additional photosensitive domains characterized. At least six of these protein classes-Type I microbial opsins, Type II animal opsins, cryptochromes, gustatory-related receptors (GRRs), transient receptor potential A1 ion channels, and euglenoid photoactivated adenylyl cylases-can be considered as playing a role in extraocular systems (e.g., expressed outside of the eye in organisms with a visual system). These six classes of extraocular photosensor proteins consist of four broad groups: (1) seven transmembrane proteins, (2) cryptochromes, (3) ion channels, and (4) adenylyl cyclases. The light-driven functions of these extraocular photoreceptors are diverse, ranging from circadian entrainment to phototactic behavior. There are surprising similarities in structural motifs, with at least three independent families-the GRRs and Types I and II opsins-evolving a seven transmembrane helical tertiary structure for light sensing. When considering all of the photosensitive proteins, particularly those in microbial lineages, an image of evolutionary flexibility is emerging, with examples of fusion proteins from multiple types of photosensors and photosensitive domains shared among diverse arrays of proteins. In general, large questions remain for most of these photosensor proteins about exactly how the protein evolved light sensitivity, how light interacts with the protein, and how the photosensitive protein is transducing the signal.

Intrinsic physiological properties of the five types of mouse ganglion-cell photoreceptors.

In the mammalian retina, some ganglion cells express the photopigment melanopsin and function as photoreceptors. Five morphological types of these intrinsically photosensitive retinal ganglion cells (ipRGCs), M1-M5, have been identified in mice. Whereas M1 specializes in non-image-forming visual functions and drives such behaviors as the pupillary light reflex and circadian photoentrainment, the other types appear to contribute to image-forming as well as non-image-forming vision. Recent work has begun to reveal physiological diversity among some of the ipRGC types, including differences in photosensitivity, firing rate, and membrane resistance. To gain further insights into these neurons' functional differences, we conducted a comprehensive survey of the electrophysiological properties of all five morphological types. Compared with the other types, M1 had the highest membrane resistance, longest membrane time constant, lowest spike frequencies, widest action potentials, most positive spike thresholds, smallest hyperpolarization-activated inwardly-rectifying current-induced "sagging" responses to hyperpolarizing currents, and the largest effects of voltage-gated K(+) currents on membrane potentials. M4 and M5 were at the other end of the spectrum for most of these measures, while M2 and M3 tended to be in the middle of this spectrum. Additionally, M1 and M2 cells generated more diverse voltage-gated Ca(2+) currents than M3-M5. In conclusion, M1 cells are significantly different from all other ipRGCs in most respects, possibly reflecting the unique physiological requirements of non-image-forming vision. Furthermore, the non-M1 ipRGCs are electrophysiologically heterogeneous, implicating these cells' diverse functional roles in both non-image-forming vision and pattern vision.

Visual photoreceptor subtypes in the chicken retina: melatonin-synthesizing activity and in vitro differentiation.

The chicken retina contains five visual photoreceptor subtypes, based on the specific opsin gene they express. In addition to the central role they play in vision, some or all of these photoreceptors translate photoperiodic information into a day-night rhythm of melatonin production. This indolic hormone plays an important role in the photoperiodic regulation of retinal physiology. Previous studies have stopped short of establishing whether melatonin synthesis takes place in all the photoreceptor spectral subtypes. Another issue that has been left unsettled by previous studies is when during development are retinal precursor cells committed to a specific photoreceptor subtype and to a melatoninergic phenotype? To address the first question, in situ hybridization of the five opsins was combined with immunofluorescent detection of the melatonin-synthesizing enzyme hydroxyindole O-methyltransferase (HIOMT, EC.2.1.1.4). Confocal microscopy clearly indicated that all photoreceptor spectral subtypes are involved in melatonin synthesis. To tackle the second question, retinal precursor cells were dissociated between embryonic day 6 (E6) and E13 and cultured in serum-free medium for 4 days to examine their ability to autonomously activate the expression of opsins and HIOMT. Real-time PCR on cultured precursors indicated that red-, green- and violet-sensitive cones are committed at E6, rods at E10 and blue-sensitive cones at E12. HIOMT gene expression was programmed at E6, probably reflecting the differentiation of early cones. The present study provides a better characterization of photoreceptor subtypes in the chicken retina and describes a combination of serum-free culture and real-time PCR that should facilitate further developmental studies.

Photoreceptor types, visual pigments, and topographic specializations in the retinas of hydrophiid sea snakes.

Sea snakes have evolved numerous anatomical, physiological, and behavioral adaptations to suit their wholly aquatic lifestyle. However, although sea snakes use vision for foraging and mate selection, little is known about their visual abilities. We used microspectrophotometry, light microscopy, and scanning electron microscopy to characterize the retinal photoreceptors of spine-bellied (Lapemis curtus) and horned (Acalyptophis peronii) sea snakes. Both species have three types of visual pigment sensitive to short (SWS; wavelength of maximum absorbance, λmax 428-430 nm), medium (MWS; λmax 496 nm), and long wavelengths of light (LWS; λmax 555-559 nm) in each of three different subtypes of cone-like single photoreceptor. They also possess a cone-like double photoreceptor subtype, both the principal and accessory member of which contain the LWS visual pigment. Conventional rods were not observed, although the MWS photoreceptor may be a "transmuted" rod. We also used stereology to measure the total number and topographic distribution of neurons in the ganglion cell layer of L. curtus, the olive sea snake (Aipysurus laevis), and the olive-headed sea snake (Disteira major). All species have a horizontal visual streak with specialized areas in the nasal and temporal retina. Both L. curtus and D. major also have a specialized area in the ventral retina, which may reflect differences in habitat usage and/or foraging behavior compared to A. laevis. Maximal spatial resolution was estimated at 1.1, 1.6, and 2.3 cycles deg⁻¹ in D. major, L. curtus, and A. laevis, respectively; the superior value for A. laevis may reflect its specialized crevice-foraging hunting technique.

Two transcription factors can direct three photoreceptor outcomes from rod precursor cells in mouse retinal development.

The typical mammalian visual system is based upon three photoreceptor types: rods for dim light vision and two types of cones (M and S) for color vision in daylight. However, the process that generates photoreceptor diversity and the cell type in which diversity arises remain unclear. Mice deleted for thyroid hormone receptor ß2 (TRß2) and neural retina leucine zipper factor (NRL) lack M cones and rods, respectively, but gain S cones. We therefore tested the hypothesis that NRL and TRß2 direct a common precursor to a rod, M cone, or S cone outcome using Nrl(b2/b2) "knock-in" mice that express TRß2 instead of NRL from the endogenous Nrl gene. Nrl(b2/b2) mice lacked rods and produced excess M cones in contrast to the excess S cones in Nrl(-/-) mice. Notably, the presence of both factors yielded rods in Nrl(+/b2) mice. The results demonstrate innate plasticity in postmitotic rod precursors that allows these cells to form three functional photoreceptor types in response to NRL or TRß2. We also detected precursor cells in normal embryonic retina that transiently coexpressed Nrl and TRß2, suggesting that some precursors may originate in a plastic state. The plasticity of the precursors revealed in Nrl(b2/b2) mice suggests that a two-step transcriptional switch can direct three photoreceptor fates: first, rod versus cone identity dictated by NRL, and second, if NRL fails to act, M versus S cone identity dictated by TRß2.

Why different regions of the retina have different spectral sensitivities: a review of mechanisms and functional significance of intraretinal variability in spectral sensitivity in vertebrates.

Vision is used in nearly all aspects of animal behavior, from prey and predator detection to mate selection and parental care. However, the light environment typically is not uniform in every direction, and visual tasks may be specific to particular parts of an animal's field of view. These spatial differences may explain the presence of several adaptations in the eyes of vertebrates that alter spectral sensitivity of the eye in different directions. Mechanisms that alter spectral sensitivity across the retina include (but are not limited to) variations in: corneal filters, oil droplets, macula lutea, tapeta, chromophore ratios, photoreceptor classes, and opsin expression. The resultant variations in spectral sensitivity across the retina are referred to as intraretinal variability in spectral sensitivity (IVSS). At first considered an obscure and rare phenomenon, it is becoming clear that IVSS is widespread among all vertebrates, and examples have been found from every major group. This review will describe the mechanisms mediating differences in spectral sensitivity, which are in general well understood, as well as explore the functional significance of intraretinal variability, which for the most part is unclear at best.

Photoreceptor coupling is controlled by connexin 35 phosphorylation in zebrafish retina.

Electrical coupling of neurons is widespread throughout the CNS and is observed among retinal photoreceptors from essentially all vertebrates. Coupling dampens voltage noise in photoreceptors and rod-cone coupling provides a means for rod signals to enter the cone pathway, extending the dynamic range of rod-mediated vision. This coupling is dynamically regulated by a circadian rhythm and light adaptation. We examined the molecular mechanism that controls photoreceptor coupling in zebrafish retina. Connexin 35 (homologous to Cx36 of mammals) was found at both cone-cone and rod-cone gap junctions. Photoreceptors showed strong Neurobiotin tracer coupling at night, extensively labeling the network of cones. Tracer coupling was significantly reduced in the daytime, showing a 20-fold lower diffusion coefficient for Neurobiotin transfer. The phosphorylation state of Cx35 at two regulatory phosphorylation sites, Ser110 and Ser276, was directly related to tracer coupling. Phosphorylation was high at night and low during the day. Protein kinase A (PKA) activity directly controlled both phosphorylation state and tracer coupling. Both were significantly increased in the day by pharmacological activation of PKA and significantly reduced at night by inhibition of PKA. The data are consistent with direct phosphorylation of Cx35 by PKA. We conclude that the magnitude of photoreceptor coupling is controlled by the dynamic phosphorylation and dephosphorylation of Cx35. Furthermore, the nighttime state is characterized by extensive coupling that results in a well connected cone network.

Beyond counting photons: trials and trends in vertebrate visual transduction.

For over 30 years, photoreceptors have been an outstanding model system for elucidating basic principles in sensory transduction and G protein signaling. Recently, photoreceptors have become an equally attractive model for studying many facets of neuronal cell biology. The primary goal of this review is to illustrate this rapidly growing trend. We will highlight the areas of active research in photoreceptor biology that reveal how different specialized compartments of the cell cooperate in fulfilling its overall function: converting photon absorption into changes in neurotransmitter release. The same trend brings us closer to understanding how defects in photoreceptor signaling can lead to cell death and retinal degeneration.

Eye and vision in the subterranean rodent cururo (Spalacopus cyanus, Octodontidae).

Subterranean mammals are generally considered to have reduced eyes and apparent blindness as a convergent adaptation to their lightless microhabitat. However, there are substantial interspecific differences. We have studied the prospect of vision in the Chilean subterranean rodent cururo (Spalacopus cyanus, Octodontidae) by analyzing the optical properties of the eye, the presence and distribution of rod and cone photoreceptors, and their spectral sensitivities. Cururo eye size is normal for rodents of similar body size, the cornea and lens are transparent from red to near-UV light, and the retina is well-structured. Electroretinography reveals three spectral mechanisms: a rod with peak sensitivity (lambda(max)) at about 500 nm, a cone with lambda(max) at about 505 nm (green-sensitive L-cone), and a cone with lambda(max) near 365 nm (UV-sensitive S-cone). This suggests dichromatic color vision. Immunocytochemistry with opsin-specific antibodies confirms the presence of rods, L-cones, and S-cones. Cururo rod density is much lower than that of nocturnal surface-dwelling rodents, and the cones form an unexpectedly high 10% proportion of the photoreceptors. Of these, S-cones constitute a regionally varying proportion from 2% in dorsal to 20% in ventral retina. The high cone proportion suggests adaptation to visual demands during the sporadic short phases of diurnal surface activity, rather than to the lightless subterranean environment. Our measurements on fresh cururo urine reveal a high UV reflectance, suggesting that scent marks may be visible to the UV-sensitive cones. The present results challenge the general view of convergent adaptive eye reduction and blindness in subterranean mammals.

Action spectrum for melatonin regulation in humans: evidence for a novel circadian photoreceptor.

The photopigment in the human eye that transduces light for circadian and neuroendocrine regulation, is unknown. The aim of this study was to establish an action spectrum for light-induced melatonin suppression that could help elucidate the ocular photoreceptor system for regulating the human pineal gland. Subjects (37 females, 35 males, mean age of 24.5 +/- 0.3 years) were healthy and had normal color vision. Full-field, monochromatic light exposures took place between 2:00 and 3:30 A.M. while subjects' pupils were dilated. Blood samples collected before and after light exposures were quantified for melatonin. Each subject was tested with at least seven different irradiances of one wavelength with a minimum of 1 week between each nighttime exposure. Nighttime melatonin suppression tests (n = 627) were completed with wavelengths from 420 to 600 nm. The data were fit to eight univariant, sigmoidal fluence-response curves (R(2) = 0.81-0.95). The action spectrum constructed from these data fit an opsin template (R(2) = 0.91), which identifies 446-477 nm as the most potent wavelength region providing circadian input for regulating melatonin secretion. The results suggest that, in humans, a single photopigment may be primarily responsible for melatonin suppression, and its peak absorbance appears to be distinct from that of rod and cone cell photopigments for vision. The data also suggest that this new photopigment is retinaldehyde based. These findings suggest that there is a novel opsin photopigment in the human eye that mediates circadian photoreception.

Vertebrate photoreceptors.

The basis of the duplex theory of vision is examined in view of the dazzling array of data on visual pigment sequences and the pigments they form, on the microspectrophotometry measurements of single photoreceptor cells, on the kinds of photoreceptor cascade enzymes, and on the electrophysiological properties of photoreceptors. The implications of the existence of five distinct visual pigment families are explored, especially with regard to what pigments are in what types of photoreceptors, if there are different phototransduction enzymes associated with different types of photoreceptors, and if there are electrophysiological differences between different types of cones.

Photoreceptor types and distributions in the retinae of insectivores.

The retinae of insectivores have been rarely studied, and their photoreceptor arrangements and expression patterns of visual pigments are largely unknown. We have determined the presence and distribution of cones in three species of shrews (common shrew Sorex araneus, greater white-toothed shrew Crocidura russula, dark forest shrew Crocidura poensis; Soricidae) and in the lesser hedgehog tenrec Echinops telfairi (Tenrecidae). Special cone types were identified and quantified in flattened whole retinae by antisera/antibodies recognizing the middle-to-long-wavelength-sensitive (M/L-)cone opsin and the short-wavelength-sensitive (S-)cone opsin, respectively. A combination of immunocytochemistry with conventional histology was used to assess rod densities and cone/rod ratios. In all four species the rods dominate at densities of about 230,000-260,000/mm2. M/L- and S-cones are present, comprising between 2% of the photoreceptors in the nocturnal Echinops telfairi and 13% in Sorex araneus that has equal diurnal and nocturnal activity phases. This suggests dichromatic color vision like in many other mammals. A striking feature in all four species are dramatically higher S-cone proportions in ventral than in dorsal retina (0.5% vs. 2.5-12% in Sorex, 5-15% vs. 30-45% in Crocidura poensis, 3-12% vs. 20-50% in Crocidura russula, 10-30% vs. 40-70% in Echinops). The functional and comparative aspects of these structural findings are discussed.

The foveal photoreceptor mosaic in the pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei).

The foveal and non-foveal retinal regions of the pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei) are examined at the level of the light and electron microscopes. The pipefish possesses a deep, pit (convexiclivate) fovea which, although lacking the displacement of the inner retinal layers as described in other vertebrate foveae, is characterised by the exclusion of rods, a marked increase in the density of photoreceptors and a regular square mosaic of four double cones surrounding a central single cone. In the perifoveal and peripheral retinal regions, the photoreceptor mosaic is disrupted by the insertion of large numbers of rods, which reduce spatial resolving power but may uniformly increase sensitivity for off-axis rays. In addition to a temporal fovea subtending the frontal binocular field, there is also a central area centralis subtending the monocular visual field. Based on morphological comparisons with other foveate teleosts, four foveal types are characterised and foveal function discussed with respect to the theoretical advantage of a regular square mosaic.