Ultrastructural localization and x-ray analysis of calcium-induced electron-dense deposits in the skate retina.
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Biomedical subjects
Publications and source records attributed to H Ripps.
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Potassium kinetics within the skate retina were monitored extracellularly with K+-selective electrodes. Two sources of K+ efflux were detected in response to photic stimulation: one in the distal retina in the region of the outer plexiform layer, and the other at a more proximal location near the border between the inner nuclear and inner plexiform layers. The magnitude of the K+ efflux at these retinal depths was affected differently by spot and full-field illumination, suggesting that the two sources originate from different classes of neuron. There is evidence that both sources are associated with current sinks provided by the Müller cells, thereby establishing radial current paths along the lengths of these elements. We have proposed a model in which asymmetries in the magnitudes of these currents give rise to the b-wave of the electroretinogram. Extracellular field potentials recorded differentially at various retinal depths, and in response to changes in stimulus configuration, were consistent with predictions of the model.
The visual pigment content of rod photoreceptors in Xenopus larvae was reduced greater than 90% through a combination of vitamin A-deficient diet and constant light. Thereafter, a dose of either all-trans-retinol or 9-cis-retinal was injected intramuscularly, leading to the formation of a rhodopsin (lambdamax 504 nm) or isorhodopsin (lambdamax 487-493 nm) pigment, respectively. Electrophysiological measurements were made of the threshold and spectral sensitivity of the aspartate-isolated PIII (photoreceptoral) component of the electroretinogram. These measures established that either rhodopsin or isorhodopsin subserved visual transduction with the same efficiency as the 519 nm porphyropsin pigment encountered normally. When animals with rhodopsin or isorhodopsin were kept in darkness or placed on a cyclical lighting regimen for 8 days, retinal densitometry showed that either pigment was being converted to porphyropsin; significantly more porphyropsin was formed as a result of cyclical lighting than after complete darkness.
Rhodopsin kinetics and visual threshold were determined in three subjects with a dominant form of retinitis pigmentosa. The retinal areas studied showed varying loss of sensitivity, which correlated well with the reduction in the measured density of rhodopsin in the test region. Rhodopsin photosensitivity was normal, and there was no evidence that either rhodopsin or the cone pigments regenerated more rapidly than normal. The findings in these cases of retinitis pigmentosa, when compared with the threshold changes induced by vitamin A deficiency or photic bleaching, suggest that the disease produces an imbalance between disc removal and new disc formation, which results in a progressive shortening of the photoreceptor outer segments and, eventually, in their complete disappearance.
The proximal negative response (PNR), a complex extracellular potential derived mainly from amacrine cell activity, was studied in the all-rod retina of the skate. Tetrodotoxin (10(-6) mg/ml) did not affect either the waveform or the latency of the response, indicating that the PNR reflects the graded, nonregenerative components of the amacrine cell potential. As regards its adaptive properties, the PNR exhibited both the extreme sensitivity to weak background light and the slow time course of light and dark adaptation that are characteristic of other responses from the proximal retina. Thus, the PNR, like the b-wave and ganglion cell discharge, appears to reflect adaptive processes located within the neural network of the inner retina.
The late photoproducts that result from the isomerization of rhodopsin have been identified in the isolated all-rod retina of the skate by means of rapid spectrophotometry. The sequence in which these intermediates form and decay could be described by a scheme that incorporates two pathways for the degradation of metarhodopsin II (MII) to retinol: one via metarhodopsin III (MIII) and the other (which bypasses MIII) through retinal. Computer simulation of the model yielded rate constants and spectral absorbance coefficients for the late photoproducts which fit experimental data obtained at temperatures ranging from 7 degrees C to 27 degrees C. Comparing the kinetics of the thermal reactions with the changes in rod threshold that occur during dark adaptation indicated that the decay of MII and the fall in receptor thresholds exhibit similarities with regard to their temperature dependence. However, the addition of 2 mM hydroxylamine to a perfusate bathing the retina greatly accelerated the photochemical reactions, but had no significant effect on the rate of recovery of rod sensitivity. It appears, therefore, that the late bleaching intermediates do not control the sensitivities of skate rods during dark adaptation.
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The photoreceptors of dark-adapted skate retinas bathed in a Ringer solution containing horseradish peroxidase (HRP) incorporate the tracer into membrane-bound compartments within the synaptic terminal of the cell; after 1 or 2 h of incubation, approx. 10-38% of the synaptic vesicles were labeled. The receptors appeared to be functioning normally throughout the incubation period, since electrical potentials of normal amplitude could be elicited in response to dimphotic stimuli. However, it was possible to block the uptake of peroxidase by a regimen of light adaptation that effectively suppressed light-induced activity in the electroretinogram. If, during incubation with peroxidase, retinas were exposed at 10-min intervals to an intense 1-ms flash from a xenon discharge tube, the receptor terminals were almost completely devoid of peroxidase; fewer than 2% of the vesicles were labeled. The suppression of HRP uptake could also be achieved in dark-adapted retinas by adding magnesium to the bathing solution, suggesting that calcium is necessary for transmitter release from vesicles in the receptor terminals. These findings are consistent with the view that vertebrate photoreceptors discharge a neurotransmitter in darkness, and that light decreases the release of this substance. It seems likely that the incorporation of peroxidase into vesicles of physiologically active receptor terminals reflects a mechanism for the retrieval of vesicle membrane after exocytosis.
1. Measurements of the aspartate-isolated PIII component of the electroretinogram (ERG) were used to estimate photoreceptor threshold in dark-adapted tadpoles of the clawed toad, Xenopus laevis raised on a normal diet. Spectral sensitivity functions established that the rod mechanism governed absolute dark thresholds from stage 40 to the end of premetamorphic development. 2. Parallel measures of rod outer-segment dimensions and visual pigment levels demonstrated a) that visual pigment concentration remained constant at all tadpole stages, and b) that the fall in threshold over the course of premetamorphic development depended exclusively on the increased probability of quantal absorption that accompanied the growth of the rod outer segments. 3. When tadpoles were obtained from vitamin A-deficient females and raised subsequently on a vitamin A-free diet, the first appearance of the ERG was delayed and its absolute threshold raised, with respect to controls. 4. Histological examination of vitamin A-deprived retinas indicated that rod outer segments retained their structural integrity in spite of up to a 75% loss of visual pigment. 5. The threshold rise which accompanied a fall in visual pigment levels, whether effected by dietary deficiency or by bleaching, was greater than could be attributed solely to a reduction in the probability of quantal absorption.
An account is given of investigations into the mechanisms of dark-adaptation in the retina of man and of the skate and other fish. Working hypotheses as to the possible sites of abnormal function in the various disorders of which night blindness is a feature are presented.
The slow PIII component of the electroretinogram (ERG) was studied in the isolated, aspartate-treated carp retina. Although the latter is richly populated with cones, slow PIII appeared to reflect almost exclusively the activity of rods; e.g. the spectral sensitivity of the potential paralleled closely the rod pigment curve, its operating range (i.e. the V-log I curve) was limited to 3 log units above absolute threshold, and raising background intensities to photopic levels produced saturation of the increment threshold function without evidence of a cone-mediated segment. Only after bleaching away a significant fraction of the porphyropsin was it possible to unmask a small photopic contribution to slow PIII, as evidenced by a displacement in the action spectrum to longer wavelengths. The spatial distribution of the slow PIII voltage within the retina (Faber, D.S. 1969. Ph.D. Thesis. State University of New York. Buffalo, N.Y.; Witkovsky, P.J. Nelson, and H. Ripps. 1973. J. Gen Physiol. 61:401) and its ability to survive aspartate treatment indicate that this potential arises in the Müller (glial) fiber. Additional support for this conclusion is provided by the slow rise time (several seconds) and long temporal integration (up to 40s) of the response. In many respects the properties of slow PIII resemble those of the c-wave, a pigment epithelial response also subserved by rod activity. On the other hand, the receptoral (fast PIII) and the b-wave components of the ERG behave quite differently. Unlike slow PIII, response saturation could not be induced, since both potentials are subserved by cones when the stimulus conditions exceed the limits of the scotopic range. Receptors appear to govern light adaptation at photopic background levels; both fast PIII and b-wave manifest identical incremental threshold values over this range of intensities. However, under scotopic conditions, the sensitivity of the b-wave is affected by luminous backgrounds too weak to alter fast PIII threshold, indicating a postreceptoral stage of adaptation.
Electrical potentials were recorded from different levels within the skate retina. Comparing the adaptive properties of the various responses revealed that the isolated receptor potential and the S-potential always exhibited similar changes in sensitivity, and that the b-wave and ganglion-cell thresholds acted in concert. However, the two sets of responses behaved differently under certain conditions. For example, a dimly iluminated background that had no measurable effect on the senitivities of either of the distal responses, raised significantly the thresholds of both the b-wave and the ganglion cell responses. In addition, the rate of recovery during the early, "neural" phase of dark adaptation was significantly faster for the receptor and S-potentials than for the b-wave or ganglion cell discharge. These results indicate that there is an adaptive ("network") mechanism in the retina which can influence significantly b-wave and gaglion cell activity and which behaves independently of the receptors and horizontal cells. We conclude that visual adaptation in the skate retina is regulated by a combination of receptoral and network mechanisms.
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The mass photoreceptor response of the isolated carp retina was studied after immersing the tissue in aspartate-Ringer solution. Two electro-retinogram components were isolated by differential depth recording: a fast cornea-negative wave, arising in the receptor layer, and a slow, cornea-negative wave arising at some level proximal to the photoreceptors. Only the fast component was investigated further. In complete dark adaptation, its action spectrum peaked near 540 nm and indicated input from both porphyropsin-containing rods (lambda(max) approximately 525 nm) and cones with longer wavelength sensitivity. Under photopic conditions a broad action spectrum, lambda(max) approximately 580 nm was seen. In the presence of chromatic backgrounds, the photopic curve could be fractionated into three components whose action spectra agreed reasonably well with the spectral characteristics of blue, green, and red cone pigments of the goldfish. In parallel studies, the carp rod pigment was studied in situ by transmission densitometry. The reduction in optical density after a full bleach averaged 0.28 at its lambda(max) 525 nm. In the isolated retina no regeneration of rod pigment occurred within 2 h after bleaching. The bleaching power of background fields used in adaptation experiments was determined directly. Both rods and cones generated increment threshold functions with slopes of +1 on log-log coordinates over a 3-4 log range of background intensities. Background fields which bleached less than 0.5% rod pigment nevertheless diminished photoreceptor sensitivity. The degree and rate of recovery of receptor sensitivity after exposure to a background field was a function of the total flux (I x t) of the field. Rod saturation, i.e. the abolition of rod voltages, occurred after approximately 12% of rod pigment was bleached. In light-adapted retinas bathed in normal Ringer solution, a small test flash elicited a larger response in the presence of an annular background field than when it fell upon a dark retina. The enhancement was not observed in aspartate-treated retinas.
Receptor potentials were recorded extracellularly from the all-rod retina of the skate after the application of sodium aspartate. This agent suppresses the responses of proximal elements, but leaves relatively unaffected the electrical activity of the photoreceptors (a-wave) and pigment epithelium (c-wave). Since the latter develops too slowly to interfere with the receptor response, it was possible to isolate receptor potentials and to compare their behavior in light and dark adaptation with earlier observations on the S-potential, b-wave, and ganglion cell discharge. The results show that the photoreceptors display the full complement of adaptational changes exhibited by cells proximal to the receptors. Thus, it appears that visual adaptation in the skate is governed primarily by the photoreceptors themselves. Of particular interest was the recovery of sensitivity in the presence of background fields that initially saturate the receptor potential. Analysis of this recovery phase indicates that a gain-control mechanism operates within the receptors, at a distal stage of the visual process.
The S-potentials recorded intracellularly from the all-rod retina of the skate probably arise from the large horizontal cells situated directly below the layer of receptors. These cells hyperpolarize in response to light, irrespective of stimulus wavelength, and the responses in photopic as well as scotopic conditions were found to be subserved by a single photopigment with lambda(max) = 500 nm. The process of adaptation was studied by recording simultaneously the threshold responses and membrane potentials of S-units during both light and dark adaptation. The findings indicate that the sensitivity of S-units, whether measured upon steady background fields or in the course of dark adaptation, exhibits changes similar to those demonstrated previously for the ERG b-wave and ganglion cell discharge. However, the membrane potential level of the S-unit and its sensitivity to photic stimulation varied independently for all the adapting conditions tested. It appears, therefore, that visual adaptation in the skate retina occurs before the S-unit is reached, i.e., at the receptors themselves.