Selective illumination of single photoreceptors in the house fly retina: local membrane turnover and uptake of extracellular horseradish peroxidase (HRP) and Lucifer Yellow

Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.     

A stage in glycolysis controls the metabolic adjustments of vertebrate rod photoreceptors upon illumination

The factors affecting the metabolic adjustments of toad rod photoreceptors were studied by monitoring the oxygen utilization of excised retinas and by measuring rod outer segment ATP and GTP concentrations. Respiratory adjustments upon illumination were observed when glucose or fructose was provided in the perfusate, but not when a glycolytic inhibitor was added to the perfusate containing glucose and pyruvate, or when a substrate beyond glycolysis or from a later stage of glycolysis was substituted for glucose. The amplitudes of the respiratory adjustments to illumination were dependent on the concentration of glucose in the perfusate. The ATP and GTP concentration changes were dependent on respiratory adjustments, including glycolytic effects, and on the levels of illumination. The data suggest a control point within glycolysis for light-induced adjustments of respiration, possibly at phosphofructokinase.     

Rapid declines in cyclic GMP of rod outer segments of intact frog photoreceptors after illumination

Considerable disagreement has resulted from experiments designed to test whether light-induced falls in cGMP in outer segment (OS) of photoreceptors precede their light-induced electrical responses. Different studies have reported initial declines at 50 ms, at s, or not at all for physiological stimuli. Such studies have employed whole retinas, isolated rod OS, or isolated rod OS with attached inner segments and involved a variety of techniques. We developed an apparatus that illuminates intact pieces of dark-adapted frog retinas at 22 degrees C for known brief durations and then rapidly (47 ms) presses their OS surface against a copper mirror cooled by liquid helium. Freezing occurs in less than 2 ms. Cyclic GMP was then assayed in cryostat sections of the OS layer. Six illumination intensities that bleached from 90 to 9 X 10(8) rhodopsin molecules per s were delivered for durations of 0.1-2 s. Compared to dark-adapted values, progressive losses of cGMP were seen with all illumination intensities. Because a significant loss in cGMP was seen after a 100 ms exposure to our dimmest stimulus, it appears that a loss of cGMP could play a role in rod visual transduction.     

Light-activated single channel currents in Limulus ventral nerve photoreceptors

Light-activated single channel currents were measured in Limulus ventral photoreceptors in the cell-attached configuration at 14°C. The results show three channel types with conductances of 6.2, 10.4 and 28.7 pS. The most active channels have the 10 pS conductance; the open time histograms of these channels could be best fitted by the sum of two exponentials with time constants (and weights) of 0.58 ms (0.78) and 4.32 ms (0.22), suggesting two populations of channels or two open states. The mean open time was 1.38 ms. The open time histogram of the channels with the 29 pS conductance could be best fitted by a single exponential with a time constant of 3.35 ms. First latencies of the 10 pS channels were between 40 and 280 ms but those of the 29 pS conductance channels were 300 ms. These findings suggest that the two channel types are gated by two different intracellular transmitters or mechanisms. Offprint requests to: K. Nagy     

Decline of electrogenic Na+/K+ pump activity in rod photoreceptors during maintained illumination

Light-evoked changes in membrane voltage were recorded intracellularly from rod photoreceptors in the isolated retina preparation of the toad, Bufo marinus, during superfusion with a solution containing pharmacological agents that blocked voltage-dependent conductances. Under these conditions, the amplitude of the hyperpolarizing photoresponse became much greater than under control conditions. The results of several experiments support the conclusion that this increase in photoresponse amplitude was due primarily to a voltage that was produced when the electrogenic current from the rods' Na+/K+ pump flowed across an increased membrane resistance (Torre, V. 1982. Journal of Physiology. 333:315). At the onset of a period of continuous illumination, the rod membrane first hyperpolarized and then began to repolarize, and after 180 s of illumination, the membrane voltage had recovered by 60-72% of its initial hyperpolarization. There did not appear to be any significant decrease in rod membrane resistance associated with this repolarization. Both the enhanced hyperpolarization at light onset and the slow repolarization during maintained illumination were blocked by superfusion with 10.0 microM strophanthidin. These data support the hypothesis that the activity of the rods' Na+/K+ pump declines progressively during maintained illumination. It is likely that the decline in pump activity produces significant changes in [K+]o in the subretinal space during maintained illumination.     

Pigment migration and spectral sensitivity in the compound eye of moths

Summary The spectral sensitivity of dark adapted eyes of the moth Manduca sexta (Lepidoptera: Sphingidae) was measured between 350 and 650 nm by determining the relative number of photons necessary to elicit a retinal potential of 50 V. The spectral sensitivity was determined in eyes with the screening pigment: a) in the extreme distal position, b) in a proximal position, and c) with the pigment removed. Sensitivity maxima were found at about 350 to 370, 450 and 530 nm, irrespective of pigment position.Pigment movement is associated with a change in light attenuation of 2 to 3 log units at all wavelengths between 350 and 650 nm. Only small variations, about 0.6 log units, in screening effect were observed throughout the measured spectrum, and therefore the effect of pigment movement on spectral sensitivity is small.There is a slightly larger decline in sensitivity of eyes with the pigment removed compared to eyes with the pigment in the distal position at wavelengths above 530 nm. This result suggests that the distally located pigments have a slight screening effect.We are grateful to Dr. Bo Lindström for advice regarding the statistical treatment of the experimental data.This work was supported partly by the Swedish Medical Research Council, Stifteisen Gustaf och Tyra Svenssons Minne, Reservationsanslaget,and partly by the Air Force Office of Scientific Research through the European Office of Aerospace Research, OAR, United States Air Force under grant number EOOAR-68-0036.     

Multiple wavelength illumination in flow cytometry using a single arc lamp and a dispersing element

The principle of a multiple wavelength illumination method for flow cytometers, based upon a combination of a helium-neon laser and an arc lamp as illumination sources is described. By using a prism, the light from the arc lamp is dispersed and the different colors are imaged at different places on the sample stream. The small angle light scattering from the helium-neon laser light is measured as a relevant parameter and serves as a trigger signal for subsequent measurements of fluorescence or scattering of light from the arc lamp. Two experimental systems are described utilizing this principle: a system where the emission is detected orthogonally with respect to the direction of the illumination beams, and an epi-illumination system. With the orthogonal set-up multiple wave-length right angle scattering measurements are possible. This is illustrated by showing that the orthogonal scattering from erythrocytes is strongly dependent on the illumination wavelength. It is further shown that the apparatus is suitable for the measurement of intracellular pH using the pH dependence of the excitation spectrum of fluorescein. The epi-illumination system allows excitation of two (or more) fluorescent dyes with different excitation spectra. In this case the emission spectra of the fluorescent dyes may overlap substantially. This is shown by simultaneous measurement of DNA and protein of Chinese hamster lung cells using mitramycin and tetramethyl rhodamin isothiocyanate (TRITC).     

Polarization and spectral sensitivity of single photoreceptors of the domestic cricket

The polarization and spectral sensitivity of single photoreceptors ofAcheta domesticus L. was measured. The morphological characteristics of the cricket rhabdome satisfy the conditions for a symmetrical model, for which the polarization sensitivity of a single photoreceptor is identically equal to the dichroism of a single microvillus. Characteristic curves of spectral sensitivity of all photoreceptors measured (24 cells) were similar and had two maxima: the principal at 500 nm and a secondary peak at 360 nm, characteristic of a pigment such as rhodopsin in the rods of the vertebrate retina. The mean value of polarization sensitivity measured was 2.28 ± 0.85 (mean ± standard deviation, 70 cells), suggesting the existence of slight preferential orientation of the dipole moments of the rhodopsin molecules along the axes of the microvilli.I. N. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 483–490, September–October, 1979.     

Screening pigment migration in a sphingid moth is triggered by light near the cornea

Summary It is possible to project a horizontal line of light onto a moth's cornea and simultaneously a vertical line onto the retina (Fig. 1). In the dark adapted eye of a sphingid moth (Theretra latreilli) this resulted in the appearance of a horizontal line of pigment across the eye (Fig. 2). This proves that the trigger for pigment migration is near the cornea, presumably in the pigment cells themselves, and not in the receptors.     

Effect of strong illumination on the ion efflux from the isolated discs of frog photoreceptors

(1) Low levels of illumination do not modify the efflux of the radioisotopes ²²Na, ⁸⁶Rb, ³⁶Cl, and ⁴⁵Ca from the isolated discs of the photoreceptors of the frog (Rana Catesbeiana). (2) The effluxes of ²²Na⁺, ⁸⁶Rb⁺ and ³⁶Cl⁻ increase when the discs are illuminated with more than 10⁴ erg/cm² per s for a few minutes. There is no effect on the efflux of ⁴⁵Ca²⁺ or of [¹⁴C]urea. (3) The effect is greater for monochromatic lights of wavelengths in the shorter region of the spectrum. (4) The effect is also present in bleached visual membranes.     

Light-adapting migration of the screening-pigment in crayfish photoreceptors is a two-stage movement comprising an all-or-nothing initial phase

The light-adapting response of the screening-pigment in crayfish retinal photoreceptors, previously described as a monophasic movement, was found to consist of two stages with different properties: (1) a rapid initial expansion that once started proceeds for at least half of the full distance, and (2) a slower and more variable continuation of the movement. The two components were resolved in isolated eyes stimulated under conditions expected to restrict Na+ influx into the photoreceptors. Only the second stage of the response to light was inhibited when Na+ was substituted with choline, or if the normal saline contained amiloride, a diuretic that hinders Na+ entry across many cell membranes. Amiloride in a medium without Na+ delayed, but did not curb, the first stage, whereas the rest of the movement was markedly restrained. Partial replacement of Na+ with Li+ blocked the second stage without affecting the rapid initial shift triggered by light. Exposure of dark-adapted eyes to high Na+ levels or to ouabain in the presence of Na+ in the dark also elicited a two-staged pigment dispersion to the light-adapted position. Low Na+ concentrations or amiloride affected the latency, but not the rate or extent, of the first stage of migration in ouabain-treated eyes. Consistent though less significant results were obtained with cyanide and the Na+ ionophore monensin. These observations suggest a differential control of pigment position over two defined domains along the photoreceptors, probably to integrate a double mechanism of light-adaptation: an all-or-nothing partial shift of the pigment screen as a safety factor against overexposure, followed by a regulated adjustment according to stimulation intensity.     

Electrophysiological measurement of the number of rhodopsin molecules in single Limulus photoreceptors

Two partly independent electrophysiological methods are described for measuring the number of rhodopsin molecules (R) in single ventral photoreceptors. Method 1 is based on measurements of the relative intensity required to elicit a quantal response and the relative intensity required to half-saturate the early receptor potential (ERP). Method 2 is based on measurements of the absolute intensity required to elicit a quantal response. Both methods give values of R approximately equal to 10(9). From these and other measurements, estimates are derived for the surface density of rhodopsin (8,000/micrometer2), the charge movement during the ERP per isomerized rhodopsin (20 X 10(-21) C), and the half-time for thermal isomerization of rhodopsin (36yr).     

Spectral sensitivity of single photoreceptors in the compound eye of a tropical butterfly (Heliconius numata)

Summary The spectral sensitivity (wavelength region 330 to 650 nm) of single photoreceptor cells in the compound eye of the butterfly Heliconius numata (Lepidoptera) was studied by intracellular recording. The configuration and amplitude of the receptor potential was similar to that reported for other insect photoreceptor cells. Maximal sensitivity was found in one of the following wavelength regions: 390 to 410 nm, 450 to 470 nm and 530 to 550 nm. Side maxima of variable amplitude were found. The results suggest three photoreceptor types with different spectral sensitivity maxima.     

Pigment migration in fish erythrophores is controlled by alpha2-adrenoceptors

• 1.1. The aggregation of erythrosomes within erythrophores of the squirrel fish (Myripristis occidentalis; belonging to the family Holocentridae) was, on pharmacological grounds, shown to be mediated by alpha₂-adrenoceptors.
• 2.2. The erythrophores were shown to be controlled by adrenergic nerves activating the alpha₂-adrenoceptors.
• 3.3. The erythrophores themselves were found to possess a K⁺-sensitive mechanism of aggregation.
• 4.4. Some similarities and differences of the alpha₂-adrenoceptor-mediated chromatosome aggregation in melanophores and erythrophores are also discussed.

     

Pigment migration in fish erythrophores is controlled by alpha 2-adrenoceptors

1. The aggregation of erythrosomes within erythrophores of the squirrel fish (Myripristis occidentalis; belonging to the family Holocentridae) was, on pharmacological grounds, shown to be mediated by alpha 2-adrenoceptors. 2. The erythrophores were shown to be controlled by adrenergic nerves activating the alpha 2-adrenoceptors. 3. The erythrophores themselves were found to possess a K+-sensitive mechanism of aggregation. 4. Some similarities and differences of the alpha 2-adrenoceptor-mediated chromatosome aggregation in melanophores and erythrophores are also discussed.