The role of cholesterol in rod outer segment membranes.

The photoreceptor rod outer segment (ROS) provides a unique system in which to investigate the role of cholesterol, an essential membrane constituent of most animal cells. The ROS is responsible for the initial events of vision at low light levels. It consists of a stack of disk membranes surrounded by the plasma membrane. Light capture occurs in the outer segment disk membranes that contain the photopigment, rhodopsin. These membranes originate from evaginations of the plasma membrane at the base of the outer segment. The new disks separate from the plasma membrane and progressively move up the length of the ROS over the course of several days. Thus the role of cholesterol can be evaluated in two distinct membranes. Furthermore, because the disk membranes vary in age it can also be investigated in a membrane as a function of the membrane age. The plasma membrane is enriched in cholesterol and in saturated fatty acids species relative to the disk membrane. The newly formed disk membranes have 6-fold more cholesterol than disks at the apical tip of the ROS. The partitioning of cholesterol out of disk membranes as they age and are apically displaced is consistent with the high PE content of disk membranes relative to the plasma membrane. The cholesterol composition of membranes has profound consequences on the major protein, rhodopsin. Biophysical studies in both model membranes and in native membranes have demonstrated that cholesterol can modulate the activity of rhodopsin by altering the membrane hydrocarbon environment. These studies suggest that mature disk membranes initiate the visual signal cascade more effectively than the newly synthesized, high cholesterol basal disks. Although rhodopsin is also the major protein of the plasma membrane, the high membrane cholesterol content inhibits rhodopsin participation in the visual transduction cascade. In addition to its effect on the hydrocarbon region, cholesterol may interact directly with rhodopsin. While high cholesterol inhibits rhodopsin activation, it also stabilizes the protein to denaturation. Therefore the disk membrane must perform a balancing act providing sufficient cholesterol to confer stability but without making the membrane too restrictive to receptor activation. Within a given disk membrane, it is likely that cholesterol exhibits an asymmetric distribution between the inner and outer bilayer leaflets. Furthermore, there is some evidence of cholesterol microdomains in the disk membranes. The availability of the disk protein, rom-1 may be sensitive to membrane cholesterol. The effects exerted by cholesterol on rhodopsin function have far-reaching implications for the study of G-protein coupled receptors as a whole. These studies show that the function of a membrane receptor can be modulated by modification of the lipid bilayer, particularly cholesterol. This provides a powerful means of fine-tuning the activity of a membrane protein without resorting to turnover of the protein or protein modification.

Microchemical analysis of retina layers in pigmented and albino rats by Fourier transform infrared microspectroscopy.

Fourier transform infrared (FT-IR) microspectroscopy is a powerful technique that can be used to collect infrared spectra from microscopic regions of tissue sections. The infrared spectra are evaluated to chemically characterize the absorbing molecules. This technique can be applied to normal or diseased tissues. In the latter case, FT-IR microspectroscopy can reveal chemical changes that are associated with discrete regions of lesion sites, which can provide insights into the chemical mechanisms of disease processes. In the present study, FT-IR microspectroscopy was used to analyze sections of retina from normal (pigmented) and albino rats. The outer segments of retinas from pigmented animals were found to have unusually strong absorption values for C&z.dbnd6;C-H unsaturation and carbonyl functional groups. Docosahexaenoic acid (DHA), a major constituent of lipids in the outer segments, also had particularly high absorption values for these functional groups, which suggests that it is responsible for those enhanced absorption values. Absorbance values for the unsaturation and carbonyl functional groups were substantially reduced in the outer segments of retinas from albino animals. This finding, together with data from other studies on light-induced oxidative events in the retina, indicates a loss of DHA by a light-induced mechanism in albino animals. The outer nuclear layer had strong absorbance values for H-C-OH and P&z. dbnd6;O functional groups, which is likely due to the sugar phosphate backbone of DNA. The outer and inner plexiform layers were found to contain greater concentrations of CH(2) and C&z.dbnd6;O functional groups than the outer and inner nuclear layers, which is due to the high concentration of synaptic connections in the former layers. In summary, FT-IR microspectroscopy revealed a unique chemical profile in the outer segments compared to other retinal layers, and this profile was altered in albino animals.

Rod outer segment length and visual sensitivity.

Detection threshold for the rod system was measured psychophysically in adult goldfish before and after exposure to constant illumination of 340 lux (91 microW/cm2) for 7 days. As shown in the previous paper, rod outer segment (ROS) length increases an average of 60% under these conditions. The present work shows that visual sensitivity also increases, in approximate proportion to the additional optical density predicted by the longer ROSs. These results are the first to show that exposure to constant light can enhance visual sensitivity. They imply further that detection threshold is related to ROS length. Apparently, the photopigment in the ROS tips that is normally shed on a daily basis retains its photon-catching ability.

Light cycle--dependent axial variations in frog rod outer segment structure.

Polarized light microscopy reveals that the structural parameters of Rana pipiens rod outer segments (ROS) are not uniform along the cell axis. In addition to a pronounced birefringence (delta n) gradient found in the basal half of most ROS, periodic delta n bands are seen in approximately 10% of intact ROS isolated by agitating retinas in frog Ringer's solution. These small delta n differences appear as very faint light and dark striations that have a period and width that depends on the duration of light and dark exposure. In ROS from frogs kept on a 14 hr light/10 hr dark cycle at 20 degrees to 22.5 degrees C, the band period for a light-dark band pair is 1.0 to 1.6 micron. Portions of ROS produced during total darkness or constant light are free of distinct periodic bands. Quantitative delta n measurements show that the ROS sections generated in the dark have a relatively higher delta n than those produced in light. Band contrast is irreversibly enhanced when ROS are treated with the calcium ionophore A23187 in the presence of calcium-free saline solution. These results indicate that the synthesis of some calcium-sensitive ROS component is different when the frog is in the dark than when exposed to light.

Lighting conditions and retinal development in goldfish: photoreceptor number and structure.

The retinas of 63 goldfish were examined after varying durations of exposure to one of three environmental lighting conditions beginning before hatching: constant light (340 lux), cyclic light (12 hr 320 lux, 12 hr dark) and constant dark. Up to 8 months, no effects of constant light or dark on photoreceptor numbers or structure were apparent. Densities of rod and cone nuclei were normal and all retinal layers appeared normal by light microscopy. Exposure to constant light for 12 months or longer resulted in a reduction in rod density by 37%. Cone numbers were unaffected by constant light, even with exposures of 3 yr, and rod and cone outer segments were normal in length at 11-20 months under all environmental conditions. Due to poor survival, only one animal was available for quantitative examination from the group reared in constant dark 12 months or longer. Photoreceptor size and number in this retina were similar to those in the constant light condition. The results suggest that the formation and maturation of rods and cones in goldfish retina is unaffected by rearing in constant light. However, long-term exposure (greater than or equal to 12 months) may disrupt maintenance of differentiated rods.

Photoreceptor characteristics in congenic strains of RCS rats.

Three congenic Royal College of Surgeons (RCS) strains of rats have been developed: the RCS-p+ strain, which is a black-eyed dystrophic strain; the pink-eyed RCS-rdy+ strain, which is wild-type (+/+) at the retinal dystrophy (rdy) genetic locus and serves as the control for the inbred, pink-eyed RCS strain; and the black-eyed RCS-rdy+p+ strain, which is +/+ at the rdy locus and serves as the control for the black-eyed RCS-p+ line. Several cytologic features of the photoreceptors were examined in the two control strains, including percentage of cone cells, rod outer segment lengths in different hemispheres of the eye and in different lighting conditions, rate of rod outer segment renewal, and the cyclic pattern of rod outer segment disc shedding. In the pink-eyed RCS-rdy+ strain these features were the same as those in previously described albino rat strains. Most of these features have not been described previously in fully pigmented rats. In the black-eyed RCS-rdy+p+ strain, rod outer segments in the superior eye hemisphere were about 50% longer than those in the inferior hemisphere. The rate of rod outer segment disc synthesis was the same in both hemispheres. Consequently, rod outer segment renewal time depended on outer segment length. This ranged from 9 to 10 days in the inferior hemisphere to 14.6 days in the superior hemisphere.

Frog rod outer segment shedding in vitro: histologic and electrophysiologic observations.

Rod outer segment shedding in the frog, Rana pipiens, has been studied using an in vitro eyecup method. Control experiments have shown that shedding responses in vitro are comparable to those in vivo and, like the situation in vivo, shedding in isolated eyecups requires a dark period followed by light onset. We found an initial, rapid and light-evoked component of the shedding response to be critically dependent upon bicarbonate concentration, supporting the initial discovery of a bicarbonate requirement for Xenopus rod shedding by Besharse et al. In Rana, in vitro shedding occurs in the presence of 20 mM aspartate, suggesting that functional integrity of the inner retina is not a prerequisite for rod shedding. Additionally, shedding was found to be suppressed completely in the presence of the local anesthetic MS-222 and the phosphodiesterase inhibitor IBMX. In the case of IBMX, electrophysiologic recording indicated changes in photoreceptor sensitivity in the presence of the drug. Such changes may play a role in the observed inhibition of shedding.

From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG.

To quantify the rate at which light in a ganzfeld produces photoisomerizations in mouse rods in situ, we measured the rate of rhodopsin bleaching in eyes of recently euthanized mice with fully dilated pupils. The amount of rhodopsin declined as a first-order (exponential) function of the duration of the exposure at the luminance of 920 scot cd m(-2): the rate constants of bleaching were 8.3 x 10(-6) and 2.8 x 10(-5) s(-1) (scot cd(-1)m2)(-1) for C57B1/6 and 129P3/J mice, respectively. When the approximately 3-fold difference in effective areas of the pupils of the mice are taken into consideration, the bleaching rates for both strains become essentially the same, 2.6 x 10(-6) fraction rhodopsin (scot Td s)(-1). Assuming 7 x 10(7) rhodopsin molecules per rod, this bleaching rate yields the result that a flash of 1 scot Td s produces 181 photoisomerizations per rod, a value close to that derived from analysis of the collecting area of the rod for axially propagating light. We measured the electroretinograms of mice of the two strains reared under controlled illumination conditions (2 and 100 lux), and compared their properties, using the calibrations to determine the absolute sensitivities of the b-wave and a-waves. The intensity that produces a half-saturating rod b-wave response is 0.3-0.6 photoisomerizations rod(-1), and the amplification constant of the rod a-wave is 5-6 s(-2) photoisomerization(-1), with little dependence on the strain.

Visual pigments and retinoids in the Mongolian jird.

The visual cells, visual pigments and major retinoids of the Mongolian jird (Meriones unguiculatus) were examined. Light and electron microscope analyses show that these jirds had mainly rod photoreceptors. Octylglucoside extracts prepared from their retinas contained only rhodopsin with a maximum absorption at 497 nm and a concentration of 0.51 nmol per retina. Employing a standard method of high performance liquid chromatography (HPLC), the pigment epithelium from each eye was found to possess 0.52 nmol of retinyl palmitate (the most abundant form of retinyl ester) along with a small amount of retinol (0.02 nmol). Most of the retinoids in the body of these animals are stored in the liver, in the form of retinyl palmitate (1228.80 nmol per gram liver). As the Mongolian jird is small, inexpensive and readily available, this animal is a mammalian species suitable for the research of the biochemistry of retinoids and vision.

Effect of bleaching on the width and index of refraction of goldfish rod and cone outer segment fragments.

Data on goldfish rod and cone fragments were obtained before and after a strong bleach by using a Zeiss Jamin-Lebedeff infrared interference microscope and computer image processing techniques. The receptor fragments were fractured in the inner segment and were immersed in goldfish aqueous humor medium. On bleaching we found that: (1) there is a small increase in rod diameter. This effect is of the same magnitude reported earlier in Rana pipiens outer segments (Vision Res 1973; 13:171). (2) There is an inferred decrease in rod refractive index. (3) There is a decrease in cone width. (4) There is a slight increase in inferred cone refractive index. These data are presented.

Effects of continuous darkness on ERG correlates of disc shedding in rabbit retina.

The electroretinogram (ERG) was recorded in the dark from photo-entrained albino rabbits, using a constant-intensity, 500-nm, 50- or 100-msec stimulus at 1-min intervals. Under these conditions, the b-wave of the ERG was previously shown to decrease in amplitude at the time of the morning rod photoreceptor disc shedding event. We have extended our observations to rabbits housed in continuous darkness. The present data show that in constant darkness the change in retinal sensitivity continues to occur, but with a period slightly less than 24 hr. Unilateral dark adaptation, achieved by eyepatching, alters the timing of the event only in the retina of the occluded eye. Thus, the change in retinal sensitivity shares the characteristics of endogenous rhythmicity and intraocular control that have been demonstrated by histologic methods for mammalian rod disc shedding. We also report elongation of rod outer segments in some regions of the albino rabbit retina after continuous darkness.

Physiologic and anatomic development of the photoreceptors of normally-reared and dark-reared rabbits.

The postnatal development of the rod photoreceptor of normally-reared and dark-reared pigmented rabbits was studied using physiological and anatomical methods. The late receptor potential (LRP) was recorded in vitro and the threshold and maximum amplitude determined. The same specimens used in the electrophysiological studies were then prepared for microscopy, and rod cell outer and inner segment dimensions and photoreceptor spacing were determined. In the light-reared animals a small LRP was first recorded at 5 days, but only at very high stimulus intensities. Thereafter, there was a rapid decrease in the threshold and an increase in the amplitude of the LRP. The threshold and amplitude of the LRP reached adult values at 14 days. Of the anatomical parameters, maturation of inter-receptor spacing (14 days) is clearly associated with the attainment of the adult LRP amplitude levels. Outer segment length was also adultlike by 14 days of age and thus paralleled physiological maturity of the photoreceptor. Changes over time in the mean diameter and length of rod cell inner segments (adultlike at 21 days) follows the pattern of ontogenetic maturation of the LRP. These findings imply a close relationship during ontogeny between the LRP and development of the outer segment. In dark-reared pups there is a delay in the maturation of the photoreceptor mosaic, but by 4 weeks of age, physiological characteristics of the photoreceptor are adultlike.

Some conjectures on the design of a rod outer segment.

Photoreceptor diameter and spacing have been extensively analyzed with respect to diffraction, wave-guiding and other phenomena and have been found to be consistent with optimal design criteria. Photoreceptor length, on the other hand, has received but little attention. It is proposed here that the outer segment length of rods is optimal with respect to the competing demands of maximal photon absorption and minimal noise. This is borne out by our calculations based on the experimental data.