Rod sensitivity and visual pigment concentration in Xenopus.

Xenopus larvae were raised on a vitamin A-free diet under constant illumination until their visual pigment content had decreased to between 8% of normal and an undetectably low level. After the intramuscular injection of 2.1 X 10(13-2.1 X 10(16) molecules of [3H]vitamin A, ocular tissue showed a rapid rate of uptake of label which reached a maximum level of incorporation by 48 h. Light-microscopic autoradiography revealed that the retinal uptake of label was concentrated within the receptor outer segments. Spectral transmissivity measurements at various times after injection were made upon intact retinas and upon digitonin extracts. They showed that visual pigment with a lambdamax of 504 nm was formed in the retina and that the amount formed was a function of incubation time and the magnitude of the dose administered. Electrophysiological measures of photoreceptor light responses were obtained from the PIII component of the electroretinogram, isolated with aspartate. The quantal flux required to elicit a criterion response was determined and related to the fraction of visual pigment present. The results showed that rod sensitivity varied linearly with the probability of quantal absorption.

Photoreceptor thresholds and visual pigment levels in normal and vitamin A-deprived Xenopus tadpoles

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.

Slow PIII component of the carp electroretinogram.

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.