Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Abstract Full Text (PDF, 4270K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Nikonov, S. S. Articles by Pugh, E. N. Search for Related Content PubMed PubMed Citation Articles by Nikonov, S. S. Articles by Pugh, E. N., Jr. Social Bookmarking                            What's this?

¹ F. M. Kirby Center for Molecular Ophthalmology, Department of Ophthalmology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
² Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, and Department of Ophthalmology and Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033
³ Department of Ophthalmology and Visual Sciences and Department of Human Genetics, University of Michigan, Ann Arbor, MI 48105

Correspondence to Edward N. Pugh Jr.: pugh{at}mail.med.upenn.edu

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl ^–/–) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl ^–/– mice were recorded with suction pipettes at 35–37°C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette ("OS in"), or in a novel configuration with a portion of the inner segment drawn in ("OS out"). Nrl ^–/– photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses t_peak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, _D = 110 ms vs. 240 ms, respectively. Nrl ^–/– photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl ^–/– outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl ^–/– cell were driven by both the short-wave (S, _max 360 nm) and the mid-wave (M, _max 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl ^–/–/Grk1^–/– photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to normal inactivation of both S- and M-mouse cone opsins, but S-opsin has access to a relatively effective, Grk1-independent inactivation pathway.

Key Words: phototransduction • GPCR signaling • cone opsin • Grk1 • spectral sensitivity

Abbreviations used in this article: OS, outer segment; WT, wild-type.

¹ The mouse genome contains the genes of two cone pigments, one from the shortwave sensitive class 1 (SWS1) homology group having _max = 359 nm (Yokoyama et al., 1998), and a second from the long/midwave sensitive homology group (MWS/LWS) with _max = 508 nm (Sun et al., 1997). The SWS1 group has _maxs ranging from 358 to 425 nm and includes the human S-cone pigment, while the MWS/LWS group has _maxs ranging from 508 to 611 nm and includes both the human M-cone and L-cone pigments (Yokoyama and Yokoyama, 2000; Ebrey and Koutalos, 2001). To reflect their homology group memberships, in this manuscript we will often refer to the mouse cone pigments with the generic labels "S-cone pigment" and "M-cone pigment." At other times, when the ultraviolet sensitivity of the mouse S-pigment is at issue, we will use the terminology "UV-cone pigment" to denote the mouse cone pigment with _max = 359 nm.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Healthy cone photoreceptor function is essential to normal human vision. The special importance of cones comes from at least three features of these cells: first, they initiate vision in the macula, the highly specialized central region of the retina whose signals map to a large portion of primary visual cortex; second, they provide the signals for color vision, allowing us to discriminate targets on the basis of their spectral content; third, they enable the retina to signal in the presence of strong ambient illumination (Burkhardt, 1994; Pugh et al., 1999; Paupoo et al., 2000).

Cone-specific disease, and disease of nearby rod and retinal pigment epithelium cells that lead to the demise of cones, have disastrous consequences for human vision (http://www.sph.uth.tmc.edu/Retnet/). More than 15 genes are currently known to be associated with autosomal dominant macular degeneration (http://www.sph.uth.tmc.edu/Retnet/); many more will certainly be discovered to be involved in age-related macular degeneration (Stone et al., 2004). To investigate the molecular mechanisms of cone disease, as well as the normal mechanisms that allow cones to perform their unique functions, it is critical to have mammalian models that allow (1) genomic analysis, and manipulation of genes expressed in cones; (2) molecular and biochemical characterization of the protein products of such genes; and (3) functional analysis of cones and their circuits. There is a striking paucity of such models when it comes to cones, and none yet that allow investigation on all three levels.

The mouse is increasingly the mammalian species of choice for the investigation of the functional properties of neuronal cell types and tissues, because of the advanced genomics, the array of tools available for its genetic manipulation, and the rapid generational time. In retinal research, definitive studies of the role of specific proteins in rod phototransduction (Xu et al., 1997; Chen et al., 1999, 2000; Mendez et al., 2000, 2001; Burns et al., 2002) and rod-based disease (for review see Pierce, 2001) have come from the genetic manipulation of rod photoreceptor-specific genes in mouse. However, in wild-type (WT) mice, cones constitute only 3% of the photoreceptors, with rods comprising the remaining 97% (Carter-Dawson and LaVail, 1979), precluding ready isolation of cone-specific proteins in the large background of their rod homologues. Furthermore, mouse cones have few morphological features that would allow them to be distinguished from rods under the infrared viewing conditions required for single-cell photoreceptor electrophysiology. Thus, despite a few studies demonstrating cone-specific effects of photoreceptor-specific gene deletions in mouse with electroretinographic responses (Lyubarsky et al., 1999, 2000, 2001; Pennesi et al., 2003), the full-scale investigation of molecularly manipulated cone-specific genes has been an elusive goal.

The Nrl ^–/– mouse is a promising mouse model for the investigation of cone function. The developing Nrl ^–/– retina produces no rods (Mears et al., 2001); rather, it is populated with photoreceptors exhibiting ultrastructural, histochemical, molecular, and kinetic features that support the hypothesis that they are indeed cones (Daniele et al., 2005). Here we establish that the single cone photoreceptors of the Nrl ^–/– mouse can be characterized in detail with single-cell suction pipette recordings.

The coexpression of two visual pigments in single vertebrate cone photoreceptor cells conflicts with the requirement for distinct neural channels to encode spectral information; the capacity to discriminate lights on the basis of their spectral content is potentially diminished when both pigments drive transduction in a single cone. Nonetheless, immunohistochemical evidence shows that cones of many mammals coexpress both an S-pigment and an M/L-pigment, including rabbits, hamsters, guinea pigs, various strains of mice (Rohlich et al., 1994; Ahnelt, 1998; Applebury et al., 2000; Lukats et al., 2002), pigs (Hendrickson and Hicks, 2002), and humans, both fetal and, to a much less extent, adult (Xiao and Hendrickson, 2000; Cornish et al., 2004). While pigment coexpression in cones is incontrovertible, its functional consequences remain largely unstudied. Cone-driven ERG b-wave evidence is consistent with the hypothesis that both S- and M-pigments¹ drive phototransduction in mouse cones (Lyubarsky et al., 1999), but this latter evidence could be explained by convergence of signals from distinct cone classes onto proximal neurons. Thus, there is of yet no incontrovertible evidence that both coexpressed photopigments drive phototransduction in individual cones. Nrl ^–/– photoreceptors now permit for the first time the characterization of the physiological consequences of coexpression, allowing detection of coexpression levels of cone M-pigment relative to UV-pigment at ratios as low as 1:10,000. Finally, the presence of two mammalian cone pigments in the same photoreceptor cell make it possible for the first time to compare responses driven by one or the other, and to assess the dependence of the inactivation of the two cone pigments on Grk1, the only G-protein receptor kinase known to be expressed in mouse cones. By examining the responses of photoreceptors of Nrl ^–/–/Grk1^–/– mice, we have determined that Grk1 is involved in the inactivation of both pigments, but that the S-pigment appears to possess an effective, Grk1-independent mechanism.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals
All experiments were performed in compliance with National Institutes of Health guidelines, as approved by the Institutional Animal Care and Use Committees of the University of Pennsylvania. Nrl ^–/– and Rho^–/– mice were generated at the University of Michigan (Mears et al., 2001) and New England Medical Center (Lem et al., 1999), respectively. Animals used for recordings were born and maintained in controlled ambient illumination on a 12-h light/dark cycle, with an illumination level of 2–3 lux. Nrl ^–/–/Grk1^–/– double knockout mice were produced at the University of Southern California (Zhu et al., 2003) by crossing the Nrl ^–/– mice with Grk1^–/– mice (Chen et al., 1999).

Histochemistry
Mouse eyes were removed and placed into 4% paraformaldehyde for at least 24 h, followed by overnight incubation in 30% sucrose/PBS. Cryosections of 20-µm thickness were made from whole Nrl ^–/– mouse eyes, embedded in tissue freezing medium (Triangle Biomedical Sciences), and kept at –80°C. Frozen sections were first washed and then incubated with normal goat serum, followed by primary antibodies at 1:5,000 dilution in PBS containing 5% BSA, 0.1% sodium azide, and 0.1% Triton X-100. After washing, sections were incubated in secondary antibodies at 1:300 dilution. The primary antibody was a rabbit polyclonal raised against residues 3–16 of the mouse M-opsin (Zhu et al., 2003). Secondary antibodies were goat anti-rabbit conjugated to FITC (Jackson ImmunoResearch Laboratories).

Tissue Preparation and Electrophysiological Methods
Mice were killed, the eyes enucleated, and whole retinas removed from eye cups under infrared illumination. Small pieces of retina were dissected in a drop of chilled Locke's solution (112.5 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl₂, 1.2 mM CaCl₂, 10 mM Hepes, 0.02 mM EDTA, 20 mM NaHCO₃, 3 mM Na₂-succinate, 0.5 mM Na-glutamate, 10 mM glucose), and placed into a recording chamber. The chamber was continuously refreshed with Locke's solution, pH 7.4, equilibrated with 95% O₂/5% CO₂, and maintained at 35–37°C with a heating system designed for microscopy (ALA Scientific). Using silanized suction pipettes, we recorded from photoreceptors embedded in 50–100-µm diameter slices of retina in either of two configurations, as we explain in the next section. In both recording configurations, once the tissue was drawn into the pipette, responses were evoked with calibrated flashes of light under control of a customized LabView (National Instruments Corp.) interface.

Novel Method of Suction Pipette Recording from the Perinuclear Region of Mouse Photoreceptors
In our initial efforts to record from Nrl ^–/– photoreceptors, we employed the only method used to date for recording from mouse photoreceptors, drawing the outer segment into the suction pipette (e.g., Baylor et al., 1979b; Chen et al., 1995, 1999; Xu et al., 1997). However, we discovered (and report below) that the functional properties of Nrl ^–/– photoreceptors, unlike those of mouse rods, deteriorated during the recording epoch. These observations led us to develop a novel method of recording, in which a portion of the perinuclear region (broadly speaking, the "inner segment") of the photoceptor is drawn into the suction pipette (Fig. 1). A long history of suction pipette recordings from amphibian rods and cones has shown that essentially the same information can be obtained by recording from either segment (e.g., Cobbs and Pugh, 1987), as expected from the nature of the circulating current whose source is primarily K⁺-selective current in the inner segment/nuclear region and whose sink is the outer segment cGMP-activated current (Hagins et al., 1970). However, mouse photoreceptors, unlike those of amphibians, are not readily isolated from one another, most likely because of much greater cell–cell adhesions in the outer nuclear layer and near the outer limiting membrane. We nonetheless found that very good recordings could be obtained with suction pipettes applied to thin retinal slices from which cells were not isolated, in effect using a "loose patch" configuration to record the portion of the overall circulating that flows through the perinuclear region (Fig. 1). To make the nomenclature of the recording methods simple, we have named the new method "outer segment out" or "OS out" in contrast to the traditional "OS in" method. In the body of the paper we document the features of each type of recording.

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Illustration of two methods of "loose patch" recording from photoreceptors of the Nrl –/– (A and B) and WT (C and D) mouse retinas, imaged with infrared illumination ( > 1000 nm). (A) Conventional "outer segment in" (OS in) method in which all or a portion of the outer segment is drawn into the suction pipette; the saturating photocurrent recorded in this case was 7 pA. Line drawings have been overlaid on the image to identify the key structures: OS, outer segment; IS, inner segment; N, nuclear region; RPE, retinal pigment epithelium; OLM, outer limiting membrane. At right one can see a array of refractive index variations perpendicular to the RPE layer that arise from the "palisade" of outer segments projecting into the RPE cell layer; one photoreceptor has been outlined on this array. Semithin plastic sections and high resolution EM images of the photoreceptor/RPE layers of the Nrl –/– retina (Daniele et al., 2005) have been used as a basis for the size and shape of the outlined cells; the images in this figure are blurred due to the limited resolution of infrared imaging. (B) OS-out method of recording in which the nuclear region of one (or more) cell is drawn into the suction pipette; in this case the saturating response was 9 pA, and data from this cell are included in the paper. The images of A and B are of the same retinal slice (though shifted in the chamber). (C) Image of WT retina with portion of rod outer segment drawn into the suction pipette (OS-in configuration); no recording was associated with this image, but the average amplitude of the saturating photocurrent in this configuration was 16 pA (Table I). (D) Image of WT retina with nuclear region of cells drawn into pipette; the saturating amplitude of the photocurrent was 34 pA, and it appeared that two nuclei had been drawn into the pipette. In each image the white bar represents 10 µm.

Light Stimulation and Calibration
Flash stimulation was provided by two light sources, a tungsten halogen lamp, whose exposure duration was controlled by an electronic shutter (Uniblitz Model 222; Vincent Associates), and a xenon flash lamp that generates flashes of 20 µs duration (Cobbs and Pugh, 1987). Flash intensity was varied by calibrated neutral density filters. Fully blocked interference filters with 10-nm bandwidth (FWHM transmittance) were used to produce monochromatic stimuli.

Flash energy densities were measured in photons µm^–2 at the image plane of the inverted microscope with calibrated photodiodes (United Detector Technology). The number of photoisomerizations per photoreceptor produced by a flash was estimated as the product of the energy density (photons µm^–2) and the outer segment collecting area, a_c (µm²), as described below.

Data Acquisition
Data acquisition, stimulus timing, as well as control of neutral density and interference filters were under the control of a computer with a customized Labview interface (National Instruments Corp.). A current-to-voltage converter (model 8900; Dagan Corp.) was used to measure membrane photocurrents of outer segments; responses were filtered with a 4-pole lowpass filter with cutoff set to 30 Hz. Signals were digitized at 200 Hz with an A-D converter (National Instruments Corp.). Custom scripts written with Matlab software (Mathworks Corp.) were used to extract single trials from stored records, and to perform sorting, averaging, and other analyses.

Estimation of Light Collecting Area of WT Rods and Nrl ^–/– Photoreceptors
The light collecting area of mouse photoreceptors illuminated transversely with unpolarized light in the recording chamber was estimated with the following formula:

(1)

where f is a factor that depends on the polarization of the incident light relative to the plane of the disc membranes, _max is the extinction coefficient at its _max of the pigment in solution, the quantum efficiency of photoisomerization, C the concentration (M) of the pigment in the outer segment, and V_OS (µm³) the envelope volume of the outer segment, and the factor 10^–4 is required for consistency with the dimensions of V_OS. This formula is essentially that of Baylor et al. (1979a), Eq. 20, except for the substitution of the product f_maxC for the specific pigment density.

Collecting Area of Rods.
For WT mouse rods, we adopted the values _max = 42,000 liter (mol cm)^–1 (Saari et al., 2001), = 0.67, C = 0.003 M, and f = 3/4. The value C = 0.003 M is derived from many microspectrophotometric (MSP) studies of rod and cone visual pigments in cells of larger diameter than mouse rods (Liebman, 1972), including "supersized" peripheral rods of some primates (Harosi, 1982). The average diameter and length of mouse rod outer segments are 1.4 and 23.6 µm, respectively (Carter-Dawson and LaVail, 1979), giving V_OS = 37 µm³. With all the parameters in Eq. 1 thus specified, the collecting area for a rod transversely illuminated in our recording chamber is estimated to be a_c = 0.54 µm². The estimate a_c = 0.48 µm² for mouse rods was provided in a recent investigation involving a similar experimental chamber (Calvert et al., 2001), and we thus adopted a_c = 0.5 µm² as a reasonable compromise.

Collecting Area of Nrl ^–/– Photoreceptors.
For Nrl ^–/– cells, we adopted the following values for the constants in Eq. 1: _max = 41,670 liter (mol cm)^–1 (Vought et al., 1999), = 0.67, and C = 0.003 M (the value used for is that widely accepted for mammalian rhodopsin, but we note that Okano et al. [1992] have estimated = 0.61 and 0.62 for chicken cone rhodopsin and iodopsin, respectively). The pigment concentration in cones expressing a UV pigment has not been estimated with MSP, and almost certainly cannot be accurately measured in WT mouse cones or Nrl ^–/– photoreceptors due to their narrow width. However, quantitative immunoblot analysis of the total UV pigment content of the eye is consistent with a concentration equal to that of rods (Daniele et al., 2005). We assume f = 3/4, as assumed for rods whose currents were recorded in the same configuration (above). Because experimenter selection might affect the length of the OS's of the cells from which we record electrically, we estimated the length of Nrl ^–/– outer segments from confocal images of pieces of live retina prepared in the same manner as for our physiological experiments, but incubated with the permeant fluorescent dye Calcein AM (Molecular Probes). This method gave abundant images of Nrl ^–/– outer segments resembling those seen under infrared illumination during physiological experiments. Outer segments in these confocal images had length 7.1 ± 0.2 µm (mean ± SEM, n = 42; unpublished data), indistinguishable from the length, 7.3 ± 0.3 µm, measured with EM (Daniele et al., 2005). The average OS volume estimated from the EM data is V_OS = 8.3 µm³. With all parameters in Eq. 1 thus specified, we obtain a_C = 0.11 µm² for the Nrl ^–/– outer segments in our experimental conditions. We assumed the photoreceptors of Rho^–/– mice to have the same collecting area as those of the Nrl ^–/–, based on the similarity of their appearance under recording conditions. We took this parallel approach to estimating a_C of rods of WT mice, and of Nrl ^–/– and Rho^–/– photoreceptors in order to make comparisons of the relative flash sensitivities and amplifications of the photoreceptor classes in the units of photoisomerizations/flash at _max, an approach that allows a comparison of the underlying transduction mechanisms in units intrinsic to photoreceptor function.

Quantitative Analysis of Response Data
The activation phase of families of normalized responses R(t) were fitted with a model of the phototransduction cascade (Lamb and Pugh, 1992; Pugh and Lamb, 1993),

(2)

In Eq. 2, "" signifies a definition, r(t) is the photoresponse, r_max its saturating amplitude, the number of photoisomerizations produced by the flash, and t_eff a brief (several ms) delay. Traces computed with Eq. 2 were convolved with digital filters to incorporate the effect of the membrane time constants of rods and cones (Smith and Lamb, 1997), set to _m = 1 or 5 ms, respectively, and the measured impulse response function of the analogue filter.

Amplitude vs. intensity functions were derived from response families and fitted with hyperbolic saturation functions of the form

(3)

where r(t_peak) is the amplitude at the time to peak, t_peak, of the response, Q is the flash intensity in photons µm^–2, and Q_1/2 the half-saturating intensity. With the amplitude–intensity function expressed in these units, the flash sensitivity S_F of the normalized response is S_F = 1/Q_1/2.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
M-opsin Is Expressed in a Dorso-ventral Gradient in the Nrl ^–/– Retina
As revealed with immunohistochemistry, rodent retinas typically express two cone visual pigments, an S-opsin and an M-opsin, and most rodents are known to coexpress these opsins in at least some of their cones (Rohlich et al., 1994; Applebury et al., 2000). Therefore, we investigated the extent of M-cone pigment expression in the Nrl ^–/– retina with immunolabeling. A clear dorso-ventral gradient of labeling is observed (Fig. 2). Comparisons of immunolabeled cells with concurrently taken DIC images indicated that most photoreceptors express M opsin at a level that can be detected with immunolabeling (unpublished data). Our physiological experiments (described below) reveal both mouse UV-cone opsin and M-cone opsin to be present in each Nrl ^–/– photoreceptor from which we have recorded and, further, allowed quantitative assessment of the relative levels of expression of M- and UV-opsin in single cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Dorso-ventral gradient of M-opsin expression in the Nrl –/– retina. Image shows a montage of a series of confocal scans from a single transverse section of a 3-mo-old mouse labeled with polyclonal rabbit anti-mouse M-opsin antibody (green) and mounted with a DAPI-containing mounting medium (blue) to show nuclei of the cells in different layers. The dashed boxes around dorsal and ventral portions of the retina illustrate the approximate retinal locations from which retinal slices were taken for the experiments described in Figs. 5 and 6.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. Comparison of the kinetics and sensitivity of responses of Nrl –/– and WT rod photoreceptors recorded in two configurations. Rods were stimulated with 501-nm flashes, while the Nrl –/– cells were stimulated with 361-nm flashes. Each row of panels presents data from one photoreceptor, recorded in one of two recording configurations described in Fig. 1. A, D, G, and J are light response families, while B, E, H, and K present the activation phases of the corresponding response family on a faster time base, with the traces fitted with the G-protein cascade model (smooth gray traces; cf. MATERIALS AND METHODS, Eq. 2); the corresponding amplification constants are given in the figures. C, F, I, and L plot the amplitude vs. intensity functions derived from the response family in the same row (left ordinate) and plot the time to 40% recovery (right ordinate) for responses to the intensities specified by the abscissa. The amplitude vs. intensity functions have been fitted with hyperbolic saturation functions (Eq. 3), whose half-saturation magnitudes, Q1/2, are given in the figures. The recovery time points to the lowermost saturating intensities have been fitted in semilog coordinates ("Pepperberg plots") with a straight line to obtain the rate of increase in recovery time per e-fold change in intensity, which estimates D, the dominant recovery time constant (Pepperberg et al., 1994; Nikonov et al., 1998). All responses were filtered during acquisition with a 4-pole low pass analogue filter set at 30 Hz and digitized at 200 Hz. At least three responses to the same flash intensity were averaged for each trace, and at least 10 individual records were used to obtain the averaged responses to the dimmest flashes. The saturating photocurrent amplitudes were 26 pA (Nrl –/– cell, OS in), 10 pA (Nrl –/– cell, OS out), 26 pA (rod, OS in), 13 pA (rod, OS out). Note that the results in D and E are presented on a twofold shorter time scale than in the other panels.

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Relative stability of the response properties of Nrl –/– photoreceptors. (A) Traces show responses of a single Nrl –/– cell to dim (2,100 photons µm–2) and bright (650,000 photons µm–2) flashes of 361 nm at three different times during a 15-min recording epoch: t1, the 1st min of recording; t2, the 2nd to 5th min; t3, the 15th min; responses got progressively slower and less sensitive. Note that dim-flash responses were not recording in the t1 time frame. (B) Amplitude vs. intensity functions and "Pepperberg plots" for the cell of panel A at various times in the recording session (ti refers to the same period as in A). At about t2, the half-saturating flash intensity, Q1/2, was 5,000 photons µm–2, but as the cell became less sensitive, Q1/2 shifted to 12,000. The dominant recovery time constant D was estimated as in the experiments of Fig. 1, and found to change little over the time course of the experiment. (C–F) Properties of the responses of 21 Nrl –/– cells (OS in, •) and 8 Nrl –/– cells (OS out, ) plotted as a function of time; the data of each cell was scaled relative to its values at 3 min (dashed line), and then the data of each scaled population dataset averaged to produce the plotted points. The minimum number of records averaged for the first time point was 8 (OS in) and 2 (OS out), while for all other time points the number of records averaged ranged from 21 to 8 for cells in the two configurations, respectively. Horizontal bars give the time bracket of data extraction from the raw records; vertical error bars are 95% confidence intervals. The amplification of phototransduction, characterized by the coefficient A (D), declined very rapidly for Nrl –/– cells recorded in the OS-in configuration, and its value early in the recording epoch was estimated from a limited number of flashes rather than from complete response families.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Spectral sensitivities of single mouse photoreceptors. The sensitivity of the cells of Nrl –/– and Rho–/– mice were normalized to unity at 361 nm, while rod sensitivities were normalized at 500 nm. (A) Data from a single Nrl –/– cell stimulated at 11 different wavelengths () and data from a Rho–/– cell stimulated at 7 different wavelengths (gray triangle); both cells were recorded in the OS-in configuration. Averaged spectral sensitivities of seven rods (OS in, ), and 2 rods (OS out, gray circle); error bars are standard deviations. The unbroken curves are pigment templates (Lamb, 1995) for the three murine pigments: max = 361 nm (purple curve; UV-cone pigment), max = 508 nm (green curve; M-cone pigment), and max = 500 nm (black; rhodopsin; for < 450 nm the rhodopsin template was extended into the short-wave region of the pigment ß-band according to Govardovskii et al. (2000). Dotted traces are the sum of M-pigment templates and UV-pigment template. (B) Results from 9 Nrl –/– cells stimulated with 361, 401, and 420-nm flashes (); error bars are 95% confidence intervals. Results from 36 different Nrl –/– cells stimulated with 361-nm and 501 or 515-nm flashes (), and from 8 Rho–/– cells (gray triangle). The gray arrow indicates the gap between the measured sensitivity of the Nrl –/– cells least sensitive at 510 nm and the predicted sensitivity of a cell expressing only the UV-cone pigment (violet curve). Data points plotted to the right of the abscissa break show sensitivity measured at 515 nm for 12 Nrl –/– cells from the dorsal part of the retina (green triangle), one cell from the central part of the retina (blue triangle) and 12 cells from the ventral part of the retina (pink triangle). All results for this analysis were recorded in the OS-in configuration.

An important question is whether the large variation in coexpression of the M-pigment manifest in the spectral sensitivity near 510 nm reflects the dorso-ventral gradient seen in Fig. 2. To address this question, we measured the spectral sensitivity of photoreceptors from the most dorsal and most ventral portions of the retina (Fig. 5 B; cf. Fig. 2); the coexpression of M-pigment in cells from the dorsal retina was on average almost 100-fold greater than in cells from the ventral retina. However, there was also substantial variation in the degree of coexpression in the dorsal and ventral retina areas probed.

Coexpression of M-pigment Is Correlated with Reduced Absolute Sensitivity of the UV-pigment–driven Response
The dorso-ventral gradient of M-pigment coexpression suggests that at least the M-cone pigment level is under the regulation of a factor that varies in a dorso-ventral manner. We examined the hypothesis that the UV-cone pigment might also be regulated in a dorso-ventral gradient, but in an "inverse" manner to that of the M-pigment. We plotted the absolute sensitivity of Nrl ^–/– photoreceptors to 361-nm flashes as a function of the "coexpression ratio," defined as s_F(515)/s_F(361), the ratio of sensitivity to 515-nm flashes relative to the sensitivity to 361-nm flashes (Fig. 6). There is a reliable negative correlation between log₁₀[s_F(361)] and log₁₀[s_F(515)/s_F(361)], with cells from the ventral retina having a higher absolute sensitivity to UV light. Interpreting the sensitivities as surrogates for the pigment expression levels, this result suggests that UV-cone pigment expression is down-regulated in cones in the regions of the retina where the M-pigment is up-regulated, and vice versa. In absolute terms, the effect is not large; thus, the sensitivity to UV light only declines (on average) about twofold over the nearly 3.5 log₁₀ domain of coexpression. That the negative correlation is manifest on a 3,000-fold scale of coexpression indicates the that underlying regulatory mechanisms do not simply act to conserve the total amount of pigment; for example, this mechanism apparently exerts an influence on UV-pigment expression level even when M-pigment levels are 1/1,000 to 1/100 that of the UV-pigment.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. M-cone pigment coexpression in Nrl –/– photoreceptors affects the absolute sensitivity to stimuli detected by the UV-cone pigment. Each point in the plot represents data from a single Nrl –/– photoreceptor; the purple circles represent data from cells in slices from the most ventral portion of the retina, while the green circles represent data from cells in slices from the most dorsal portion of the retina, and the white circle one cell from a slice near the optic disc (see Fig. 2, colored boxes); these cells were recorded from in the OS-in configuration in a simplified protocol that focused only on the dim-flash responses. The gray circles plot data from cells recorded from in the OS-out configuration, in slices from unknown retinal locations. The abscissa plots the "coexpression ratio," defined as the ratio of the sensitivities (sF) to dim flashes of 515 and 361-nm stimulation; the ordinate plots the absolute sensitivity to 361-nm stimulation, sF(361). The black line was determined from Pearson product-moment correlation analysis of the bivariate data: its slope is –0.08 log10 units of sF(361) per log10 units of the coexpression ratio; the slope is significantly different from zero (tdf=36 = –2.27, P < 0.02).

View larger version (55K): [in this window] [in a new window]   FIGURE 7. Dim-flash responses of individual photoreceptors from Nrl –/– mice (A) or Nrl –/–/Grk1–/– mice (B) driven by either the UV-cone pigment (purple traces) or the M-cone pigment (green). Each panel presents averaged responses of a different single cell in response to a 361-nm flash (purple traces) and a 501 or 515-nm flash (green traces). In each experiment, the thicker purple trace was obtained first, then the green trace, and finally the thinner purple trace. The average number of responses taken for each trace was 31 (range, 10–70). The fraction of current suppressed by the flashes was 0.20 ± 0.11 (mean ± SD taken over the population) of the maximum, and did not differ for the responses to the UV and midwave flashes. Each trace is scaled to unity at its peak.

To quantify the comparisons of the recovery kinetics of the responses driven by the two cone pigments in individual cells, we measured a simple feature of the dim-flash response, its full-width at half-maximum, or T₅₀ (Fig. 8 A). T₅₀s for the UV- and M-cone pigment–driven responses for animals of the two genotypes are summarized in the bar chart of Fig. 8 B. While the deletion of Grk1 slowed the recovery of responses driven by both pigments, the effect on the M-pigment response was much larger; thus, in the absence of Grk1, the average T₅₀ of the response to the M-pigment increased 7.5-fold (from 0.33 to 2.44 s), while the T₅₀ of the UV-pigment response increased 2.8-fold (from 0.25 to 0.69 s); both these differences in T₅₀ are highly significant (P < 0.0004), arguing strongly that Grk1 is necessary for normal inactivation of both pigments. Since the recordings used for comparison of dim-flash responses were made with the outer segment drawn into the suction pipette, caution is called for in interpreting the absolute numerical values of the T₅₀s. Nonetheless, as comparisons were only made when the UV-pigment–driven response was unchanged throughout the recording epoch, it can be concluded that the inactivation of the M-cone pigment is more severely affected by the absence of Grk1 than that of the UV-cone pigment.

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Quantitative comparison of the recoveries of dim-flash responses of photoreceptors of Nrl –/– mice or Nrl –/–/Grk1–/– mice, driven by either the UV-cone pigment (purple) or M-cone pigment (green). (A) Method of quantification: the full width half-maximum (T50) of the normalized dim-flash response was determined from the intersection of the traces with a line at 50% amplitude; three intersections are illustrated by cross-hairs superimposed on the traces. T50 is the interval between the initial intersection with the response rising phase and the second intersection with the recovery phase. (B) Histograms of the T50s for the UV-cone pigment (purple bars) and M-cone pigment–driven responses (green bars) from populations of cells from animals of the two genotypes (Nrl –/–, n = 17 cells; Nrl –/–/Grk1–/–, n = 9 cells); the error bars are 95% confidence intervals. Statistical analysis showed that all the values of T50 represented in Fig. 6 B are significantly different from one another on the basis of t tests (two samples, unequal variance, one-tail): for Nrl –/– cells, UV vs. M-opsin–driven responses, P < 0.032; for Nrl –/–/Grk1–/– cells, UV vs. M-opsin–driven responses, P < 10–4; for UV-opsin–driven responses of Nrl –/– cells vs. Nrl –/–/Grk1–/– cells, P < 10–3; for M-opsin–driven responses of Nrl –/– cells vs. Nrl –/–/Grk1–/– cells, P < 10–4. There was no statistically reliable difference between the T50s of the "pre" and "post" UV-driven responses, and so the T50s of these responses were averaged for the histogram analysis. In other words, the traces of all cells obeyed the reversibility criterion illustrated in Fig. 7 and in panel A of this figure.

View larger version (16K): [in this window] [in a new window]   FIGURE 9. Comparison of response properties of WT rods, Nrl –/– and Rho–/– photoreceptors. Responses of WT rods were recorded with the outer segment either in the suction electrode (OS in, black bars, n = 10) or with a portion of the inner segment drawn into the pipette (OS out, gray bars, n = 13). Similarly, Nrl –/– photoreceptors were recorded in both configurations (OS out, red bars, n = 8; OS in, green, n = 12; cyan, n = 13; and blue, n = 5), with the latter groups subdivided based on the time-to-peak of the dim-flash response (A). Rho–/– cells were recorded only the OS-in configuration (clear bars, n = 8).

Dominant Time Constant of Recovery (_D).
The dominant recovery time constant for just-saturating flashes (cf. Fig. 3) was reliably shorter in Nrl ^–/– photoreceptors recorded in the OS-out configuration as compared with rods in the configuration that gave the shorter value: _D = 110 ± 40 ms vs. _D = 210 ± 40 ms (Fig. 9 B; P < 0.0004, one-tailed t test).

Half-saturating Flash Intensity (Q_1/2). The flash intensities that produced half-saturating responses of Nrl ^–/– photoreceptors were close to 100-fold higher than those that half saturated rod responses. Our rod results (Q_1/2 = 40 ± 10 photons µm^-2, mean ± s.d.) are in good agreement with previous reports: Q_1/2 = 30 ± 6 photons µm^–2 (Howes et al., 2002); 49 ± 3 (Xu et al., 1997), 55 ± 5 (Calvert et al., 2000), 60 ± 10 (Calvert et al., 2001), and 67 ± 6 (Chen et al., 1999).

Flash Sensitivity, Expressed as Fraction of Circulating Current Suppressed per Photoisomerization.
For interpretation in terms of the intrinsic properties of phototransduction, flash sensitivity is best expressed in terms of fraction circulating current suppressed per photoisomerization. To do this, flash intensities expressed in photons µm^–2 are multiplied by the collecting area, a_C expressed in µm² (MATERIALS AND METHODS). At the peak of the dim-flash response, 5.4 ± 1.3% of the rod circulating current was suppressed per photoisomerization; in contrast, only 0.24% of the circulating current of the Nrl ^–/– photoreceptors was suppressed per photoisomerization (Table I), a 40-fold lower sensitivity than rods. Comparable differences in rod and cone sensitivities expressed in photoisomerizations have been reported for other mammalian species, e.g., 5 ± 1.5% for primate rods (Baylor et al., 1984) vs. 0.2% for primate cones (Schnapf et al., 1990).

Amplification Coefficient (A).
The amplification coefficient of Nrl ^–/– photoreceptors in the OS-out configuration was 2.2-fold lower than that of rods: A = 3.5 ± 1.4 s^–2 vs. A = 8.0 ± 1.0 s^–2. As mentioned above, Nrl ^–/– photoreceptors recorded in the OS-in configuration had considerably reduced amplification (Fig. 3 B; Fig. 9 D).

Recordings from Rho^–/– Cones
Despite a great effort, we were only able to make a small number of recordings from the cones of rhodopsin knockout (Rho^–/–) mice, which like Nrl ^–/– mice have no rod outer segments (Table I; Fig. 9). Remarkably, slices of these retinas appear to have a large number of cone outer segments, but very few generate a measurable photocurrent, and those that did decayed even more rapidly in their function than Nrl ^–/– photoreceptors recorded in the OS-in configuration. At present, no strong conclusions can be drawn, but it is notable that Rho^–/– outer segments appeared to be even more fragile and labile that those of Nrl ^–/– photoreceptors.

Final Perspectives
To provide a final perspective on the comparison of the kinetics of WT rods and Nrl ^–/– photoreceptors, we have plotted the average dim-flash responses of the two cell types (Fig. 10). Nrl ^–/– photoreceptors in the OS-out configuration (red trace in Fig. 10, A and B) are faster in their recovery kinetics than in the OS-in configuration (green trace in Fig. 10 B), whereas the dim-flash responses of rods were very similar in the two recording configurations (Fig. 10 B). Clearly, Nrl ^–/– photoreceptors in the OS-out configuration have much faster response recoveries than do rods, but a subpopulation of Nrl ^–/– photoreceptors recorded in the OS-in configuration also had reliably faster recoveries (green traces, Fig. 10 B).

View larger version (36K): [in this window] [in a new window]   FIGURE 10. Comparison of the normalized dim-flash responses of Nrl –/– photoreceptors and WT rods. (A) Responses of the population of Nrl –/– photoreceptors whose responses were recorded in the OS-out configuration are all shown (gray traces), along with their average (red trace). (B) Dim-flash responses of rods recorded in the two configurations are compared with those of Nrl –/– photoreceptors; the green trace presents the average response of the subpopulation of Nrl –/– photoreceptors recorded in the OS-in configuration whose data are summarized in the histogram of Fig. 9 with green bars; the red trace is the same as that illustrated in A.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Functional Coexpression of UV- and M-cone Pigments in Nrl ^–/– and Rho^–/– Photoreceptors
The results presented here establish incontrovertibly for the first time that the photoreceptors of Nrl ^–/– and Rho^–/– mice functionally coexpress both the UV- and M-cone pigments (Fig. 5); that is, both cone pigments drive phototransduction in the individual cells, consistent with expectations from WT mice and other rodents based on immunohistochemistry (Rohlich et al., 1994; Applebury et al., 2000; Dkhissi-Benyahya et al., 2001; Lukats et al., 2002). The results further establish that the coexpression ratio of M- to UV-pigments varies 1000-fold, reflecting a positional gradient in the retina (Fig. 2; Fig. 5 B), well documented in WT mouse cones (Applebury et al., 2000; Fei and Hughes, 2001). Functional coexpression of a UV-pigment and one or more midwave to longwave pigments has been previously shown in recordings of single salamander cones (Makino and Dodd, 1996), but the work presented here provides the first demonstration of functional coexpression in mammalian cones.

The dim-flash responses driven by the two pigments in Nrl ^–/– photoreceptors differ little in kinetics (Fig. 7 A), implying that phototransduction activated by the two pigments is quite similar. Nonetheless, responses driven by the M-pigment recover reliably if only slightly more slowly than responses driven by the UV-pigment in some cells (Fig. 7 A; Fig. 8). Genomic information on the mouse indicates that (as in other mammals) there is only one cone transducin (Gnat2), and certainly the remaining proteins of the transduction cascade are the same regardless of which pigment is activated in a cell. Hence, the most likely explanation of this slight difference in recovery kinetics is that the UV- and M-pigments are inactivated differently.

Functional Coexpression of S- and M-cone Pigments in Rodent Photoreceptors and Color Vision
A basic insight of color science is that "color vision," operationally defined as the ability to discriminate of stimuli on the basis of their spectral content, requires at least two visual pigments and, further, requires that the signals generated by the photoreceptors containing the two pigments be encoded neurally in a manner that preserves their distinct information. The functional coexpression of S- and M-cone pigments in rodent photoreceptors appears to be at odds with this basic requirement for color vision proper and seems to pose a problem as to why rodent cones would express two pigments. The solution to this conundrum is that the view that "cones are for color vision" is misguided. The most important function of cones in vision is to provide useful visual signals in daylight, even under conditions when most of their visual pigment is bleached. Indeed, the steady-state response of cones never saturates in steady illumination, no matter how intense (Burkhardt, 1994). Beyond the prime directive of cones to give signals in bright light there is additional survival value to sampling different portions of the spectrum. Such spectral sampling by two pigments needn't be encoded in separate neural channels to be useful to the organism. Functional coexpression of S- and M-opsin, with the latter expressed in a dorso-ventral gradient (Fig. 1) no doubt samples the spectrum of the sky and the ground in a manner that provides information of survival value to rodents. Moreover, it seems possible that despite cone pigment coexpression, mice could learn to discriminate spectrally pure stimuli (e.g., 400 vs. 500 nm) independent of relative intensity (and thus appear to have a rudimentary form of color vision) based on the unique dorso-ventral pattern that each stimulus would produce. Thus, independent of its absolute intensity, a 400-nm stimulus of large angular extent would produce a higher level of excitation in the ventral than in the dorsal retina, while a 500-nm stimulus would produce the opposite. The encoding of the differing dorso-ventral gradients resulting of the two stimuli could be done with a single neural channel, in effect generating a distinct retinotopic pattern of neural excitation for stimuli of different wavelengths, even though the physical stimuli are spatially homogeneous. Discrimination based on such retinotopic gradients would not qualify as color vision proper, which requires that such stimuli be discriminable when the retina is stimulated locally. However, such a mechanism could conceivably underlie the discriminability found in a recent behavioral investigation by Jacobs et al. (2004).

Both the UV-cone Pigment and S-cone Pigment Require Grk1 for Normal Inactivation
Genomic analysis and histochemistry (Chen et al., 2001; Weiss et al., 2001; Caenepeel et al., 2004) indicate that only one GPCR kinase known to phosphorylate opsins, Grk1 (alias "rhodopsin kinase"), is expressed in mouse photoreceptors. In particular GRK7, an apparently more ancient kinase that is strongly expressed in the cones of many species, is completely absent in mouse (Chen et al., 2001; Weiss et al., 2001; Tachibanaki et al., 2001), although evolutionary analysis of genomic information suggests that it was present in the common ancestor of primates and mice (Caenepeel et al., 2004). Recent work has shown that the light-exposed UV-pigment is phosphorylated in the Nrl ^–/– retina, but not in retinas of Nrl ^–/–/Grk1^–/– (Zhu et al., 2003), providing indirect evidence that Grk1 is involved in mouse S-pigment inactivation. Our recordings from Nrl ^–/–/Grk1^–/– photoreceptors (Fig. 7 B; Fig. 8) show that the recovery phase of dim-flash responses driven by either the UV- or M-cone pigment is slowed by the deletion of Grk1, establishing definitively that Grk1 is necessary for the normal inactivation of the mouse cone photoresponse, as previously inferred from cone-driven electroretinographic responses of Grk1^–/– mice (Lyubarsky et al., 2000).

The Inactivation of the M-cone Pigment Is More Severely Slowed by Grk1 Deletion
The possibility of recording dim-flash responses from individual photoreceptors driven by either the UV- or the M-cone pigment produced a major surprise: the M-cone pigment–driven responses recover much more slowly than the UV-cone pigment–driven responses in the absence of Grk1 (Figs. 7 and 8). This result implies that there are important differences between the two cone pigments in their requirement for Grk1 for inactivation and, in particular, leads to the hypothesis that the UV-cone pigment has a Grk1-independent mechanism of inactivation that is faster than that of the M-cone pigment. One example of such an hypothesis is that the spontaneous hydrolysis of the all-trans chromophore from its Schiff-base attachment in the Metarhodopsin II/Metarhodopsin III states is faster in the UV- pigment than in the M-pigment (Vought et al., 1999). Another example would be the presence of an as yet undetected Grk or other kinase, although such a kinase would have to have a strong preference for S-opsin over M-opsin as a substrate. In this context, it bears mention that in fish, Grk7 appears to be more efficient in inactivating rhodopsin than Grk1 (Tachibanaki et al., 2001); given that S-opsins have greater homology with rhodopsin than with M-cone opsins, if another kinase is responsible for the relatively faster inactivation of S-opsin in the absence of Grk1, it would have an unusual substrate specificity.

The Rate of Activation of Phosphodiesterase per Photoisomerization in Nrl ^–/– Photoreceptors Is Reduced Relative to that in WT Rods
The amplification constant, extracted from analysis of the activation phase of the response of photoreceptors, quantifies the accelerating closure of cGMP channels as the photoactivated pigment (R*) activates its transducin, transducins activate phosphodiesterase (PDE), and cGMP declines (Lamb and Pugh, 1992; Pugh and Lamb, 1993; Leskov et al., 2000). The average amplification constant of WT rods (A = 8.0 s^–2) is 2.3-fold higher than that of Nrl ^–/– photoreceptors (A = 3.5 s^–2; OS-out configuration) (Table I). Theory predicts A to be inversely proportional to the OS cytoplasmic volume, V_cyto (Lamb and Pugh, 1992), and analysis of rods having different OS volumes has confirmed this prediction (Pugh and Lamb, 1993). Given that the Hill coefficient of the cGMP-activated channels in both types of photoreceptors is the same (Yau, 1994), and that the kinetic parameters (k_cat and K_m) of the cone PDE are comparable to those of the rod PDE (Gillespie and Beavo, 1988; Gillespie, 1990), it follows from the inverse dependence of A on V_cyto and the volume ratio of the WT rod OS to the Nrl ^–/– OS, 4.5, that the rate of activation of PDEs per R* is 10-fold lower in Nrl ^–/– cells than in rods.

The amplification coefficients of both rods and Nrl ^–/– photoreceptors estimated from the single-cell recordings presented here are close to estimates obtained from ERG a-wave data; for rods, A = 8.0 s^–2 (single cells, Table I) vs. 7.4 s^–2 (WT ERG a-waves, 8 wk olds; Lyubarsky et al., 2004); A = 3.5 s^–2 (single Nrl ^–/– cells, OS out, Table I), vs. 4.0 s^–2 (ERG a-waves, 4–6-wk-old Nrl ^–/– mice; Daniele et al., 2005). It bears mention that the end-on collecting area of Nrl ^–/– photoreceptors in vivo, required to estimate amplification from a-wave data, was hypothesized to be increased fourfold by light funneling in the inner segment. This hypothesis is supported by the dimensions of the Nrl ^–/– photoreceptor inner segment, which electron microscopy reveals to taper steeply to the outer segment (Daniele et al., 2005).

The Decreased Amplification and Speed of Nrl ^–/– Photoreceptors in the OS-in Configuration Suggests that Mouse Cones Removed from their Matrix Sheath Deteriorate
The changes in the properties of the responses of Nrl ^–/– photoreceptors recorded with the OS drawn into the suction electrode (Fig. 4) suggest that these outer segments are more labile than those of WT rods. One explanation is that the suction pipette itself may damage the outer segment in the OS-in configuration. An alternative, and not mutually exclusive, hypothesis is that damage may begin before drawing the cell into the suction pipette. Like WT mouse cones, each Nrl ^–/– photoreceptor has a PNA-stained extracellular matrix, which appears to be attached to the inner segment and which ensheaths the outer segment (Daniele et al., 2005). Since the cone matrix sheath makes adhesions with both the cone and the RPE apical surface, the removal of the retina from the RPE necessarily disrupts the attachment of the cone sheath to the RPE, and likely also disrupts attachment of the sheath to cones (Johnson et al., 1986; Hageman et al., 1995). Damage to or loss of these attachments could compromise the cone disc membranes.

The Nrl ^–/– Retina as a Preparation for the Investigation of Mammalian Cone Function
Because of the power of mouse genetics and molecular biology, the Nrl ^–/– retina holds much promise as a preparation for the investigation of cone physiological and biochemical function, and for the identification of molecules involved in cone-specific genetic disease. The recent characterization of the dependence of mouse UV-cone pigment phosphorylation and cone arrestin binding on Grk1 (Zhu et al., 2003) constitutes an initial realization of the first promise, and the genetic characterization of a large set of genes whose expression is enriched in the Nrl ^–/– retina (Yoshida et al., 2004) provides a foundation for the second. The degree to which the gene expression profiles and physiological response properties of Nrl ^–/– photoreceptors correspond to those of WT mouse cones will ultimately be resolved by physiological experiments and gene expression analyses. Meanwhile, the conclusion that Nrl ^–/– photoreceptors are indeed a species of cones opens the door to many investigations of fundamental importance to the understanding of molecular function in cones, and ways in which mutations of genes expressed specifically in cones and nearby cells leads to their demise.

	ACKNOWLEDGMENTS

 
We thank Dr. J. Lem (Tufts University School of Medicine, Boston, MA) for providing rhodopsin^–/– mice, and Dr. C.-K. Chen (University of Utah Health Sciences Center, Salt Lake City, UT) for providing Grk1^–/– mice.

E.N. Pugh Jr. is supported by a Jules and Doris Stein Research to Prevent Blindness Professorship, and C.M. Craft is supported by the Mary D. Allen Chair in Vision Research of the Doheny Eye Research Institute. This work was also supported by NIH-EY-02660; EY-11115; EY-00395, The Foundation Fighting Blindness; and Research to Prevent Blindness Foundation.

Lawrence G. Palmer served as editor.

Submitted: 4 November 2004
Accepted: 2 February 2005

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

Ahnelt, P.K. 1998. The photoreceptor mosaic. Eye. 12:531–540.[Medline]

Applebury, M.L., M.P. Antoch, L.C. Baxter, L.L. Chun, J.D. Falk, F. Farhangfar, K. Kage, M.G. Krzystolik, L.A. Lyass, and J.T. Robbins. 2000. The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning. Neuron. 27:513–523.[CrossRef][Medline]

Baylor, D.A., T.D. Lamb, and K.-W. Yau. 1979a. Responses of retinal rods to single photons. J. Physiol. 288:613–634.[Abstract/Free Full Text]

Baylor, D.A., T.D. Lamb, and K.-W. Yau. 1979b. The membrane current of single rod outer segments. J. Physiol. 288:589–611.[Abstract/Free Full Text]

Baylor, D.A., B.J. Nunn, and J.L. Schnapf. 1984. The photocurrent, noise and spectral sensitivity of rods of the monkey Macaca fascicularis. J. Physiol. 357:575–607.[Abstract/Free Full Text]

Blanks, J.C., G.S. Hageman, L.V. Johnson, and C. Spee. 1988. Ultrastructural visualization of primate cone photoreceptor matrix sheaths. J. Comp. Neurol. 270:288–300.[CrossRef][Medline]

Blanks, J.C., and L.V. Johnson. 1983. Selective lectin binding of the developing mouse retina. J. Comp. Neurol. 221:31–41.[CrossRef][Medline]

Burkhardt, D.A. 1994. Light adaptation and photopigment bleaching in cone photoreceptors in situ in the retina of the turtle. J. Neurosci. 14:1091–1105.[Abstract]

Burns, M.E., A. Mendez, J. Chen, and D.A. Baylor. 2002. Dynamics of cyclic GMP synthesis in retinal rods. Neuron. 36:81–91.[CrossRef][Medline]

Caenepeel, S., G. Charydczak, S. Sudarsanam, T. Hunter, and G. Manning. 2004. The mouse kinome: discovery and comparative genomics of all mouse protein kinases. Proc. Natl. Acad. Sci. USA. 101:11707–11712.[Abstract/Free Full Text]

Calvert, P.D., V.I. Govardovskii, N. Krasnoperova, R.E. Anderson, J. Lem, and C.L. Makino. 2001. Membrane protein diffusion sets the speed of rod phototransduction. Nature. 411:90–94.[CrossRef][Medline]

Calvert, P.D., N.V. Krasnoperova, A.L. Lyubarsky, T. Isayama, M. Nicolo, B. Kosaras, G. Wong, K.S. Gannon, R.F. Margolskee, R.L. Sidman, et al. 2000. Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha-subunit. Proc. Natl. Acad. Sci. USA. 97:13913–13918.[Abstract/Free Full Text]

Carter-Dawson, L.D., and M.M. LaVail. 1979. Rods and cones in the mouse retina. I. Structural analysis using light and electron microscopy. J. Comp. Neurol. 188:245–262.[CrossRef][Medline]

Chen, C.K., M.E. Burns, W. He, T.G. Wensel, D.A. Baylor, and M.I. Simon. 2000. Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1. Nature. 403:557–560.[CrossRef][Medline]

Chen, C.K., M.E. Burns, M. Spencer, G.A. Niemi, J. Chen, J.B. Hurley, D.A. Baylor, and M.I. Simon. 1999. Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase. Proc. Natl. Acad. Sci. USA. 96:3718–3722.[Abstract/Free Full Text]

Chen, C.K., K. Zhang, J. Church-Kopish, W. Huang, H. Zhang, Y.J. Chen, J.M. Frederick, and W. Baehr. 2001. Characterization of human GRK7 as a potential cone opsin kinase. Mol. Vis. 7:305–313.[Medline]

Chen, J., C.L. Makino, N.S. Peachey, D.A. Baylor, and M.I. Simon. 1995. Mechanisms of rhodopsin inactivation in vivo as revealed by a COOH-terminal truncation mutant. Science. 267(5196):374–377.[Abstract/Free Full Text]

Cobbs, W.H., and E.N. Pugh Jr. 1987. Kinetics and components of the flash photocurrent of isolated retinal rods of the larval salamander, Ambystoma tigrinum. J. Physiol. 394:529–572.[Abstract/Free Full Text]

Cornish, E.E., M. Xiao, Z. Yang, J.M. Provis, and A.E. Hendrickson. 2004. The role of opsin expression and apoptosis in determination of cone types in human retina. Exp. Eye Res. 78:1143–1154.[CrossRef][Medline]

Daniele, L.L., C. Lillo, A.L. Lyubarsky, S.S. Nikonov., N. Philp, A.J. Mears, A. Swaroop, D.S. Williams, and E.N. Pugh Jr. 2005. Cone-like morphological, molecular and electrophysiological features of the photoreceptors of the Nrl knockout mouse. Investigative Ophthalmology and Visual Science. In press.

Dkhissi-Benyahya, O., A. Szel, W.J. Degrip, and H.M. Cooper. 2001. Short and mid-wavelength cone distribution in a nocturnal Strepsirrhine primate (Microcebus murinus). J. Comp. Neurol. 438:490–504.[CrossRef][Medline]

Ebrey, T., and Y. Koutalos. 2001. Vertebrate photoreceptors. Prog. Retin. Eye Res. 20:49–94.[CrossRef][Medline]

Fei, Y., and T.E. Hughes. 2001. Transgenic expression of the jellyfish green fluorescent protein in the cone photoreceptors of the mouse. Vis. Neurosci. 18:615–623.[CrossRef][Medline]

Gillespie, P.G. 1990. Phosphodiesterases in visual transduction by rods and cones. Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action. J. Beavo and M.D. Houslay, editors. Wiley, New York. 163–184.

Gillespie, P.G., and J.A. Beavo. 1988. Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-sepharose chromatography. J. Biol. Chem. 263:8133–8141.[Abstract/Free Full Text]

Govardovskii, V.I., N. Fyhrquist, T. Reuter, D.G. Kuzmin, and K. Donner. 2000. In search of the visual pigment template. Vis. Neurosci. 17:509–528.[CrossRef][Medline]

Hageman, G.S., M.F. Marmor, X.Y. Yao, and L.V. Johnson. 1995. The interphotoreceptor matrix mediates primate retinal adhesion. Arch. Ophthalmol. 113:655–660.[Abstract/Free Full Text]

Hagins, W.A., R.D. Penn, and S. Yoshikami. 1970. Dark current and photocurrent in retinal rods. Biophys. J. 10:380–412.[Medline]

Harosi, F.I. 1982. Polarized microspectrophotometry for pigment orientation and concentration. Methods Enzymol. 81:642–647.[Medline]

Hendrickson, A., and D. Hicks. 2002. Distribution and density of medium- and short-wavelength selective cones in the domestic pig retina. Exp. Eye Res. 74:435–444.[CrossRef][Medline]

Howes, K.A., M.E. Pennesi, I. Sokal, J. Church-Kopish, B. Schmidt, D. Margolis, J.M. Frederick, F. Rieke, K. Palczewski, S.M. Wu, et al. 2002. GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice. EMBO J. 21:1545–1554.[CrossRef][Medline]

Jacobs, G.H., G.A. Williams, and J.A. Fenwick. 2004. Influence of cone pigment coexpression on spectral sensitivity and color vision in the mouse. Vision Res. 44:1615–1622.[CrossRef][Medline]

Johnson, L.V., G.S. Hageman, and J.C. Blanks. 1986. Interphotoreceptor matrix domains ensheath vertebrate cone photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 27:129–135.[Abstract/Free Full Text]

Kraft, T.W. 1988. Photocurrents of cone photoreceptors of the golden-mantled ground squirrel. J. Physiol. 404:199–213.[Abstract/Free Full Text]

Lamb, T.D. 1995. Photoreceptor spectral sensitivities: common shape in the long-wavelength region. Vision Res. 35:3083–3091.[CrossRef][Medline]

Lamb, T.D., and E.N. Pugh Jr. 1992. A quantitative account of the activation steps involved in phototransduction in amphibian photoreceptors. J. Physiol. 449:719–758.[Abstract/Free Full Text]

Lem, J., N.V. Krasnoperova, P.D. Calvert, B. Kosaras, D.A. Cameron, M. Nicolo, C.L. Makino, and R.L. Sidman. 1999. Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc. Natl. Acad. Sci. USA. 96:736–741.[Abstract/Free Full Text]

Leskov, I.B., V.A. Klenchin, J.W. Handy, G.G. Whitlock, V.I. Govardovskii, M.D. Bownds, T.D. Lamb, E.N. Pugh Jr., and V.Y. Arshavsky. 2000. The gain of rod phototransduction: reconciliation of biochemical and electrophysiological measurements. Neuron. 27:525–537.[CrossRef][Medline]

Liebman, P.A. 1972. Microspectrophotometry of photoreceptors. Handbook of Sensory Physiology. H.J.A. Dartnall, editor. Springer, New York. 481–528.

Lukats, A., O. Dkhissi-Benyahya, Z. Szepessy, P. Rohlich, B. Vigh, N.C. Bennett, H.M. Cooper, and A. Szel. 2002. Visual pigment coexpression in all cones of two rodents, the Siberian hamster, and the pouched mouse. Invest. Ophthalmol. Vis. Sci. 43:2468–2473.[Abstract/Free Full Text]

Lyubarsky, A.L., C. Chen, M.I. Simon, and E.N. Pugh Jr. 2000. Mice lacking G-protein receptor kinase 1 have profoundly slowed recovery of cone-driven retinal responses. J. Neurosci. 20:2209–2217.[Abstract/Free Full Text]

Lyubarsky, A.L., B. Falsini, M.E. Pennesi, P. Valentini, and E.N. Pugh Jr. 1999. UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments. J. Neurosci. 19:442–455.[Abstract/Free Full Text]

Lyubarsky, A.L., F. Naarendorp, X. Zhang, T. Wensel, M.I. Simon, and E.N. Pugh Jr. 2001. RGS9-1 is required for normal inactivation of mouse cone phototransduction. Mol. Vis. 7:71–78.[Medline]

Lyubarsky, A.L., L.L. Daniele, and E.N. Pugh. 2004. From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG. Vision Research. 44:3235–3251.[CrossRef][Medline]

Makino, C.L., and R.L. Dodd. 1996. Multiple visual pigments in a photoreceptor of the salamander retina. J. Gen. Physiol. 108:27–34.[Abstract/Free Full Text]

Mears, A.J., M. Kondo, P.K. Swain, Y. Takada, R.A. Bush, T.L. Saunders, P.A. Sieving, and A. Swaroop. 2001. Nrl is required for rod photoreceptor development. Nat. Genet. 29:447–452.[CrossRef][Medline]

Mendez, A., M.E. Burns, A. Roca, J. Lem, L.W. Wu, M.I. Simon, D.A. Baylor, and J. Chen. 2000. Rapid and reproducible deactivation of rhodopsin requires multiple phosphorylation sites. Neuron. 28:153–164.[CrossRef][Medline]

Mendez, A., M.E. Burns, I. Sokal, A.M. Dizhoor, W. Baehr, K. Palczewski, D.A. Baylor, and J. Chen. 2001. Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors. Proc. Natl. Acad. Sci. USA. 98:9948–9953.[Abstract/Free Full Text]

Nikonov, S., N. Engheta, and E.N. Pugh Jr. 1998. Kinetics of recovery of the dark-adapted salamander rod photoresponse. J. Gen. Physiol. 111:7–37.[Abstract/Free Full Text]

Okano, T., Y. Fukada, Y. Shichida, and T. Yoshizawa. 1992. Photosensitivities of iodopsin and rhodopsins. Photochem. Photobiol. 56:995–1001.[Medline]

Paupoo, A.A., O.A. Mahroo, C. Friedburg, and T.D. Lamb. 2000. Human cone photoreceptor responses measured by the electroretinogram a-wave during and after exposure to intense illumination. J. Physiol. 529:469–482.[Abstract/Free Full Text]

Pennesi, M.E., K.A. Howes, W. Baehr, and S.M. Wu. 2003. Guanylate cyclase-activating protein (GCAP) 1 rescues cone recovery kinetics in GCAP1/GCAP2 knockout mice. Proc. Natl. Acad. Sci. USA. 100:6783–6788.[Abstract/Free Full Text]

Pepperberg, D.R., J. Jin, and G.J. Jones. 1994. Modulation of transduction gain in light adaptation of retinal rods. Vis. Neurosci. 11:53–62.[Medline]

Pierce, E.A. 2001. Pathways to photoreceptor cell death in inherited retinal degenerations. Bioessays. 23:605–618.[CrossRef][Medline]

Pugh, E.N., Jr., and T.D. Lamb. 1993. Amplification and kinetics of the activation steps in phototransduction. Biochim. Biophys. Acta. 1141:111–149.[Medline]

Pugh, E.N., Jr., S. Nikonov, and T.D. Lamb. 1999. Molecular mechanisms of vertebrate photoreceptor light adaptation. Curr. Opin. Neurobiol. 9:410–418.[CrossRef][Medline]

Rohlich, P., T. van Veen, and A. Szel. 1994. Two different visual pigments in one retinal cone cell. Neuron. 13:1159–1166.[CrossRef][Medline]

Saari, J.C., M. Nawrot, B.N. Kennedy, G.G. Garwin, J.B. Hurley, J. Huang, D.E. Possin, and J.W. Crabb. 2001. Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation. Neuron. 29:739–748.[CrossRef][Medline]

Schnapf, J.L., B.J. Nunn, M. Meister, and D.A. Baylor. 1990. Visual transduction in cones of the monkey Macaca fascicularis. J. Physiol. 427:681–713.[Abstract/Free Full Text]

Smith, N.P., and T.D. Lamb. 1997. The a-wave of the human electroretinogram recorded with a minimally invasive technique. Vision Res. 37:2943–2952.[CrossRef][Medline]

Stone, E.M., T.A. Braun, S.R. Russell, M.H. Kuehn, A.J. Lotery, P.A. Moore, C.G. Eastman, T.L. Casavant, and V.C. Sheffield. 2004. Missense variations in the fibulin 5 gene and age-related macular degeneration. N. Engl. J. Med. 351:346–353.[Abstract/Free Full Text]

Sun, H., J.P. Macke, and J. Nathans. 1997. Mechanisms of spectral tuning in the mouse green cone pigment. Proc. Natl. Acad. Sci. USA. 94:8860–8865.[Abstract/Free Full Text]

Tachibanaki, S., S. Tsushima, and S. Kawamura. 2001. Low amplification and fast visual pigment phosphorylation as mechanisms characterizing cone photoresponses. Proc. Natl. Acad. Sci. USA. 98:14044–14049.[Abstract/Free Full Text]

Vought, B.W., A. Dukkipatti, M. Max, B.E. Knox, and R.R. Birge. 1999. Photochemistry of the primary event in short-wavelength visual opsins at low temperature. Biochemistry. 38:11287–11297.[CrossRef][Medline]

Weiss, E.R., M.H. Ducceschi, T.J. Horner, A. Li, C.M. Craft, and S. Osawa. 2001. Species-specific differences in expression of G-protein-coupled receptor kinase (GRK) 7 and GRK1 in mammalian cone photoreceptor cells: implications for cone cell phototransduction. J. Neurosci. 21:9175–9184.[Abstract/Free Full Text]

Xiao, M., and A. Hendrickson. 2000. Spatial and temporal expression of short, long/medium, or both opsins in human fetal cones. J. Comp. Neurol. 425(4):545–559.[CrossRef][Medline]

Xu, J., R.L. Dodd, C.L. Makino, M.I. Simon, D.A. Baylor, and J. Chen. 1997. Prolonged photoresponses in transgenic mouse rods lacking arrestin. Nature. 389:505–509.[CrossRef][Medline]

Yau, K.-W. 1994. Cyclic nucleotide-gated channels: an expanding new family of ion channels. Proc. Natl. Acad. Sci. USA. 91:3481–3483.[Free Full Text]

Yokoyama, R., and S. Yokoyama. 2000. Comparative molecular biology of visual pigments. Molecular Mechanisms in Visual Transduction. D.G. Stavenga, W.J. de Grip, and E.N. Pugh II, editors. Elsevier Science Publishing Co. Inc., New York. 257–296.

Yokoyama, S., F.B. Radlwimmer, and S. Kawamura. 1998. Regeneration of ultraviolet pigments of vertebrates. FEBS Lett. 423:155–158.[CrossRef][Medline]

Yoshida, S., A.J. Mears, J.S. Friedman, T. Carter, S. He, E. Oh, Y. Jing, R. Farjo, G. Fleury, C. Barlow, et al. 2004. Expression profiling of the developing and mature Nrl^–/– mouse retina: identification of retinal disease candidates and transcriptional regulatory targets of Nrl. Hum. Mol. Genet. 13:1487–1503.[Abstract/Free Full Text]

Zhu, X., B. Brown, A. Li, A.J. Mears, A. Swaroop, and C.M. Craft. 2003. GRK1-dependent phosphorylation of S- and M-opsins and their binding to cone arrestin during cone phototransduction in the mouse retina. J. Neurosci. 23:6152–6160.[Abstract/Free Full Text]

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				C. Chen, L. R. Blakeley, and Y. Koutalos Formation of all-trans Retinol after Visual Pigment Bleaching in Mouse Photoreceptors Invest. Ophthalmol. Vis. Sci., August 1, 2009; 50(8): 3589 - 3595. [Abstract] [Full Text] [PDF]

				L. L. Daniele, B. Sauer, S. M. Gallagher, E. N. Pugh Jr, and N. J. Philp Altered visual function in monocarboxylate transporter 3 (Slc16a8) knockout mice Am J Physiol Cell Physiol, August 1, 2008; 295(2): C451 - C457. [Abstract] [Full Text] [PDF]

				D.-G. Luo, T. Xue, and K.-W. Yau How vision begins: An odyssey PNAS, July 22, 2008; 105(29): 9855 - 9862. [Abstract] [Full Text] [PDF]

				V. A. Smyth, D. Di Lorenzo, and B. N. Kennedy A Novel, Evolutionarily Conserved Enhancer of Cone Photoreceptor-specific Expression J. Biol. Chem., April 18, 2008; 283(16): 10881 - 10891. [Abstract] [Full Text] [PDF]

				K. L. Feathers, A. L. Lyubarsky, N. W. Khan, K. Teofilo, A. Swaroop, D. S. Williams, E. N. Pugh Jr, and D. A. Thompson Nrl-Knockout Mice Deficient in Rpe65 Fail to Synthesize 11-cis Retinal and Cone Outer Segments Invest. Ophthalmol. Vis. Sci., March 1, 2008; 49(3): 1126 - 1135. [Abstract] [Full Text] [PDF]

				G. Shi, K.-W. Yau, J. Chen, and V. J. Kefalov Signaling Properties of a Short-Wave Cone Visual Pigment and Its Role in Phototransduction J. Neurosci., September 19, 2007; 27(38): 10084 - 10093. [Abstract] [Full Text] [PDF]

				M. C. Cornwall and P. Ala-Laurila A Perfect Marriage: Molecular Genetics Ties the Knot with Electrophysiology in Studies of Visual Transduction J. Gen. Physiol., July 1, 2007; 130(1): 7 - 10. [Full Text] [PDF]

				K. Sakurai, A. Onishi, H. Imai, O. Chisaka, Y. Ueda, J. Usukura, K. Nakatani, and Y. Shichida Physiological Properties of Rod Photoreceptor Cells in Green-sensitive Cone Pigment Knock-in Mice J. Gen. Physiol., July 1, 2007; 130(1): 21 - 40. [Abstract] [Full Text] [PDF]

				M. A. Raven, E. C. T. Oh, A. Swaroop, and B. E. Reese Afferent Control of Horizontal Cell Morphology Revealed by Genetic Respecification of Rods and Cones J. Neurosci., March 28, 2007; 27(13): 3540 - 3547. [Abstract] [Full Text] [PDF]

				A. Wenzel, J. von Lintig, V. Oberhauser, N. Tanimoto, C. Grimm, and M. W. Seeliger RPE65 Is Essential for the Function of Cone Photoreceptors in NRL-Deficient Mice Invest. Ophthalmol. Vis. Sci., February 1, 2007; 48(2): 534 - 542. [Abstract] [Full Text] [PDF]

				T. D. Lamb and E. N. Pugh Jr Phototransduction, Dark Adaptation, and Rhodopsin Regeneration The Proctor Lecture Invest. Ophthalmol. Vis. Sci., December 1, 2006; 47(12): 5138 - 5152. [Full Text] [PDF]

				M. E. Estevez, P. Ala-Laurila, R. K. Crouch, and M. C. Cornwall Turning Cones Off: the Role of the 9-Methyl Group of Retinal in Red Cones J. Gen. Physiol., December 1, 2006; 128(6): 671 - 685. [Abstract] [Full Text] [PDF]

				L. Yin, R. G Smith, P. Sterling, and D. H. Brainard Chromatic Properties of Horizontal and Ganglion Cell Responses Follow a Dual Gradient in Cone Opsin Expression. J. Neurosci., November 22, 2006; 26(47): 12351 - 12361. [Abstract] [Full Text] [PDF]

				L. G. Glushakova, A. M. Timmers, J. Pang, J. T. Teusner, and W. W. Hauswirth Human blue-opsin promoter preferentially targets reporter gene expression to rat s-cone photoreceptors. Invest. Ophthalmol. Vis. Sci., August 1, 2006; 47(8): 3505 - 3513. [Abstract] [Full Text] [PDF]

				R. Farjo, J. S. Skaggs, B. A. Nagel, A. B. Quiambao, Z. A. Nash, S. J. Fliesler, and M. I. Naash Retention of function without normal disc morphogenesis occurs in cone but not rod photoreceptors J. Cell Biol., April 10, 2006; 173(1): 59 - 68. [Abstract] [Full Text] [PDF]

				B. E. Knox and E. Solessio Shedding Light On Cones J. Gen. Physiol., March 27, 2006; 127(4): 355 - 358. [Full Text] [PDF]

				S. S. Nikonov, R. Kholodenko, J. Lem, and E. N. Pugh Jr. Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings J. Gen. Physiol., March 27, 2006; 127(4): 359 - 374. [Abstract] [Full Text] [PDF]

				S Nymark, H Heikkinen, C Haldin, K Donner, and A Koskelainen Light responses and light adaptation in rat retinal rods at different temperatures J. Physiol., September 15, 2005; 567(3): 923 - 938. [Abstract] [Full Text] [PDF]

				A. Wenzel, V. Oberhauser, E. N. Pugh Jr., T. D. Lamb, C. Grimm, M. Samardzija, E. Fahl, M. W. Seeliger, C. E. Reme, and J. von Lintig The Retinal G Protein-coupled Receptor (RGR) Enhances Isomerohydrolase Activity Independent of Light J. Biol. Chem., August 19, 2005; 280(33): 29874 - 29884. [Abstract] [Full Text] [PDF]

				S. Tachibanaki, D. Arinobu, Y. Shimauchi-Matsukawa, S. Tsushima, and S. Kawamura Highly effective phosphorylation by G protein-coupled receptor kinase 7 of light-activated visual pigment in cones PNAS, June 28, 2005; 102(26): 9329 - 9334. [Abstract] [Full Text] [PDF]

				Highlights from the Literature Physiology, June 1, 2005; 20(3): 149 - 151. [Full Text] [PDF]

				L. L. Daniele, C. Lillo, A. L. Lyubarsky, S. S. Nikonov, N. Philp, A. J. Mears, A. Swaroop, D. S. Williams, and E. N. Pugh Jr Cone-like Morphological, Molecular, and Electrophysiological Features of the Photoreceptors of the Nrl Knockout Mouse Invest. Ophthalmol. Vis. Sci., June 1, 2005; 46(6): 2156 - 2167. [Abstract] [Full Text] [PDF]

				S. Haverkamp, H. Wassle, J. Duebel, T. Kuner, G. J. Augustine, G. Feng, and T. Euler The Primordial, Blue-Cone Color System of the Mouse Retina J. Neurosci., June 1, 2005; 25(22): 5438 - 5445. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF, 4270K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Nikonov, S. S. Articles by Pugh, E. N. Search for Related Content PubMed PubMed Citation Articles by Nikonov, S. S. Articles by Pugh, E. N., Jr. Social Bookmarking                            What's this?