Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion.

The relaxation of fluorescence from diffraction-limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo-conversion was measured in solutions of varied viscosity (eta), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point-spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, D(ap). The model incorporates two novel features that make it of broad utility. First, the model includes the no-flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of D(ap). Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between D(ap) and eta(-1) were obtained for the two proteins: for PAGFP, D(aq)= 89 +/- 2.4 microm2 s(-1) (mean +/- 95% confidence interval), and for EGFP D(aq)= 91 +/- 1.8 microm2 s(-1). In CHO cells, the application of the model yielded D(ap)= 20 +/- 3 microm2 s(-1) (PAGFP) and 19 +/- 2 microm2 s(-1) (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked 'bound fraction' explanation of the deviation of the terminal fluorescence recovery from its pre-bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488-nm excitation, and the requirement for approximately 1/200th the cumulative light energy to produce data of comparable signal/noise.

The distribution, concentration, and toxicity of enhanced green fluorescent protein in retinal cells after genomic or somatic (virus-mediated) gene transfer.

PURPOSE: The concentration of enhanced green fluorescent protein (EGFP) in individual photoreceptor cells of live mouse retina was quantified and correlated with physiological measurements of cell function. METHODS: EGFP protein levels in the retinas of mice injected subretinally by either one of two serotypes of adeno-associated virus (AAV; AAV2/5.CMV.EGFP; AAV2/2.CMV.EGFP) were quantified with a photon-counting confocal laser scanning microscope and compared with those of transgenic mice whose retinas expressed EGFP under the beta-actin (pbetaAct) or human L/M-cone opsin (pLMCOps) promoter. Single-cell suction pipette recordings of single rods and whole-field electroretinograms (ERGs) were performed to assess retinal cell function. RESULTS: The highest levels of EGFP (680 microM) were in the retinal pigment epithelium (RPE) cells of the AAV-transduced eyes. Living photoreceptors of pbetaAct.EGFP mice contained 270 microM EGFP, while their bipolars had 440 microM. The cones of pLMCOps.EGFP mice expressed 60 microM protein. The amplitudes of the major components of ERGs were within the normal range for all transgenic animals examined, and single cell recordings from living pbetaAct.EGFP rods were indistinguishable from those of controls. CONCLUSIONS: EGFP levels in individual cells of live mouse retinas can be quantified, so that the efficacy of gene transfer methods can be quantified. Concentrations of several hundred microM are not deleterious to normal function of photoreceptors and bipolar cells. This approach can also be used to quantify levels of biologically active EGFP fusion proteins.

Deficiency of Bax and Bak protects photoreceptors from light damage in vivo.

Photoreceptors of bax(-/-)bak(-/-) but neither bax(-/-) mice nor bak(-/-) mice are protected from developmental apoptosis, suggesting that bax(-/-)bak(-/-) photoreceptors may also be protected from pathologic apoptosis. To test this possibility, we exposed bax(-/-)bak(-/-) and bax(-/-) mice to bright light, which normally induces photoreceptor death. Photoreceptors in bax(-/-)bak(-/-) mice were protected from death compared to bax(-/-) mice as indicated by a reduction in the number of TUNEL-positive photoreceptor nuclei 24 h following light damage and almost complete preservation of photoreceptors 7 days following light damage. These results provide the first in vivo evidence that combined deficiency of Bax and Bak can rescue cells from a pathologic stimulus more effectively than Bax deficiency and suggest that combined deficiency of Bax and Bak may also protect cells from other insults.

Dark adaptation and the retinoid cycle of vision.

Following exposure of our eye to very intense illumination, we experience a greatly elevated visual threshold, that takes tens of minutes to return completely to normal. The slowness of this phenomenon of "dark adaptation" has been studied for many decades, yet is still not fully understood. Here we review the biochemical and physical processes involved in eliminating the products of light absorption from the photoreceptor outer segment, in recycling the released retinoid to its original isomeric form as 11-cis retinal, and in regenerating the visual pigment rhodopsin. Then we analyse the time-course of three aspects of human dark adaptation: the recovery of psychophysical threshold, the recovery of rod photoreceptor circulating current, and the regeneration of rhodopsin. We begin with normal human subjects, and then analyse the recovery in several retinal disorders, including Oguchi disease, vitamin A deficiency, fundus albipunctatus, Bothnia dystrophy and Stargardt disease. We review a large body of evidence showing that the time-course of human dark adaptation and pigment regeneration is determined by the local concentration of 11-cis retinal, and that after a large bleach the recovery is limited by the rate at which 11-cis retinal is delivered to opsin in the bleached rod outer segments. We present a mathematical model that successfully describes a wide range of results in human and other mammals. The theoretical analysis provides a simple means of estimating the relative concentration of free 11-cis retinal in the retina/RPE, in disorders exhibiting slowed dark adaptation, from analysis of psychophysical measurements of threshold recovery or from analysis of pigment regeneration kinetics.

Functionally rodless mice: transgenic models for the investigation of cone function in retinal disease and therapy.

Two genetically engineered strains of mice were used to characterize murine cone function electroretinographically, without interference of rod-driven responses: (1) mice with a deletion of the gene for the rod transducin alpha-subunit (transducin alpha-/-), and (2) mice with rod arrestin deleted (arrestin -/-). In the first three months of age, both strains have a normal complement of rods and normal rod structure, but transducin alpha-/- mice have no rod-driven responses to light, while rod-driven activity of arrestin -/- mice can be suppressed by a single intense flash for hours. In response to intense flashes the electroretinograms of these strains of mice showed a readily identifiable, pure-cone a-wave of approximately 10 microV saturating amplitude. A 530 nm background that saturates rod responses of wild type mice was found to desensitize the b-wave responses of mice of both transgenic lines, whether the b-waves were driven by photons captured by M- or UV-cone pigments. The desensitizing effect of the 530 nm background on UV-pigment driven responses provides new evidence in support of the hypothesis of functional co-expression of the M-pigment in cones expressing primarily the UV-pigment.

A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival.

A cadherin family member, prCAD, was identified in retina cDNA by subtractive hybridization and high throughput sequencing. prCAD is expressed only in retinal photoreceptors, and the prCAD protein is localized to the base of the outer segment of both rods and cones. In prCAD(-/-) mice, outer segments are disorganized and fragmented, and there is progressive death of photoreceptor cells. prCAD is unlikely to be involved in protein trafficking between inner and outer segments, since phototransduction proteins appear to be correctly localized and the light responses of both rods and cones are only modestly compromised in prCAD(-/-) mice. These experiments imply a highly specialized cell biological function for prCAD and suggest that localized adhesion activity is essential for outer segment integrity.

Rods are rods and cones cones, and (never) the twain shall meet.

More than 100 photopigment G protein-coupled receptors (opsins) have been sequenced and organized into six classes. Rod photoreceptors in various species have been found to express an opsin from one of the two rhodopsin classes, while cones express an opsin from one of the four remaining classes. It has now been discovered that salamander short-wavelength sensitive cones and green rods express the same opsin, while manifesting other features that classically distinguish rods from cones.

RGS9-1 is required for normal inactivation of mouse cone phototransduction.

PURPOSE: To test the hypothesis that Regulator of G-protein Signaling 9 (RGS9-1) is necessary for the normal inactivation of retinal cones. METHODS: Mice having the gene RGS9-1 inactivated in both alleles (RGS9-1 -/-) were tested between the ages 8-10 weeks with electroretinographic (ERG) protocols that isolate cone-driven responses. Immunohistochemistry was performed with a primary antibody against RGS9-1 (anti-RGS9-1c), with the secondary conjugated to fluorescein isothiocyanate, and with rhodamine-conjugated peanut agglutinin. RESULTS: (1) Immunohistochemistry showed RGS9-1 to be strongly expressed in the cones of wildtype (WT is C57BL/6) mice, but absent from the cones of RGS9-1 mice. (2) Cone-driven b-wave responses of dark-adapted RGS9-1 -/- mice had saturating amplitudes and sensitivities in the midwave and UV regions of the spectrum equal to or slightly greater than those of WT (C57BL/6) mice. (3) Cone-driven b-wave and a-wave responses of RGS9-1 -/- mice recovered much more slowly than those of WT after a strong conditioning flash: for a flash estimated to isomerize 1.2% of the M-cone pigment and 0.9% of the UV-cone pigment, recovery of 50% saturating amplitude was approximately 60-fold slower than in WT. CONCLUSIONS: (1) The amplitudes and sensitivities of the cone-driven responses indicate that cones and cone-driven neurons in RGS9-1 -/- mice have normal generator currents. (2) The greatly retarded recovery of cone-driven responses of RGS9-1 -/- mice relative to those of WT mice establishes that RGS9-1 is required for normal inactivation of the cone phototransduction cascades of both UV- and M-cones.

The light response of ON bipolar neurons requires G[alpha]o.

ON bipolar neurons in retina detect the glutamate released by rods and cones via metabotropic glutamate receptor 6 (mGluR6), whose cascade is unknown. The trimeric G-protein G(o) might mediate this cascade because it colocalizes with mGluR6. To test this, we studied the retina in mice negative for the alpha subunit of G(o) (Galpha(o)-/-). Retinal layering, key cell types, synaptic structure, and mGluR6 expression were all normal, as was the a-wave of the electroretinogram, which represents the rod and cone photocurrents. However, the b-wave of the electroretinogram, both rod- and cone-driven components, was entirely missing. Because the b-wave represents the massed response of ON bipolar cells, its loss in the Galpha(o) null mouse establishes that the light response of the ON bipolar cell requires G(o). This represents the first function to be defined in vivo for the alpha subunit of the most abundant G-protein of the brain.

The role of steady phosphodiesterase activity in the kinetics and sensitivity of the light-adapted salamander rod photoresponse.

We investigated the kinetics and sensitivity of photocurrent responses of salamander rods, both in darkness and during adaptation to steady backgrounds producing 20-3,000 photoisomerizations per second, using suction pipet recordings. The most intense backgrounds suppressed 80% of the circulating dark current and decreased the flash sensitivity approximately 30-fold. To investigate the underlying transduction mechanism, we expressed the responses as a fraction of the steady level of cGMP-activated current recorded in the background. The fractional responses to flashes of any fixed intensity began rising along a common trajectory, regardless of background intensity. We interpret these invariant initial trajectories to indicate that, at these background intensities, light adaptation does not alter the gain of any of the amplifying steps of phototransduction. For subsaturating flashes of fixed intensity, the fractional responses obtained on backgrounds of different intensity were found to "peel off" from their common initial trajectory in a background-dependent manner: the more intense the background, the earlier the time of peeling off. This behavior is consistent with a background-induced reduction in the effective lifetime of at least one of the three major integrating steps in phototransduction; i.e., an acceleration of one or more of the following: (1) the inactivation of activated rhodopsin (R*); (2) the inactivation of activated phosphodiesterase (E*, representing the complex G(alpha)-PDE of phosphodiesterase with the transducin alpha-subunit); or (3) the hydrolysis of cGMP, with rate constant beta. Our measurements show that, over the range of background intensities we used, beta increased on average to approximately 20 times its dark-adapted value; and our theoretical analysis indicates that this increase in beta is the primary mechanism underlying the measured shortening of time-to-peak of the dim-flash response and the decrease in sensitivity of the fractional response.

Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit.

Retinal photoreceptors use the heterotrimeric G protein transducin to couple rhodopsin to a biochemical cascade that underlies the electrical photoresponse. Several isoforms of each transducin subunit are present in the retina. Although rods and cones seem to contain distinct transducin subunits, it is not known whether phototransduction in a given cell type depends strictly on a single form of each subunit. To approach this question, we have deleted the gene for the rod transducin alpha-subunit in mice. In hemizygous knockout mice, there was a small reduction in retinal transducin alpha-subunit content but retinal morphology and the physiology of single rods were largely normal. In homozygous knockout mice, a mild retinal degeneration occurred with age. Rod-driven components were absent from the electroretinogram, whereas cone-driven components were retained. Every photoreceptor examined by single-cell recording failed to respond to flashes, with one exception. The solitary responsive cell was insensitive, as expected for a cone, but had a rod-like spectral sensitivity and flash response kinetics that were slow, even for rods. These results indicate that most if not all rods use a single transducin type in phototransduction.

The gain of rod phototransduction: reconciliation of biochemical and electrophysiological measurements.

We have resolved a central and long-standing paradox in understanding the amplification of rod phototransduction by making direct measurements of the gains of the underlying enzymatic amplifiers. We find that under optimized conditions a single photoisomerized rhodopsin activates transducin molecules and phosphodiesterase (PDE) catalytic subunits at rates of 120-150/s, much lower than indirect estimates from light-scattering experiments. Further, we measure the Michaelis constant, Km, of the rod PDE activated by transducin to be 10 microM, at least 10-fold lower than published estimates. Thus, the gain of cGMP hydrolysis (determined by kcat/Km) is at least 10-fold higher than reported in the literature. Accordingly, our results now provide a quantitative account of the overall gain of the rod cascade in terms of directly measured factors.

Mice lacking G-protein receptor kinase 1 have profoundly slowed recovery of cone-driven retinal responses.

G-Protein receptor kinase 1 (GRK1) ("rhodopsin kinase") is necessary for the inactivation of photoactivated rhodopsin, the light receptor of the G-protein transduction cascade of rod photoreceptors. GRK1 has also been reported to be present in retinal cones in which its function is unknown. To examine the role of GRK1 in retinal cone signaling pathways, we measured in mice having null mutations of GRK1 (GRK1 -/-) cone-driven electroretinographic (ERG) responses, including an a-wave component identified as the field potential generated by suppression of the circulating current of the cone photoreceptors. Dark-adapted GRK1 -/- animals generated cone-driven ERGs having saturating amplitudes and sensitivities in both visible and UV spectral regions similar to those of wild-type (WT) mice. However, after exposure to a bright conditioning flash, the cone-driven ERGs of GRK1 -/- animals recovered 30-50 times more slowly than those of WT mice and similarly slower than the cone-driven ERGs of mice homozygously null for arrestin (Arrestin -/-), whose cone (but not rod) response recoveries were found to be as rapid as those of WT. Our observations argue that GRK1 is essential for normal deactivation of murine cone phototransduction and provide the first functional evidence for a major role of a specific GRK in the inactivation of vertebrate cone phototransduction.

Molecular mechanisms of vertebrate photoreceptor light adaptation.

An important recent advance in the understanding of vertebrate photoreceptor light adaptation has come from the discovery that as many as eight distinct molecular mechanisms may be involved, and the realization that one of the principal mechanisms is not dependent on calcium. Quantitative analysis of these mechanisms is providing new insights into the nature of rod photoreceptor light adaptation.

UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments.

Molecular biological, histological and flicker electroretinographic results have established that mice have two cone photopigments, one peaking near 350 nm (UV-cone pigment) and a second near 510 nm [midwave (M)-cone pigment]. The goal of this investigation was to measure the action spectra and absolute sensitivities of the UV-cone- and M-cone-driven b-wave responses of C57BL/6 mice. To achieve this goal, we suppressed rod-driven signals with steady or flashed backgrounds and obtained intensity-response relations for cone-driven b-waves elicited by narrowband flashes between 340 and 600 nm. The derived cone action spectra can be described as retinal1 pigments with peaks at 355 and 508 nm. The UV peak had an absolute sensitivity of approximately 8 nV/(photon microm2) at the cornea, approximately fourfold higher than the M peak. In an attempt to isolate UV-cone-driven responses, it was discovered that an orange conditioning flash (lambda > 530 nm) completely suppressed ERG signals driven by both M pigment- and UV pigment-containing cones. Analysis showed that the orange flash could not have produced a detectable response in the UV-cone pathway were their no linkage between M pigment- and UV pigment-generated signals. Because cones containing predominantly the UV and M pigments have been shown to be located largely in separate parts of the mouse retina (), the most probable linkage is coexpression of M pigment in cones primarily expressing UV pigment. New histological evidence supports this interpretation (). Our data are consistent with an upper bound of approximately 3% coexpression of M pigment in the cones that express mostly the UV pigment.

Extreme responsiveness of the pupil of the dark-adapted mouse to steady retinal illumination.

PURPOSE: To measure the dependence of the size of the pupils of mice on steady retinal illumination. METHODS: Anesthetized C57BL/6 mice aged 7 to 8 weeks were placed in a ganzfeld chamber in darkness, and in monochromatic (510 nm) and white light whose intensity was varied more than 6 log units. The pupils of the mice were photographed with an infrared video camera and recorded on videotape and the pupil areas determined by digital image analysis of the video recordings. RESULTS: Fully dark-adapted murine pupils had an area of 2.29 +/- 0.35 mm2. The minimum pupil size at saturating intensity was 0.10 +/- 0.05 mm2. The steady state pupil area declined to half its dark-adapted maximum when ganzfeld luminance was 10(-5) scotopic candela (scot. cd) per meter squared. Pupil area declined to 20% of the dark-adapted magnitude at approximately 10(-3) scot. cd/m2. CONCLUSIONS: The mouse pupil can regulate retinal illumination by a factor exceeding 20. The neural circuitry that determines steady state murine pupil size is extremely sensitive to retinal illumination and under these experimental conditions is controlled almost exclusively by rod signals. This follows, because the ganzfeld illuminance (10(-5) scot. cd/m2) that causes the pupil to constrict to half its dark-adapted value corresponds to only approximately 0.01 photoisomerization per rod per second, whereas 80% reduction in pupil area occurs at approximately 1 photoisomerization per rod per sec. Based on this extreme responsiveness to steady illumination, the hypothesis is proposed that the murine pupil functions to protect a retinal circuit that can become saturated at extremely low photon capture rates. General principles of dark-adapted retinal circuitry support the identification of the first three neurons in the circuit as the rod, the rod bipolar, and the AII-amacrine. The rod and rod bipolar neurons do not approach saturation at the intensities at which the pupil constricts, however, and it seems unlikely that the AII-amacrine does. Thus the retinal neurons protected from saturation by the mouse pupil constrictions are probably ganglion cells with large receptive fields that have sustained responses.

Kinetics of recovery of the dark-adapted salamander rod photoresponse.

The kinetics of the dark-adapted salamander rod photocurrent response to flashes producing from 10 to 10(5) photoisomerizations (Phi) were investigated in normal Ringer's solution, and in a choline solution that clamps calcium near its resting level. For saturating intensities ranging from approximately 10(2) to 10(4) Phi, the recovery phases of the responses in choline were nearly invariant in form. Responses in Ringer's were similarly invariant for saturating intensities from approximately 10(3) to 10(4) Phi. In both solutions, recoveries to flashes in these intensity ranges translated on the time axis a constant amount (tauc) per e-fold increment in flash intensity, and exhibited exponentially decaying "tail phases" with time constant tauc. The difference in recovery half-times for responses in choline and Ringer's to the same saturating flash was 5-7 s. Above approximately 10(4) Phi, recoveries in both solutions were systematically slower, and translation invariance broke down. Theoretical analysis of the translation-invariant responses established that tauc must represent the time constant of inactivation of the disc-associated cascade intermediate (R*, G*, or PDE*) having the longest lifetime, and that the cGMP hydrolysis and cGMP-channel activation reactions are such as to conserve this time constant. Theoretical analysis also demonstrated that the 5-7-s shift in recovery half-times between responses in Ringer's and in choline is largely (4-6 s) accounted for by the calcium-dependent activation of guanylyl cyclase, with the residual (1-2 s) likely caused by an effect of calcium on an intermediate with a nondominant time constant. Analytical expressions for the dim-flash response in calcium clamp and Ringer's are derived, and it is shown that the difference in the responses under the two conditions can be accounted for quantitatively by cyclase activation. Application of these expressions yields an estimate of the calcium buffering capacity of the rod at rest of approximately 20, much lower than previous estimates.