Isolated Frog Retina Research Articles

Endogenous Nitric Oxide Content in the Cellular Layers of the Retina

Nitric oxide is a universal molecule which regulates numerous functions in the body. In the retina, nitric oxide plays a regulatory role modulating synaptic transmission between photoreceptors and bipolar cells; it also has a toxic role and is a necessary inducer of the development of apoptosis in the outer nuclear layer and the ganglion cell layer. We report here studies of the endogenous nitric oxide concentration in vivo in the cellar layers of the retina. Nitric oxide concentrations were determined by two independent methods – using a spin trap and electron paramagnetic resonance (EPR) and an NO-sensitive microelectrode, to obtain in situ measurements in the live isolated frog retina. Significant nitric oxide concentrations were seen only in the ganglion cell layer (~0.25 μM) and the inner segments of photoreceptors (~0.6 μM). NO synthase was present in the same locations; the activity and kinetic parameters of this enzyme were also determined.

Two-photon excited autofluorescence imaging of freshly isolated frog retinas

The purpose of this study was to investigate cellular sources of autofluorescence signals in freshly isolated frog (Rana pipiens) retinas. Equipped with an ultrafast laser, a laser scanning two-photon excitation fluorescence microscope was employed for sub-cellular resolution examination of both sliced and flat-mounted retinas. Two-photon imaging of retinal slices revealed autofluorescence signals over multiple functional layers, including the photoreceptor layer (PRL), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Using flat-mounted retinas, depth-resolved imaging of individual retinal layers further confirmed multiple sources of autofluorescence signals. Cellular structures were clearly observed at the PRL, ONL, INL, and GCL. At the PRL, the autofluorescence was dominantly recorded from the intracellular compartment of the photoreceptors; while mixed intracellular and extracellular autofluorescence signals were observed at the ONL, INL, and GCL. High resolution autofluorescence imaging clearly revealed mosaic organization of rod and cone photoreceptors; and sub-cellular bright autofluorescence spots, which might relate to connecting cilium, was observed in the cone photoreceptors only. Moreover, single-cone and double-cone outer segments could be directly differentiated.

High spatiotemporal resolution imaging of fast intrinsic optical signals activated by retinal flicker stimulation

High resolution monitoring of stimulus-evoked retinal neural activities is important for understanding retinal neural mechanisms, and can be a powerful tool for retinal disease diagnosis and treatment outcome evaluation. Fast intrinsic optical signals (IOSs), which have the time courses comparable to that of electrophysiological activities in the retina, hold the promise for high resolution imaging of retinal neural activities. However, application of fast IOS imaging has been hindered by the contamination of slow, high magnitude optical responses associated with transient hemodynamic and metabolic changes. In this paper we demonstrate the feasibility of separating fast retinal IOSs from slow optical responses by combining flicker stimulation and dynamic (temporal) differential image processing. A near infrared flood-illumination microscope equipped with a high-speed (1000 Hz) digital camera was used to conduct concurrent optical imaging and ERG measurement of isolated frog retinas. High spatiotemporal resolution imaging revealed that fast IOSs could follow flicker frequency up to at least 6 Hz. Comparable time courses of fast IOSs and ERG kinetics provide evidence that fast IOSs are originated from stimulus activated retinal neurons.

Intrinsic optical signal imaging of retinal activation

Fast intrinsic optical signals (IOSs) correlated with stimulus-activated retinal responses are reviewed. Fast IOSs have a time course comparable to the stimulus-evoked electrophysiological kinetics of the retina, and thus promise a new methodology for high-resolution evaluation of the physiological health of the retina. However, practical application of fast IOSs for retinal study and diagnosis is challenging because of their low sensitivity and limited specificity. Using isolated amphibian retinas, a series of experiments to optimize and characterize fast IOSs has been conducted. Fast, high-resolution nearinfrared light imaging disclosed both positive (increasing) and negative (decreasing) optical responses in adjacent retinal areas, which satisfied spatial resolution essential to the differentiation of IOSs from opposite polarities. At the subcellular (approximately microm) level, fast IOSs often exceeded 5% DeltaI/I, where I is the dynamic optical change, and I is the background light intensity. Experiments with isolated frog retinas suggest that negative IOSs stem primarily from the photoreceptor layer, while positive IOSs come from inner retinal layers.

Intrinsic optical imaging of stimulus-modulated physiological responses in amphibian retina

We demonstrated near-infrared light imaging of stimulus-modulated physiological activities in an isolated frog retina using transient intrinsic optical responses (TIORs). While low-strength visible-light stimuli evoked TIORs with positive polarity, strengthened stimuli elicited additional negative TIORs. Our experimental study and physiological analysis suggest that the negative TIORs are associated with photoreceptor response, and the positive TIORs result primarily from dynamic changes of postphotoreceptor response during retinal activation. In coordination with the sophisticated design of visible-light stimulation protocols, optical imaging of TIORs promises an important application for noninvasive assessment of retinal photoreceptor and inner neurons.

Dynamic neuroimaging of retinal light responses using fast intrinsic optical signals

Transient intrinsic optical responses associated with neural activation offer an attractive strategy for dynamic imaging of neural activity, and may provide a noninvasive methodology for imaging of retinal function. Here we demonstrate the feasibility of near infrared imaging of fast intrinsic optical changes in isolated frog retina activated by visible light. Using a photodiode detector in a transmitted light geometry, we routinely measured dynamic transmitted optical responses in single passes, at the level of one part in 10 4 of background light. Rapid CCD image sequences acquired with transmitted light (bright field) illumination disclosed larger fractional responses and showed evidence of multiple response components with both negative- and positive-going signals with different timecourses. Dark field imaging further enhanced the contrast and sensitivity of optical measures of neural activation. High-resolution imaging disclosed optical responses in single pixels often exceeding 5%, of background light, allowing dynamic imaging at the resolution of single cells, in single passes. Fast optical signals are closely related to identified response components of the electroretinogram. Optical responses showed complex but consistent spatial organization from frame to frame. Our experimental results and theoretical analysis suggest that the optical responses may result from dynamic volume changes corresponding to ion and water flow across the cell membrane, directly associated with the electrophysiological response.

Rapid optical coherence tomography and recording functional scattering changes from activated frog retina

Optical coherence tomography (OCT) has important potential advantages for fast functional neuroimaging. However, dynamic neuroimaging poses demanding requirements for fast and stable acquisition of optical scans. Optical phase modulators based on the electro-optic effect allow rapid phase modulation; however, applications to low-coherence tomography are limited by the optical dispersion of a broadband light source by the electro-optic crystal. We show that the optical dispersion can be theoretically estimated and experimentally compensated. With an electro-optic phase modulator-based, no-moving-parts OCT system, near-infrared scattering changes associated with neural activation were recorded from isolated frog retinas activated by visible light.

Characterization of Strychnine-sensitive Glycine Receptor in the Intact Frog Retina: Modulation by Protein Kinases

We studied 3H-glycine and 3H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a KD of 2.5 and 2.0 microM, respectively. Specific binding of glycine was displaced by beta-alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.

Light-evoked oscillatory discharges in retinal ganglion cells are generated by rhythmic synaptic inputs.

In the visual system, optimal light stimulation sometimes generates gamma-range (ca. 20 approximately 80 Hz) synchronous oscillatory spike discharges. This phenomenon is assumed to be related to perceptual integration. Applying a planar multi-electrode array to the isolated frog retina, Ishikane et al. demonstrated that dimming detectors, off-sustained type ganglion cells, generate synchronous oscillatory spike discharges in response to diffuse dimming illumination. In the present study, applying the whole cell current-clamp technique to the isolated frog retina, we examined how light-evoked oscillatory spike discharges were generated in dimming detectors. Light-evoked oscillatory ( approximately 30 Hz) spike discharges were triggered by rhythmic ( approximately 30 Hz) fluctuations superimposed on a depolarizing plateau potential. When a suprathreshold steady depolarizing current was injected into a dimming detector, only a few spikes were evoked at the stimulus onset. However, repetitive spikes were triggered by a gamma-range sinusoidal current superimposed on the steady depolarizing current. Thus the light-evoked rhythmic fluctuations are likely to be generated presynaptically. The light-evoked rhythmic fluctuations were suppressed not by intracellular application of N-(2,6-dimethyl-phenylcarbamoylmethyl)triethylammonium bromide (QX-314), a Na(+) channel blocker, to the whole cell clamped dimming detector but by bath-application of tetrodotoxin to the retina. The light-evoked rhythmic fluctuations were suppressed by a GABA(A) receptor antagonist but potentiated by a GABA(C) receptor antagonist, whereas these fluctuations were little affected by a glycine receptor antagonist. Because amacrine cells are spiking neurons and because GABA is one of the main transmitters released from amacrine cells, amacrine cells may participate in generating rhythmically fluctuated synaptic input to dimming detectors.

The role of taurine in osmotic, mechanical, and chemical protection of the retinal rod outer segments.

The ability of the photoreceptor cell to resist osmotic stress was examined by incubating isolated frog retina in medium of varying osmolality. An electron microscopic analysis of the rod outer segment following a severe hypoosmotic insult revealed connections between adjacent disks and between disk rims and the plasma membrane, which presumably provide mechanical stability to the rod outer segment. One surprising result was the extent of the damage incurred by the electrical signaling pathway of the photoreceptor cells subjected to a 50 mOsm insult; only the distal P111 component of the ERG remained unaffected. Thus, the rod outer segment is particularly resistant to osmotic-induced injury, presumably because of the effective osmoregulatory actions of taurine. Incubation of retina with tauret, retinylidentaurine, uncovered rose-like hexagonal structures on the surface of the rod outer segment. These structures purportedly consist of connections between disk rims and the plasma membrane of the rod outer segments. Based on the influence of tauret, it is likely that the calcium dependence of these channels is selective for retinoids. These data are discussed relative to taurine's role in the process of rhodopsin regeneration and in the protection of the rod outer segments against osmotic, mechanical and light induced damage.

The c-wave of the electroretinogram possesses a third component from the proximal retina

ERG and light-induced extracellular potassium ([K +] o) changes have been measured in isolated retinas of both Rana esculenta and Rana temporaria. The conditions of the preparations have been varied. Isolated frog retinas kept receptor side-upward in a moist chamber without perfusion showed the well-known slow PIII in the ERG. Retinas superfused from the receptor side, with O 2 enrichment at their vitreal surface, however, exhibit a slow cornea-positive potential in the ERG. The slow ERG-potentials relate to different light-induced potassium changes in the proximal retina. There was a long lasting and larger proximal potassium increase in adequately maintained retinas but a smaller and shorter one in preparations lacking superfusion and oxygen. There was no significant difference between the size of potassium decrease around receptors of retinas supervised from their vitreal side and those supervised from receptor side. A reduction of slow PIII should therefore not be responsible for the slow cornea-positive potential. The long lasting and larger (by 59%) potassium increase in the proximal retina may counteract the potential in the Mu¨ller cells caused by the potassium decrease around receptors and thereby cancel slow PIII and generate a third component of the electroretinogram c-wave.

Alpha A- and alpha B-crystallin in the retina. Association with the post-Golgi compartment of frog retinal photoreceptors

alpha A- and alpha B-Crystallins are significant contributors to maintaining the transparency of the vertebrate lens. We have found that both alpha A- and alpha B-crystallins are also present, at approximately equimolar concentrations, in frog retinal cells. They were identified by sequencing portions of each polypeptide, by immunochemical cross-reactivity with antibodies to bovine alpha-crystallins, and by their relative mobility in two-dimensional gel electrophoresis. Retinal alpha-crystallins form macromolecular multimeric complexes similar to those found in the lens, and they are abundant both in soluble and membrane-associated forms. A surprising finding is that alpha-crystallins bind specifically to the photoreceptor post-Golgi membranes that mediate transport of newly synthesized rhodopsin. Upon treatment of post-Golgi membranes with urea or Triton X-114, a portion of the bound alpha B-crystallin remains tightly associated, indicating that the alpha B-form may mediate membrane binding of an alpha-crystallin multimeric complex. Both subunits are synthesized in vitro by isolated frog retinas, but alpha B-crystallin appears to have a higher renewal rate. Newly synthesized alpha-crystallins become associated with the post-Golgi membranes concurrently with newly synthesized rhodopsin. Association of alpha-crystallins with newly synthesized rhodopsin suggests that they may participate in photoreceptor outer segment membrane renewal. Our findings implicate an important function for both alpha A- and alpha B-crystallins in the same, extralenticular, tissue.

The neural retina of the frog contributes a slow cornea-positive potential to the electroretinogram.

A c-wave-like cornea-positive potential in the isolated rabbit retina has been described. In this study, frog retinas were investigated to see if the neural retina contributes a slow cornea-positive component to the c-wave of the electroretinogram. The eye cups of both Rana esculenta and Rana temporaria exhibited a normal electroretinogram with c-wave, a larger proximal light-induced extracellular potassium increase, a small distal extracellular potassium increase and an extracellular potassium decrease around photoreceptors. Isolated frog retinas kept receptor side-upward in a moist chamber without perfusion showed the well-known slow PIII generated by the potassium decrease around receptors. If the isolated retinas were well perfused, the slow PIII was not seen, but a cornea-positive d.c. potential sometimes appeared after the b-wave. The different slow potentials seemed to relate to different light-induced potassium changes in the proximal retina. There was a long-lasting proximal potassium increase in the superfused retinas but a quick return of the proximal potassium increase to the baseline in the retinas lacking oxygen at the vitreal side. The lasting proximal potassium increase in adequately maintained retinas may counteract the potassium decrease around receptors and cancel slow PIII.

Simulation of the electroretinographic slow PIII component by injection of K+ -free Ringer's solution in the frog retina

The light-induced hyperpolarization of the photoreceptor results in a decrease in extracellular potassium ion concentration, [K+]. A decrease in [K+] in the photoreceptor layer was caused by injecting a small amount of K+-free Ringer's solution in the isolated frog retina, and the distribution of the field potential as induced by K+-free Ringer's solution was mapped. The depth profile of the response resembled that of the slow PIII component of the electroretinogram. These results support the notion that the slow PIII is generated by the Müller cells as they hyperpolarize in response to a light-induced decrease in [K+] in the photoreceptor layer.

Time-dependent differential effects of cobalt ions on rod- and cone-driven responses in the isolated frog retina

The effects of cobalt ions on 502-nm rod- and 575-nm cone-driven components of the b-wave of the electroretinogram were studied in the isolated frog retina. Addition of 100-150 microM cobalt initially caused a suppression of rod-driven responses and an enhancement of cone-driven responses. In the continued presence of cobalt, however, the rod-driven responses gradually recovered and the cone-driven responses became suppressed. These concentrations of cobalt had no effect on the rod- and cone-driven mass receptor potentials which were isolated in the presence of 4 mM glutamate. At higher concentrations of cobalt (1 mM or greater), both rod- and cone-driven b-wave responses were eliminated and there was no recovery in the continued presence of cobalt. The results suggest that cobalt has markedly different, time-dependent effects on signal transmission from rods and cones to second-order cells.

Protective effect of insulin on suppressed electrical activity caused by ouabain in isolated frog retina

The reduction in amplitude of the a-wave of the frog ERGs in response to light at seven different intensities recorded from isolated frog retinas (Rana catesbeiana) caused by ouabain (10(-5) M) was partially prevented dose-dependently by the insulin pre-treatment (between 6.6 x 10(-8) and 6.6 x 10(-5) M). Inversely, however, the already suppressed a-waves by ouabain could not be recovered by the application of insulin. The preventive effect of insulin pretreatment was also significantly dependent upon the stimulus intensity, but not upon the presence of glucose in the external media. The effect of insulin itself on the amplitude of the a-wave was below significance level over the range of its concentrations employed in the present experiments. It was suggested that this effect of insulin on the a-wave can be explained by assuming that insulin enhanced the Na-K pump activity under its suppressed conditions in visual cell.

Suppression of cone-driven responses by rods in the isolated frog retina

The sensitivity of rod- and cone-driven responses was studied in the isolated frog retina during the period of rapid dark adaptation following a conditioning flash which bleached a negligible amount of visual pigment. Following a conditioning flash, cone-driven b-wave responses were first enhanced and then depressed. The time courses of the enhancement and subsequent depression of cone-drive responses varies greatly with the intensity and wavelength of the conditioning flash, but were identical when the conditioning flashes were matched for equal excitation of 502 nm rods. These changes in cone-driven response sensitivity were correlated with the desensitization and recovery of rods following the conditioning flash. When signal transmission from rods to second-order cells was interrupted by the addition of L-glutamate, the conditioning flash did not produce the above-described enhancement and subsequent depression of long-wavelength receptor potential responses. The suppression of cone-driven response therefore appears to be due to a synaptically mediated influence from 502 nm rods which is maximal when the rods are in the dark-adapted state, with little or no contribution from 433 nm rods, and no involvement of the pigment epithelium.

Microelectrode depth study of the electroretinographic oscillatory potentials in the frog retina.

The depth profile of the electroretinographic oscillatory potentials was studied in the isolated frog retina. The intraretinal electrode was introduced from the receptor side, and the reference electrode was placed on the vitreal side. The electroretinogram, recorded either transretinally or with the electrode tip in the receptor layer, showed 4 to 10 oscillatory potential wavelets. As the electrode was advanced proximally, the wavelets disappeared as a function of retinal depth. The wavelets with longer peak latencies disappeared earlier, and only the first one or two wavelets could be identified when the electrode was in the inner plexiform layer. These findings indicate that the oscillatory potentials are generated between the inner and outer plexiform layers and that the earlier wavelets originate in the more proximal retina. The results are consistent with the notion that the oscillatory potentials are generated by synaptic feedback circuits.

Effect of induced lipid peroxidation on dark adaptation of photoreceptors of the isolated frog retina

Acute experiments on anesthetized cats under extracorporeal circulation were made to study the effects of papaverine, phentolamine and dihydroergotamine on the blood flow-pressure relations of the vascular bed of the greater circulation. It was shown that the drug-induced change in the vascular resistance were dependent on the perfusion flow and on the initial distensibility of arterial vessels as well. A mathematical simulating model that permits fore-telling alterations in the vascular resistance is described.

Generation of currents accompanying the off-response of the phororeceptors in the isolated frog retina

A prolonged weak illumination has been reported to induce an off-response in transretinal potential of bullfrog retina immersed in a low Ca 2+-, Ba 2+ and Na-aspartate-containing solution. Changes in the interstitial current accompanying the generation of the off-response were studied by inserting a pair of micropipettes into the bullfrog retina. A transient increase in proximal-to-distal current above the dark level was observed when the off-response was developing (off-current). The current divergence of the off-current indicated that the source of the off-current was located about 75 μm below the receptor surface. In the absence of Ba 2+, similar changes in currents were also observed, although no detectable off-response was observed in the transretinal potential. Findings suggest that regardless of the presence of Ba 2+, current flowing outward through the plasma membrane of the rod inner segments increases transiently when the off-response is developing.