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.

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.

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.

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.

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.

Recovery phase of the murine rod photoresponse reconstructed from electroretinographic recordings.

The activation and recovery phases of the murine rod photo-response were determined from corneal electroretinograms (ERGs) obtained in response to pairs of full-field flashes producing 50-10(5) photoisomerized rhodopsins (R*) per rod. The a-wave component of the ERG in response to the initial flash provided a well established measure of the activation phase of the rod response. The amplitude of the a-wave response to an intense second flash (45,000 R*) delivered 0.2-5 seconds (s) after the first flash was used to reconstruct the recovery phase of the response. For 160-3000 R* rod-1, recovery curves were isomorphic, translating on the time axis such that each e-fold increase in R* produced an incremental recovery delay of tau c = 210 +/- 50 ms (mean +/- SD). For initial flashes producing > 3000 R*, recovery curves lost their initial isomorphism and half-times had intensity dependence exceeding 1 s per e-fold increase in R*. We conclude that for flashes producing < 3000 R*, the effective lifetime of these R* is not > 210 ms. Two extant and non-mutually exclusive hypotheses are discussed that can account for the sharp increase in recovery times from flashes producing > 3000 R*. They are as follows: (1) approximately 0.03% of R* have a lifetime exceeding 1 s; and (2) the gamma subunit of phosphodiesterase (PDE gamma) serves as a GTPase-activating factor, and 3000 R* produce sufficient activated G-protein (G*) to exceed the total quantity of PDE gamma subunits such that excess G* must wait for unoccupied PDE gamma to inactivate via GTP hydrolysis.

Regulation of cGMP-dependent conductance in cytoplasmic membrane of rod outer segments by transducin.

A preparation of the photoreceptor G-protein, transducin, containing mainly the T alpha-subunit in a GTP-gamma-S-bound form, has been used for perfusion of the intracellular surface of excised patches of rod outer segment cytoplasmic membrane from frog retina. The preparation has been shown to result in the complete suppression of the cGMP-activated ionic conductance of the cytoplasmic membrane patch. The effect is entirely reversible after the protein has been washed out and is not observed in the absence of cGMP. The degree of conductance inhibition depends on the protein concentration, half-maximal inhibition occurring at 1 microM T alpha-GTP-gamma-S.

The effect of ATP, GTP and cAMP on the cGMP-dependent conductance of the fragments from frog rod plasma membrane.

Using a 'patch-clamp' method in the 'inside-out' configuration, ATP, ADP, AMP-PCP and AMP-PNP have been shown to increase the cGMP-dependent component of the rod plasma membrane conductance 2-4-fold and GTP, GDP but not GMP or nonhydrolyzable GTP analogs GMP-PNP and GTP-gamma-S to abolish the ATP action. The ATP and GTP effects were observed at [EDTA] = 1 mM when magnesium and calcium ions were absent. In about half of the experiments the cGMP-dependent conductance was shown to be increased by cAMP in the micromolar concentration range by 10-50%, the cAMP action did not depend on the presence of nucleoside triphosphates. In vivo ATP, GTP and cAMP are assumed to modulate the sensitivity of the photoreceptor plasma membrane to cGMP.

Direct action of cGMP on the conductance of retinal rod plasma membrane.

In order to identify the intracellular transmitter in the phototransduction process in the retinal rod, the action of cGMP, 2',3'cGMP, cAMP, GMP and Ca2+ on the isolated inside-out patches of the plasma membrane of retinal rods of the frog (Rana temporaria) was studied. cGMP applied at the intracellular membrane surface markedly increased the conductance of patches. The action of cGMP took place in the absence of nucleoside triphosphates and, hence, was not mediated by protein phosphorylation. The dependence of cGMP-induced component of conductance on cGMP concentration was S-shaped, with half-saturation within 10-30 microM and a Hill coefficient of about 1.7-1.8. cAMP, 2',3'cGMP, GMP (1 mM) did not exhibit any action on the membrane. Ca2+ did not affect the patch conductance in the absence of cGMP. In the presence of cGMP, lowering Ca2+ concentration from 10(-3) to 10(-8) M decreased the cGMP-dependent component of conductance by 20-30%. The approximate value of the elementary event underlying the cGMP-induced conductance estimated from the magnitude of the variance of the cGMP-induced current is within 100-250 fS. We suppose that the cGMP-activated channels found by us provide the light-sensitive conductance of the rod plasma membrane in vivo and that cGMP is the intracellular transmitter acting in the phototransduction process.

Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment.

Vertebrate rod photoreceptors hyperpolarize when illuminated, due to the closing of cation-selective channels in the plasma membrane. The mechanism controlling the opening and closing of these channels is still unclear, however. Both 3',5'-cyclic GMP and Ca2+ ions have been proposed as intracellular messengers for coupling the light activation of the photopigment rhodopsin to channel activity and thus modulating light-sensitive conductance. We have now studied the effects of possible conductance modulators on excised 'inside-out' patches from the plasma membrane of the rod outer segment (ROS), and have found that cyclic GMP acting from the inner side of the membrane markedly increases the cationic conductance of such patches (EC50 30 microM cyclic GMP) in a reversible manner, while Ca2+ is ineffective. The cyclic GMP-induced conductance increase occurs in the absence of nucleoside triphosphates and, hence, is not mediated by protein phosphorylation, but seems rather to result from a direct action of cyclic GMP on the membrane. The effect of cyclic GMP is highly specific; cyclic AMP and 2',3'-cyclic GMP are completely ineffective when applied in millimolar concentrations. We were unable to recognize discrete current steps that might represent single-channel openings and closings modulated by cyclic GMP. Analysis of membrane current noise shows the elementary event to be 3 fA with 110 mM Na+ on both sides of the membrane at a membrane potential of -30 mV. If the initial event is assumed to be the closure of a single cyclic GMP-sensitive channel, this value corresponds to a single-channel conductance of 100 fS. It seems probable that the cyclic GMP-sensitive conductance is responsible for the generation of the rod photoresponse in vivo.

Photoinduced isochromic rearrangement in rhodopsin.

Two tests have been used to detect and to study conformational rearrangements of cattle rhodopsin, occurring in the process of rhodopsin photolysis and resulting in no change in the visual pigment absorption spectrum. The first test concerns the ability of retinal to react with hydroxylamine. This ability occurs after photoisomerization of retinal with a time constant of 0.3 s at 20 degrees C reflecting this way a conformational transition demasking the retinal-opsin NC-bond. The other test takes advantage of the ability of rhodopsin to modulate the conductance of artificial lipid membranes. After a bleaching flash such a rhodopsin containing membrane shows a transient change in conductance. One of its characteristic time constants is that of NC-bond demasking. It shows that the "demasking" rearrangement is not an artefact due to presence of hydroxylamine and that it occurs in native rhodopsin. It has been shown that the "demasking" rearrangement is isochromic, not associated with known rhodopsin conformational transitions and, judging by its time characteristics, it may be of a functionally importance. The common scheme of rhodopsin photolysis has been modified to include a new conformational transition.

Single anion channels of frog rod plasma membrane.

Anion channels have been found in the plasma membrane of the outer and inner segment of the isolated retinal rod by means of the patch voltage-clamp technique. The permeability of the channels for different anions follows a sequence: Cl- greater than F- greater than NO3- greater than propionate; the channel conductance in the fully open state is 200 +/- 30 pS measured in 108 mM NaCl. The non-linear character of the current-voltage relationship at membrane potentials from -40 to -20 mV suggests that these anion channels may be involved in receptor potential generation through an electrogenic mechanism.