Early Receptor Potential Research Articles

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors.

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Mechanisms of Light-Induced Deformations in Photoreceptors

Biological cells deform on a nanometer scale when their transmembrane voltage changes, an effect that has been visualized during the action potential using quantitative phase imaging. Similar changes in the optical path length have been observed in photoreceptor outer segments after a flash stimulus via phase-resolved optical coherence tomography. These optoretinograms reveal a fast, millisecond-scale contraction of the outer segments by tens of nanometers, followed by a slow (hundreds of milliseconds) elongation reaching hundreds of nanometers. Ultrafast measurements of the contractile response using line-field phase-resolved optical coherence tomography show a logarithmic increase in amplitude and a decreasing time to peak with increasing stimulus intensity. We present a model that relates the early receptor potential to these deformations based on the voltage-dependent membrane tension—the mechanism observed earlier in neurons and other electrogenic cells. The early receptor potential is caused by conformational changes in opsins after photoisomerization, resulting in the fractional shift of the charge across the disk membrane. Lateral repulsion of the ions on both sides of the membrane affects its surface tension and leads to its lateral expansion. Because the volume of the disks does not change on a millisecond timescale, their lateral expansion leads to an axial contraction of the outer segment. With increasing stimulus intensity and the resulting tension, the area expansion coefficient of the disk membrane also increases as thermally induced fluctuations are pulled flat, resisting further expansion. This leads to the logarithmic saturation observed in measurements as well as the peak shift in time. This imaging technique therefore relates the structural changes in the photoreceptor to the underlying neurological function of transducing light into electrical signals. Such label-free optical monitoring of neural activity using fast interferometry may be applicable not only to optoretinography but also to neuroscience in general.

Rhodopsin in the rod surface membrane regenerates more rapidly than bulk rhodopsin in the disc membranes in vivo.

Sustained vertebrate vision requires that opsin chromophores isomerized by light to the all-trans form be replaced with 11-cis retinal to regenerate the visual pigment. We have characterized the early receptor potential (ERP), a component of the electroretinogram arising from photoisomerization-induced charge displacements in plasma membrane visual pigment, and used it to measure pigment bleaching and regeneration in living mice. The mouse ERP was characterized by an outward 'R2' charge displacement with a time constant of 215 μs that discharged through a membrane with an apparent time constant of ∼0.6 ms. After complete bleaching of rhodopsin, the ERP recovered in two phases. The initial, faster phase had a time constant of ∼1 min, accounted for ∼20% of the total, and was not dependent on the level of expression of the retinal pigment epithelium isomerase, Rpe65. The slower, complementary phase had a time constant of 23 min in wild-type (WT) mice (C57Bl/6) and was substantially slowed in Rpe65(+/-) mice. Comparison of the ERPs of a mouse line expressing 150% of the normal level of cone M-opsin with those of WT mice revealed that M-opsin contributed 26% of the total WT ERP in these experiments, with the remaining 74% arising from rhodopsin. Thus, the fast regenerating fraction (20%) corresponds approximately to the fraction of the total ERP independently estimated to arise from M-opsin. Because both phases of the ERP recover substantially faster than previous measurements of bulk rhodopsin regeneration in living mice, we conclude that delivery of the highly hydrophobic 11-cis retinal to the interior of rod photoreceptors appears to be retarded by transit across the cytoplasmic gap between plasma and disc membranes.

Absorption Spectra of Planarian Visual Pigments and Two States of the Metarhodopsin Intermediates

Abstract— Absorption spectra measurements of isolated planarian ocelli by a microspectrophotometer (MSP) and intra‐ocellar recordings of the early receptor potential (ERP) were carried out in order to characterize in situ planarian rhodopsin (pRh) and its photoproducts. The MSP spectra of the isolated ocelli revealed Λmax at about 500 nm. The ERP evoked by a test stimulus was a positive monophasic waveform in the dark but became negative during exposure to violet light. During subsequent darkness, the ERP rapidly reverted to a positive waveform but with a smaller amplitude than before exposure. The ERP amplitude recovered to its initial level upon exposure to red light. The ERP experiments suggest that pRh produces two metarhodopsin intermediates, with Λmax longer than that of pRh: the metastate responsible for the negative ERP converts to another metastate that results in a smaller ERP in the dark‐adapted ocellus.

Absorption Spectra of Planarian Visual Pigments and Two States of the Metarhodopsin Intermediates

Abstract— Absorption spectra measurements of isolated planarian ocelli by a microspectrophotometer (MSP) and intra-ocellar recordings of the early receptor potential (ERP) were carried out in order to characterize in situ planarian rhodopsin (pRh) and its photoproducts. The MSP spectra of the isolated ocelli revealed Λmax at about 500 nm. The ERP evoked by a test stimulus was a positive monophasic waveform in the dark but became negative during exposure to violet light. During subsequent darkness, the ERP rapidly reverted to a positive waveform but with a smaller amplitude than before exposure. The ERP amplitude recovered to its initial level upon exposure to red light. The ERP experiments suggest that pRh produces two metarhodopsin intermediates, with Λmax longer than that of pRh: the metastate responsible for the negative ERP converts to another metastate that results in a smaller ERP in the dark-adapted ocellus.

Role of the functional state of the central nervous system in the mechanism forming responses from the peripheral and central parts of the visual analyzer.

Chronic experiments on rabbits showed that changes in the functional state of the central nervous system induced by Nembutal and changes in the brainstem reticular formation induced by aminazine had significant effects on the formation of the early receptor potential, a-wave, and oscillatory potentials of retinal ERG signals, as well as on oscillatory potentials in the superior colliculi and visual cortex. Nembutal inhibited the formation of the early receptor potential, the a-wave, and the second and third ERG oscillatory potentials, and had similar effects on the oscillatory potentials of the superior colliculi and the visual cortex. Only the first oscillatory potential was enhanced in all visual system structures. Aminazine had long-lasting enhancing effects on the formation of the early receptor potential and the a-wave and on the oscillatory potentials of the retina, superior colliculi, and visual cortex. This enhancement may be associated with "release" of the retina and central structures from the constant inhibitory influences of the brainstem reticular formation.

Screening-Pigment Migration in the Octopus Retina Includes Control by Dopaminergic Efferents

ABSTRACT The extent of screening-pigment (SP) migration in the intact octopus retina and the amplitude of the early receptor potential (ERP) correspond with the degree of adaptation to light or darkness. The light-adapted retina has SP granules concentrated in an apical layer, at the tips of the photoreceptor rhabdoms and supporting cells, and the ERP is barely detectable. In the fully dark-adapted retina, the SP granules are mostly at the base of the rhabdoms, and the ERP is at its maximum. Retinae at intermediate stages, between the fully dark-and light-adapted states, show corresponding intermediate stages of SP migration and ERP amplitude. A series of experiments demonstrates the effects on SP migration of the efferent nerves, which form a subset of fibres in the optic nerves. When the optic nerves to one half of the retina have been severed, there is a dramatic difference in the distribution of SP in areas of the retina (of the dark-adapted eye) connected with severed or intact nerves: apical versus basal, respectively. On incubation of a light-adapted retina with 5 μmol l−1 dopamine, but not with other catecholamines or other putative neurotransmitter substances, SP migrates basally and the ERP is significantly larger than for controls. In octopuses treated with reserpine, SP stays in an apical location and the ERP remains very small, regardless of the state of adaptation and of whether the optic nerves are intact. It is concluded that dopaminergic efferents from the optic lobes effect dark-adaptational SP migration in the cephalopod retina. The arrival in the retina of efferent signals that effect adaptational changes through the mediation of dopamine is a remarkable analogue of the vertebrate system.

Flash photolysis of caged compounds in Limulus ventral photoreceptors.

Rapid concentration jumps of Ins(1,4,5)P3 or ATP were made inside Limulus ventral photoreceptors by flash photolysis of the parent caged compounds. In intact ventral photoreceptors, the photolysis flash evokes a maximum amplitude light-activated current; therefore, a procedure was developed for uncoupling phototransduction by blocking two of the initial reactions in the cascade, rhodopsin excitation and G protein activation. Rhodopsin was inactivated by exposure to hydroxylamine and bright light. This procedure abolished the early receptor potential and reduced the quantum efficiency by 325 +/- 90-fold (mean +/- SD). G protein activation was blocked by injection of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). GDP beta S injection reduced the quantum efficiency by 1,881 +/- 1,153-fold (mean +/- SD). Together hydroxylamine exposure and GDP beta S injection reduced the quantum efficiency by 870,000 +/- 650,000-fold (mean +/- SD). After the combined treatment, photoreceptors produced quantum bumps to light that was approximately 10(6) times brighter than the intensity that produced quantum bumps before treatment. Experiments were performed with caged compounds injected into photoreceptors in which phototransduction was largely uncoupled. Photolysis of one compound, myo-inositol 1,4,5-triphosphate P4(5)-1-(2-nitrophenyl)ethyl ester (caged IP3), increased the voltage clamp current in response to the flashlamp by more than twofold without changing the latency of the response. The effect was not seen with photolysis of either adenosine-5'-triphosphate P3-1-(2-nitrophenyl)ethyl ester (caged ATP) or caged IP3 in cells preloaded with either heparin or (1,2-bis-(o-amino-phenoxy)ethane-N-N-N'-N' tetraacetic acid tetrapotassium salt (BAPTA). The results suggest that photoreleased IP3 releases calcium ions from intracellular stores and the resulting increase in [Ca2+]i enhances the amplification of the phototransduction cascade.

Retinal Early Receptor Potential in Retinitis pigmentosa: Correlations with Visual Field and Fluoroangiography Estimates

The flash induced electroretinogram (ERG) and early receptor potential (ERP) were recorded in a population of patients with definite diagnosis of retinitis pigmentosa in the context of a study protocol including a complete diagnostic screening. ERPs were evoked by full-field 20-joule stimuli (5 stimuli at 10 min of interval in dark adaptation); recording was by skin electrodes positioned at the inferior orbital margin; amplifiers were set at 10-10,000 Hz. The flash ERG was absent or markedly abnormal in all cases, even at an early stage of development of the retinitis, while ERPs (with a normal morphology, latency of the R1 component increased and a reduced amplitude compared to normal controls) could be identified unambiguously in all cases. The amplitude reduction was highly correlated with the severity of the disease as determined by kinetic perimetry and fluorangiography.

Enigma of early receptor potential in fly eyes

The early receptor potential (ERP) of fly photoreceptors R1–6 has been recorded in the blowfly Calliphora erythrocephala, and quantitatively analyzed. Photoconversion of the native visual pigment (P) into the meta-state (M) does not induce a measurable ERP. Photoconversion of M into P induces a biphasic ERP, presumably due to the slow thermal decay of intermediate N, recently discovered by Roebroek, Gagné and Stavenga (1989). The experimentally measured ERP's are interpreted with an RC-model which incorporates the time constant of the flash, the time constant of the thermal decay of intermediate N, the electrical properties of the photoreceptor cell and the charge displacements within the visual pigment molecules occurring upon photoconversion. The model satisfactorily describes the ERP for the M to P conversion by assuming that effectively 0.03 electron charges are moved across the cell membrane during conversion of the M to the N state and that 0.04 electron charges are moved in the opposite direction during conversion of N to P. The net 0.01 electron charge movement during conversion of P to M would, according to the simple RC-model, induce a measurable ERP, in conflict with the experimental results. The longstanding enigma of the ERP of fly photoreceptors appears largely but not completely resolved.

Disorders of retinal function in long-term therapy with retinoid etretinate

Four patients receiving etretinate as long-term therapy were examined. The accumulated dosage was 135 g for Patient 1, about 80 g for Patients 2 and 3, and 31 g etretinate and 4.8 g etretine (Ro 10-1670; main metabolite of etretinate) for Patient 4. Clinical examinations, visual fields (Haitz, Amsler, Goldmann), and EOGs (two patients) were normal. Patient 1 (135 g) showed a decreased level of rod sensitivity at dark adaptation (Goldmann-Weekers), whereas the cone responses were regular in all cases. Furthermore, Patient 1 showed reduced scotopic ERG amplitudes. Aside from this, photopic and scotopic ERGs were normal as far as a- and b-wave amplitudes and peak latencies were concerned. The flicker fusion frequencies were found to be regular. The peak latencies of the R2-component of the early receptor potentials seemed rather short. In all four patients alterations of color sense were analyzed in the Farnsworth Munsell 100 Hue test. The color sense defect seems to originate in the blue-yellow spectrum and--at higher dosages--extends to the red-green spectrum. Examinations with the anomaloscope revealed deuteranomalous settings. Ophthalmological checks for patients receiving long-term aromatic retinoid therapy are recommended, especially with regard to dark adaptation and color sense.

The early receptor potential (ERP).

The ERP contains information on the function of the outer segments of the retinal photoreceptors. The authors have established normative values of the ERP and the factors which might be of influence hereupon. ERP findings in patients suffering from various eye diseases are given. Value and limitation of ERP recording are discussed in relation to the other clinically available electrodiagnostic tests.

Spectrally selective flash early receptor potential (ERP) in dichromats.

The human spectrally selective flash early receptor potential (ERP) was studied in 12 dichromats: 6 protanopes (12 eyes) and 6 deuteranopes (12 eyes). Color filters used were Kodak Wratten filters No.23A, No.57, and No.47 for the red, green, and blue flash ERPs, respectively. The ERP amplitude was measured between the summits of R1 and R2. Mean amplitudes of the red flash ERP and green flash ERP were highly significantly decreased in the protanopes (p less than 0.001) and deuteranopes (p less than 0.01) as compared with the corresponding data in 10 normal subjects (20 eyes). The mean amplitude of the blue flash ERP was significantly lower than normal (p less than 0.001) in the deuteranopes. The mean ratio of the blue flash ERP amplitude to the red flash ERP amplitude showed a highly significant increase in the protanopes (p less than 0.001) and a highly significant decrease in the deutoranopes (p less than 0.001) compared with the mean ratio in the normal subjects, indicating a new, useful index for the objective clinical detection of congenital color defects.

Spatial properties of the prolonged depolarizing afterpotential in barnacle photoreceptors. I. The induction process.

In invertebrate photoreceptors, when the light stimulus results in substantial net transfer of the visual pigment from the rhodopsin (R) to the metarhodopsin (M) state, the ordinary late receptor potential (LRP) is followed by a prolonged depolarizing afterpotential (PDA). The dependence of the amplitude of the PDA on the amount of pigment conversion is strongly supralinear, and the PDA duration also depends on this amount. These observations indicate an interaction among the elements of the PDA induction process and also make possible a test of the range of this interaction. The test consists of a comparison of the PDA after localized pigment conversion, obtained by strong spot illumination, to that after weaker diffuse illumination converting a comparable total amount of pigment. The experiment was performed on the barnacle lateral eye. The effective spot size was measured by the early receptor potential (ERP), in seawater saturated with CO2, which considerably reduced the electrical coupling between the photoreceptors. The ERP was also used to determine whether there is diffusion of R molecules into the illuminated spot. The spot illumination induced a PDA with small amplitude and long duration, while no detectable PDA was induced by the diffuse light. This indicates that the range of the PDA interaction is much smaller than the entire cell. In addition, the ERP results showed that there was no detectable diffusion of R molecules into the illuminated spot area over 30 min. This measurement, with a calculated correction for the microvillar geometry of the photoreceptor, enabled us to put an upper limit on the diffusion coefficient of the pigment molecules in the inact, unfixed barnacle photoreceptor of D less than 6 X 10(-9) cm2 s-1.

Ontogeny of the visual system in the cuttlefish, Sepiella japonica. II. Intramembrane particles, histofluorescence, and electrical responses in the developing retina.

To study molecular and functional differentiation of photoreceptive membranes, the development of the retina in embryos of the cuttlefish, Sepiella japonica, was examined by the freeze-fracture technique, by histo-fluorescence of retinal-related protein, and by electrical responses to photic stimulation. P-face particles in the microvillar plasma membrane of the receptor cell increased in density from about 2,000/micron2 to about 6,000/micron2 during the early stages of rhabdomere formation (from stage 31 to stage 36). The size distribution of P-face particles in the microvillar and the cell body plasma membranes showed a similar monomodal pattern with a peak at 8 nm in diameter from stage 31 to 33. In the cell body plasma membrane, the size distribution pattern hardly changed throughout later stages, but in the microvillar membrane, the peak shifted progressively to 9 nm (stages 35 and 36) and then to 10 nm (stage 37). The histofluorescence of reduced rhodopsin was first visible at stage 36 in the layer of growing apical processes. The fluorescence of reduced retinochrome first appeared faintly at stage 40 on both sides of the basal lamina. Electroretinograms (ERGs) were first obtained at stage 34 when many microvilli appeared from the apical processes, increasing rapidly in amplitude with increase in regularity of the microvillar arrangement from stage 35 to 36. Early receptor potentials (ERPs) elicited by a bright light flash were first recorded at the earliest stage of the apical process formation (stage 30), increasing gradually in amplitude during development. The increase was correlated with an increase in the total number of P-face particles in the microvillar membrane within the retina. These findings suggest that the P-face particles in the microvillar membrane are associated with rhodopsin, that the receptor cells have a small amount of rhodopsin before rhabdomere formation, and that differentiation of the apical plasma membrane of the receptor cells into the functional photoreceptive membrane occurs in conjunction with its morphological differentiation into rhabdomeres.

Multiple Evanescent White Dot Syndrome

We performed electrophysiologic studies of photoreceptor function in three patients with multiple evanescent white dot syndrome. During the acute stage, while the visual acuity was impaired, the electroretinogram (ERG) a-wave and the early receptor potential (ERP) amplitudes were profoundly decreased. The ERP regeneration times, determined for one subject, were prolonged. These findings suggest that photoreceptor function was impaired (abnormal a-wave), the effective visual pigment optical density of the outer segments was markedly reduced (ERP amplitude), and visual pigment regeneration was abnormal (ERP regeneration kinetics). During the recovery stage, the ERG and ERP amplitudes and visual acuity returned to normal. Our patients' disease seemed to be primarily of the retinal pigment epithelium (RPE). The decreased visual pigment density and prolonged regeneration kinetics emphasize the physiologic dependence of the sensory retina on the RPE.

Rapid Mechanical Responses of the Dark-Adapted Squid Retina to Light Pulses

Dark-adapted squid retinas respond to brief light pulses with early and late mechanical responses. These responses are mechanical counterparts of the early and late receptor potentials.

Early receptor potential in rod and in cone dominated eyes.

Early receptor potential in rod and in cone dominated eyes.

Colour dependence of the early receptor potential and late receptor potential in scallop distal photoreceptor.

1. Intracellular voltage and current responses to short (blue) and long (red) wave-length lights were measured in the distal hyperpolarizing photoreceptor (;off receptor') of the isolated and perfused scallop (Pecten irradians) retina.2. The early receptor potential (e.r.p.) was isolated by holding membrane potential at the reversal potential for the late receptor potential (l.r.p.) or by working at temperatures (< 5.0 degrees C) that abolished the l.r.p.3. The e.r.p., measured using intense flashes of white light, consisted of a positive phase followed by a negative phase, but was converted to a monophasic, negative-going wave following pre-adaptation with red light and to a monophasic, positive-going wave following pre-adaptation with blue light.4. The spectral sensitivity curve for the negative e.r.p. was maximum at 500 nm, whereas the spectral sensitivity curve for the positive e.r.p. was maximum at 575 nm.5. The positive or negative e.r.p.s approached their maximum amplitude exponentially when tested with red or blue flashes of increasing intensity. The results suggest that the positive (or negative) e.r.p. is proportional to the number of photopigment molecules photo-isomerized.6. The photosensitivity maximum of rhodopsin calculated at 500 nm, using the exponential constant and the spectral sensitivity data, was estimated to be 2.1 x 10(-16) cm(2) photon(-1), whereas the photosensitivity maximum of metarhodopsin calculated at 575 nm was estimated to be 2.6 x 10(-16) cm(2) photon(-1).7. In cells pre-adapted with white light, stimulation with blue light caused a hyperpolarizing l.r.p. which was followed by a prolonged hyperpolarizing after-potential (p.h.a.). Stimulation with red light under similar conditions caused an initial hyperpolarization which was followed by a small depolarization during the stimulus, but no after-potential.8. The duration of the p.h.a. was increased by pre-adaptation with a red light, which caused the maximum net transfer of metarhodopsin to rhodopsin; however, its decay was always complete in 5 min or less.9. The photo-isomerization of metarhodopsin by red light suppressed the p.h.a. and caused an after-depolarizing response that decayed in less than 1 min.10. The spectral sensitivity curve for the induction of the p.h.a. was maximum at 500 nm and corresponded to the spectral sensitivity for the negative e.r.p. and for the l.r.p. studied in the dark-adapted retina, whereas the spectral sensitivity curve for the suppression of the p.h.a. and for the induction of the after-depolarization was maximum at 575 nm and corresponded to the spectral sensitivity for the positive e.r.p.11. In photoreceptors clamped to the resting potential in normal ASW, the photo-isomerization of rhodopsin, in the absence of light absorption by metarhodopsin, activated a persistent outward current that had the same time course of decay as the p.h.a. The photo-isomerization of metarhodopsin suppressed the persistent outward current and activated an inward current whose decay took longer than the decay of the after-depolarizing response.12. In the absence of external Ca(2+) and Na(+) ions, the persistent outward current produced by light absorption by rhodopsin, and the inward current produced by light absorption by metarhodopsin, both reversed at the K(+) equilibrium potential. The results show that the induction of the prolonged hyperpolarizing after-potential and the after-depolarizing response involve only the movement of K(+) ions through the same light-dependent K(+) channels that determine the hyperpolarizing l.r.p. of the distal cells.

Early receptor potential of pineal organ and lateral eye of the pike.

Early receptor potential of pineal organ and lateral eye of the pike.