Effects of carteolol on the electroretinogram in the perfused cat eye

Carteolol, a nonselective beta-antagonist, was administered intra-arterially in perfused cat eyes. Carteolol increased both photopic and scotopic electroretinogram b-wave amplitude dose-dependently and reversibly, but carteolol failed to induce significant changes in the flow rate of perfusate. This study suggests that carteolol may increase selectively the retinal perfusion flow rate, though it did not reflect the total perfusion flow, or carteolol may have an interaction with retinal beta-adrenergic receptors related to the origin of the b-wave. These ideas are supported by carteolol's intrinsic sympathomimetic activity and effects on endothelium of vessels.     

The products of photoreversing rhodopsin bleaching by microsecond flashes in the isolated vertebrate retina

Bleaching experiments were carried out at room temperature on rhodopsin in isolated rat, frog and rat retinas with blue and orange laser flashes of very high energy and duration less than 3 microseconds. Blue flashes bleached a maximum of about 50% at the highest energies; orange ones bleached about 30% at intermediate energies but the value decreased to below 20% as the energy increased. This bleaching behaviour can be explained in terms of a kinetic model which assumes that bleaching is photoreversed during the flashes and which incorporates the relevant properties of rhodopsin, isorhodopsin, bathorhodopsin and lumirhodopsin.     

Long-term behavior and intra-individual stability of the direct current electroretinogram and of the standing potential in the albino rabbit eye

The direct current electroretinogram (ERG) and the standing potential (SP) were studied in seven albino rabbits under general anesthesia. Identical experiments were performed on 2 consecutive days. After 30 min of dark adaptation, repeated light stimuli of maximal intensity of the system were presented to the eyes. The interstimulus interval was 70 s, and stimulus duration 10 s. Each experiment lasted for almost 3 h. In the first experiment, the b- and c-wave amplitudes measured in response to the second light stimulus were markedly reduced compared to those recorded in response to the first stimulus. Both amplitudes then recovered. The b-wave attained a peak about 20 min after the start of light stimulation. The peak was followed by a trough about 20 min later, and the amplitude then slowly increased. Following the minimum recorded during the second light stimulus, the c-wave amplitude reached a peak about 14 min after the start of stimulation. A trough in the amplitude occurred 20 min later. The amplitude then slowly increased to the end value, which was higher than the initial level. The a-wave behaved similarly to the b-wave, but the changes in most cases did not attain statistical significance. A minimum in the SP occurring at the second light stimulus was followed by a peak about 13 min after the start of light stimulation, and then by a trough about 17 min later. In the second experiment, performed one day after the first, the development of the a-, b-, and c-wave amplitudes and of the SP was similar to that observed during the first experiment, and no statistically significant differences between the two experiments were found. The reactions of the ERG and the SP were thus very stable between identical experiments performed on two consecutive days.     

Contributions of the mouse UV photopigment to the ERG and to vision

The mouse retina contains two classes of cone photopigment with respective peak sensitivities in the middle (M) wavelengths and in the ultraviolet (UV) portion of the spectrum. To examine the functional roles subserved by the UV pigment, the absorption of light by the mouse lens was measured and voltage versus intensity (V-log I) functions were derived from recordings of the flicker ERG made under test conditions designed to maximize the relative sensitivities of the two pigment types. These V-log I data accurately predict ERG-based spectral sensitivity functions, but they fail to provide a similarly accurate account of behaviorally based measurements of spectral sensitivity in that the ERG spectral sensitivity function has much higher sensitivity in the UV wavelengths than does the behavioral spectral sensitivity function. The disparity between these two is argued to be a consequence of the widespread receptor co-expression of the two types of cone pigment in the mouse and of the pattern of retinal wiring that is thought to be characteristic of all mammalian retinas.     

Comparing the retinal structures and functions in two species of gulls (Larus delawarensis and Larus modestus) with significant nocturnal behaviours

Ring-billed gulls (Larus delawarensis) and gray gulls (Larus modestus) are two species active both by day and night. We have investigated the retinal adaptations that allow the diurnal and nocturnal behaviours of these two species. Electroretinograms and histological analyses show that both species have a duplex retina in which cones outnumber rods, but the number of rods appears sufficient to provide vision at night. Their retinas respond over the same scotopic dynamic range of 3.4logcdm(-2), which encompasses all of the light levels occurring at night in their photic environment. The amplitudes of the scotopic saturated a- and b-wave responses as well as the photopic saturated b-wave response and the photopic sensitivity parameter S are however higher in ring-billed gulls than in gray gulls. Moreover, the process of dark adaptation is about 30min faster in gray gulls than in ring-billed gulls. Our results suggest that both species have acquired in the course of their evolution functional adaptations that can be related to their specific photic environment.     

Extracellular glucose dependence of rhodopsin regeneration in the excised mouse eye.

To study the process of rhodopsin regeneration a superfused excised whole eye preparation of the albino mouse was developed. With this preparation, complete regeneration could be observed after each of the first two illuminations (bleaching 15-20%), and incomplete regeneration after a third illumination. Regeneration was minimal at extracellular glucose concentrations of 0 or 1 mM with improved regenerations at higher concentrations. Maximum regenerations were observed at glucose concentrations of 4-10 mM. First-bleach regenerations were as follows: 0 mM glucose, 20%; 1 mM, 8%; 2 mM, 45%; 3 mM, 82%; 4 mM, 115%; 5.1 mM, 121%; 7 mM, 120%; and 10 mM, 126%. The effects of reduced glucose were reversible. After an initial bleach with 0 or 1 mM extracellular glucose that exhibited minimal regeneration, the re-addition of glucose (5.1 mM) restored the ability of the eye to regenerate rhodopsin following a second bleach, but only to the level prior to that bleach. Mitochondrial substrates fumarate (10 mM) or pyruvate (10 mM) partly substituted for glucose, exhibiting first-bleach regenerations of 56 and 85%, respectively.     

An adaptive ERG technique to measure normal and altered dark adaptation in the mouse

The time-course of dark adaptation provides valuable insights into the function and interactions between the rod and cone pathways in the retina. Here we describe a technique that uses the flash electroretinogram (ERG) response to probe the functional integrity of the cone and rod pathways during the dynamic process of dark adaptation in the mouse. Retinal sensitivity was estimated from the stimulus intensity required to maintain a 30 μV criterion b-wave response during a 40 min period of dark adaptation. When tracked in this manner, dark adaptation functions in WT mice depended upon the bleaching effects of initial background adaptation conditions. Altered dark adaptation functions, commensurate with the functional deficit were recorded in pigmented mice that lacked cone function (Gnat2 ^cplf3 ) and in WT mice injected with a toxin, sodium iodate (NaIO₃), which targets the retinal pigment epithelium and also has downstream effects on photoreceptors. These data demonstrate that this adaptive tracking procedure measures retinal sensitivity and the contributions of the rod and/or cone pathways during dark adaptation in both WT control and mutant mice.     

Contribution of rod and cone pathways to the dark-adapted electroretinogram (ERG) b-wave following retinal degeneration in RCS rats

Although the RCS rat is widely used as a model of progressive photoreceptor loss, it is unclear how the relative rod and cone functions change with age. Rod and cone b-waves were isolated using a double flash ERG paradigm. In contrast to cones, rods never reached normal functional maturity levels, and the ERG b-wave changed from being predominantly rod-driven to being purely cone-driven by age 74 days, at which point, b-waves were progressively replaced by negative STR-like (scotopic threshold response) waves that persisted up to age 180 days. A double flash commonly abolished this wave and unveiled a b-wave.     

Availability of 11-cis retinal and opsins without chromophore as revealed by small bleaches of rhodopsin in excised albino mouse eyes

Small bleaches were used to study the rhodopsin regeneration process. At bleaches from 5.2% to 24.7%, the rhodopsin regenerations were consistent with a one-for-one recovery of bleached molecules. At response saturation rod photoreceptors exhibit a bleach level of only 5%. Major increases in rhodopsin regeneration were observed at bleach levels between 1.3% and 5.2%. The rhodopsin regenerations exhibited a linear relationship that was 4-times the bleach (dark adaptations of 0.75 and 1.5 h). The data show that the bleach initiates the availability, and possibly production, of 11-cis retinal in amounts that are 4-times the number of bleached molecules within the functional range of the rod photoreceptors. Rhodopsin regeneration also requires the presence of opsins without chromophore. Regenerations beyond the bleach indicate the presence of such opsins prior to the bleach. The opsin amounts were 8.1%, 8.6%, 3.1% and 0% of the total visual pigment at dark adaptation times of 0.75, 1.5, 24 and 48 h, respectively. Those opsins, as well as the ones produced by the bleach, may be regenerated to rhodopsin following a small bleach or with additional time in the dark.     

Studies on the effects of bleaching amphibian rod pigments in situ: II. The kinetics of the slow bleaching reactions in axolotl red rods

The kinetics of the slow bleaching reactions in axolotl retinas either rich in rhodopsin or porphyropsin were studied by measuring flash-induced changes in absorbance with ultraviolet (380 or 400 nm) and blue light (470 and 480 nm). The results were analyzed in terms of four kinetic models, three of which could satisfactorily simulate the data. The models require that the extinction coefficients of some of the bleaching products in situ should differ from their reported solution values.For the most plausible model, the rate constants for rhodopsin bleaching at 19–20°C are: metarhodopsin II → metarhodopsin III, 0·32 min^?1; metarhodopsin II → “retinal”, 0·77 min^?1; metarhodopsin III → retinol, 0·19 min^?1; “retinal” → retinol, 0·087 min^?1; the rate constants for porphyropsin bleaching at 22–23°C are: metaporphyropsin II → metaprophyropsin III, 0·46 min^?1; metaporphyropsin II → “3-dehydroretinal”, 0·72 min^?1; metaporphyropsin III,→ 3-dehydroretinol, 0·20 min^?1; “3-dehydroretinal” → 3-dehydroretinol, 0·13 min^?1.     

The specific inhibition of 11-cis-retinyl palmitate formation in the frog eye by diaminophenoxypentane, an inhibitor of rhodopsin regeneration

The antischistosomal drug 1,5-di-(p-aminophenoxy) pentane (DAPP), an inhibitor of rhodopsin regeneration in the vertebrate retina, is shown to completely block the production of 11-cis-retinyl palmitate in the frog eye. An untreated frog generates a large amount of 11-cis-retinyl palmitate during 1-2 days in the dark after a strong bleach. Also, it is demonstrated that DAPP can deplete the stores of 11-cis-retinyl palmitate in the dark-adapted frog eye. The specificity of DAPP's inhibition of dark-adaptation is explored, and the usefulness of employing retinotoxic drugs to investigate the physiology and biochemistry of rhodopsin regeneration is discussed.     

Studies on the effects of bleaching amphibian rod pigments in situ: I. The absorbance spectra of axolotl and tiger salamander rhodopsin and porphyropsin

Spectrophotometric measurements on retinal folds of axolotl larvae show that the visual pigments of the red rods are rhodopsin with a λ_max of ca. 502 nm and porphyropsin with a λ_max of ca. 526 nm. The spectral changes associated with the bleaching products of these pigments are described. When the larvae metamorphose, following the addition of thyroxine to their water, more than two-thirds of the pigment in their rods is rhodopsin. The same appears to be true for the red rod pigments of naturally metamorphosed tiger salamanders.     

Studies on the effects of bleaching amphibian rod pigments in situ: III. Linear dichroism in axolotl red rods before and during bleaching

The linear dichroism of axolotl porphyropsin and rhodopsin was studied spectrophotometrically using transverse illumination of retinal folds. Partial bleaching experiments on retinas whose rods contained roughly equal mixtures of the pigments showed that the dichroism of the porphyropsin was lower than that of the rhodopsin. The orientations of the long-lived photoproducts observed in retinas containing predominantly porphyropsin or rhodopsin were determined from absorbance changes at selected wavelengths following flash bleaches. The dichroism of metaporphyropsin II and metaporphyropsin III was found to be higher than that of the porphyropsin they derived from, while metarhodopsin II and III showed similar dichroism to their parent rhodopsin. Kinetic analyses of the near u.v. absorbance changes during bleaching indicated that both “retinal” and “3-dehydroretinal” intermediates were also aligned predominantly parallel to the plane of the disc membrane. In contrast, the final in situ bleaching products, retinol and 3-dehydroretinol, were found to be oriented at a large mean angle to the plane of the disc membrane.     

Results from screening over 9000 mutation-bearing mice for defects in the electroretinogram and appearance of the fundus

Random mutagenesis combined with phenotypic screening using carefully crafted functional tests has successfully led to the discovery of genes that are essential for a number of functions. This approach does not require prior knowledge of the identity of the genes that are involved and is a way to ascribe function to the nearly 6000 genes for which knowledge of the DNA sequence has been inadequate to determine the function of the gene product. In an effort to identify genes involved in the visual system via this approach, we have tested over 9000 first and third generation offspring of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) for visual defects, as evidenced by abnormalities in the electroretinogram and appearance of the fundus. We identified 61 putative mutations with this procedure and outline the steps needed to identify the affected genes.     

Suitability of retinol, retinal and retinyl palmitate for the regeneration of bleached rhodopsin in the isolated frog retina

The all-trans, 9-cis and 11-cis isomers of retinol, retinal and retinyl palmitate were incorporated into unilamellar dioleoyl-lecithin vesicles (liposomes). Isolated frog retinas were inserted into a perfusion chamber and their absorption spectra were recorded (i) in the dark-adapted state, (ii) after exposure to intense light that bleached greater than 95% of rhodopsin and (iii) after liposomes had been added to the perfusate of the bleached preparations. In each experiment, one of the above isomers of the retinol analogs was tested. Regeneration was promoted by the 9-cis and the 11-cis isomer of retinol and of retinal whereas the trans isomers of all compounds and the cis isomers of retinyl palmitate were inactive.     

Effects of intravitreal perfusion with dopamine in different concentrations on the DC electroretinogram and the standing potential of the albino rabbit eye

The direct current electroretinogram and the standing potential were recorded from both eyes of 23 albino rabbits during intraocular perfusion of one of the eyes, which was vitrectomized, with a physiologic reference solution (PHS). PHS was then replaced by a test solution containing dopamine dissolved in PHS. The fluids were subsequently alternated (PHS-dopamine-PHS). During irrigation with 0.25–0.5 mM dopamine (11 rabbits) the c-wave amplitude was 140% higher (p < 0.001) and during irrigation with 25 mM dopamine (6 rabbits) 85% lower (p < 0.01) than it was during the corresponding initial perfusion with PHS. The simultaneously recorded b-wave amplitude was reduced (0.25–0.5 mM: -22%, p < 0.001; 25 mM: - 69%, p < 0.001) and the SP level increased (0.25–0.5 mM: +2375 V, p < 0.01; 25 mM: +2530 V, p < 0.05) compared with the values obtained during the corresponding preceding irrigation with PHS. Thus the changes in the b- and c-wave amplitudes during perfusion with dopamine were dependent on the concentration of the drug. In the contralateral control eye (23 rabbits) the c-wave amplitude was 21% higher (p < 0.001), the b-wave amplitude 14% higher (p < 0.001) and the standing potential 1007 V higher (p < 0.001) during intravitreal perfusion with dopamine in the other eye than during the preceding irrigation with PHS in that eye, possibly as a result of increasing dark adaptation.     

Ultraviolet radiation transmittance of the mouse eye and its individual media components

Recently, the mouse has become the preferred animal model in ophthalmic research. Therefore, there is a need for enhanced understanding of the mouse eye to validate its use in different experimental setting. The purpose of this study was to determine the ocular transmittance of the whole mouse eye, the cornea and the crystalline lens, particularly in the ultraviolet radiation (UVR) wavebands. This was carried out using a non-cuvette based fiber optic spectrometer system and the resulting transmittance curves were compared with published cone spectral response curves and mouse ocular transmittance data. First, transmittance curves of the whole mouse eye were measured by removing a small disc of sclera from the posterior pole to provide an anterior to posterior optical path. No statistical difference was found between left and right eye in each of the four mice sampled, therefore, all eight eyes were included in the final statistical analysis. The average of five test measurements from each left and right eye for the four test mice showed a transmittance cut off at approximately 310 nm. Secondly, the cornea with a scleral rim was excised and transmittance curves obtained for all eight eyes. This data showed an average transmittance cut off at 280 nm for the cornea. Similarly measured data for the excised crystalline lens showed UVR transmittance down to 310 nm. The good correlation between total ocular UVR transmittance and the sum of the individually measured components (i.e. the cornea and the crystalline lens) supported the validity of our method and its findings. This experiment demonstrated that the mouse cornea transmits more UV-B (280-315 nm) than the rabbit and the human corneal transmittance. The mouse crystalline lens on the other hand showed a cut off in the UV-B at 310 nm, which is at a much lower UV-B wavelength than the approximate UV-A (315-400 nm) cut off for the human crystalline lens at around 390 nm. The increased transmittance of UVR in the mouse eye serves its vision, since the mouse has a cone photopigment peaking at approximately 350 nm. Due to the above stated differences between the mouse and the human it is concluded that the mouse is not an ideal model for the human eye in experiments involving UVR.     

The c-wave of the direct-current electroretinogram and the standing potential of the albino rabbit eye in response to repeated series of light stimuli with different interstimulus intervals

The direct-current electroretinogram and the standing potential of the eye of seven albino rabbits were recorded in response to repeated light stimuli, which were presented in four consecutive series. The intervals between the beginning of succeeding stimuli were 8 minutes, 4 minutes, 2 minutes and 70 seconds (series 1, 2, 3 and 4, respectively). Stimulus duration (10 seconds) and light intensity (6.8 × 10⁴ lux) were constant during the experiments. The series lasted for 36–40 minutes, and each was preceded by 30 minutes of dark adaptation. During series 1, the end amplitudes of the a-, b- and c-waves were not significantly changed compared with the initial levels. During series 2, 3 and 4, the a-, b- and c-wave amplitudes were markedly reduced immediately after the first electroretinogram recording. The a- and b-waves then recovered to a limited extent, but the c-wave was more fully restored. A slight peak in the c-wave amplitude could be discerned 16–20 minutes after the start of recording. A decrease in the standing potential was seen 50–54 seconds after the start of light stimulation during all four series, and a peak occurred 12–16 minutes after the start of recording. The similarity in behavior between the c-wave and the standing potential suggests the operation of a pigment epithelial mechanism behind the more complete recovery of the c-wave amplitude. When electroretinogram amplitudes and standing potential levels are discussed, and when one experiment is compared with another one, it is important that adaptational and stimulus conditions, as well as time course, are well controlled and clearly specified.Abbreviations RPE retinal pigment epithelium - SP standing potential