Dopaminergic amacrine neurons of rat retinas with photoreceptor degeneration continue to respond to light

Exposure of albino rats to continuous light of low intensity (350–700 lux) for 4 months produces massive degeneration of the photoreceptor segments and cell bodies of the outer nuclear layer of the retina. Only a few heterochromatic, receptor cell nuclei remain, and no photoreceptor segments are present. On the other hand, the inner layers of these retinas remain morphologically intact. The inner nuclear layer of the normal rat retina contains a group of amacrine cells which contain the putative neurotransmitter, dopamine (DA). Short term exposure to light (30 or 60 min) markedly stimulates the rate of DA turnover in these cells in normal, previously dark-adapted rats. Such enhancement of the rate of neurotransmitter turnover in the brain has been correlated with an increase in nerve impulse activity. The present study was undertaken to determine if the dopaminergic amacrine cells of the inner nuclear layer were still responsive to light in the retinas of rats whose photoreceptors were previously destroyed by long term exposure to continuous illumination. One week before sacrifice, the animals which had been housed in continuous light for 4 months were returned to normal 14 hr light: 10 hr dark lighting conditions. At the end of this time they and a group of control rats which had been housed in cyclic lighting conditions for the entire 4 months were dark adapted for approximately 15 hr. Then the rate of retinal DA turnover was estimated from the depletion of DA following inhibition of DA synthesis by α methyl para-tyrosine. The turnover of DA in the dark-adapted retinas of the control rats and of experimental rats with photoreceptor degeneration was dramatically enhanced 2–4 fold by short term exposure (up to 1 hr) to light. Since rats are nocturnal and avoid light, we tested the light aversion of another group of rats which had been exposed to light for 4 months and then returned to cyclic lighting conditions for one week. These rats and control animals which had been maintained in cyclic lighting conditions for 4 months both chose the dark side of a light-dark box over 80% of the time. This behavior of the rats with retinal degeneration was taken as a crude indication of their continued ability to detect light. The light-induced increase in DA activity in retinas with photoreceptor degeneration may play a role in the continued ability of these rats to perceive light.     

Expressions of visual pigments and synaptic proteins in neonatal chick retina exposed to light of variable photoperiods

Light causes damage to the retina, which is one of the supposed factors for age-related macular degeneration in human. Some animal species show drastic retinal changes when exposed to intense light (e.g. albino rats). Although birds have a pigmented retina, few reports indicated its susceptibility to light damage. To know how light influences a cone-dominated retina (as is the case with human), we examined the effects of moderate light intensity on the retina of white Leghorn chicks (Gallus g. domesticus). The newly hatched chicks were initially acclimatized at 500 lux for 7 days in 12 h light: 12 h dark cycles (12L:12D). From posthatch day (PH) 8 until PH 30, they were exposed to 2000 lux at 12L:12D, 18L:6D (prolonged light) and 24L:0D (constant light) conditions. The retinas were processed for transmission electron microscopy and the level of expressions of rhodopsin, S- and L/M cone opsins, and synaptic proteins (Synaptophysin and PSD-95) were determined by immunohistochemistry and Western blotting. Rearing in 24L:0D condition caused disorganization of photoreceptor outer segments. Consequently, there were significantly decreased expressions of opsins and synaptic proteins, compared to those seen in 12L:12D and 18L:6D conditions. Also, there were ultrastructural changes in outer and inner plexiform layer (OPL, IPL) of the retinas exposed to 24L:0D condition. Our data indicate that the cone-dominated chick retina is affected in constant light condition, with changes (decreased) in opsin levels. Also, photoreceptor alterations lead to an overall decrease in synaptic protein expressions in OPL and IPL and death of degenerated axonal processes in IPL.     

Photochemical Damage in the Albino Rat Retina: Morphological Changes and Endogenous Amino Acids

Abstract: Endogenous amino acids were measured in retinas of rats exposed for up to 48 h to fluorescent light. Typical light damage was seen in photo–receptor cells after 30 h exposure to a maximum luminance of 1544 scotopic lux; and, from this time, taurine levels were significantly reduced. In contrast, the concentrations of other amino acids increased. After 18 h exposure to light, GABA, glycine, glutamate, and aspartate levels were raised in the photo-receptor cells, and GABA, glutamate, and glutamine levels in the inner retina. When ‘exposed’ animals were returned to their normal environment for 72 h, photoreceptor degeneration progressed and taurine concentrations were further reduced: the results suggest that the loss was from damaged photo–receptor cells. At this time the concentrations of the other amino acids measured had, in general, returned to normal     

Effect of light history on the rat retina: Timecourse of morphological adaptation and readaptation

Sprague Dawley rats were born and raised under either 5 or 800 lux cyclic light (12L:12D) and were sacrificed at 1, 2, 3, 6, 12, 16, and 28 weeks of age. At each time point outer nuclear layer (ONL) area and rod outer segment (ROS) length were measured. The former is an estimation of photoreceptor number, and the latter is an estimation of the photon-catching integrity of the retina, both of which are known to be dependent on the light environment. Regression analysis revealed an ONL area reduction with time of 0.003 mm²/wk for 5-lux-reared rats and 0.009 mm²/wk for 800-lux-reared rats. ROS length was relatively constant in the dim light group, but showed a decline in 800 lux rats of 0.5 m/wk. Rats moved from 800 to 5 lux at 9 and 21 wks of age showed no significant change in ONL area after 3 wks. ROS length in these rats increased at a prodigious rate, and in the 12-wk-olds (9 wks at 800 lux, followed by 3 wks at 5 lux), ROS length exceeded that of age-matched rats raised in 5 lux for life.Special issue dedicated to Dr. Frederick E. Samson     

Preventive and therapeutic effects of SkQ1-containing Visomitin eye drops against light-induced retinal degeneration

The human retina is constantly affected by light of varying intensity, this being especially true for photoreceptor cells and retinal pigment epithelium. Traditionally, photoinduced damages of the retina are induced by visible light of high intensity in albino rats using the LIRD (light-induced retinal degeneration) model. This model allows study of pathological processes in the retina and the search for retinoprotectors preventing retinal photodamage. In addition, the etiology and mechanisms of retina damage in the LIRD model have much in common with the mechanisms of the development of age-related retinal disorders, in particular, with age-related macular degeneration (AMD). We have studied preventive and therapeutic effects of Visomitin eye drops (based on the mitochondria-targeted antioxidant SkQ1) on albino rat retinas damaged by bright light. In the first series of experiments, rats receiving Visomitin for two weeks prior to illumination demonstrated significantly less expressed atrophic and degenerative changes in the retina compared to animals receiving similar drops with no SkQ1. In the second series, the illuminated rats were treated for two weeks with Visomitin or similar drops without SkQ1. The damaged retinas of the experimental animals were repaired much more effectively than those of the control animals. Therefore, we conclude that Visomitin SkQ1-containing eye drops have pronounced preventive and therapeutic effects on the photodamaged retina and might be recommended as a photoprotector and a pharmaceutical preparation for the treatment of AMD in combination with conventional medicines.     

Melanin protects choroidal blood vessels against light toxicity

Low ocular pigmentation and high long-term exposure to bright light are believed to increase the risk of developing age-related macular degeneration (ARMD). To investigate the role of pigmentation during bright light exposure, cell damage in retinae and choroids of pigmented and non-pigmented rats were compared. Pigmented Long Evans (LE) rats and non-pigmented (albino) Wistar rats were exposed to high intensity visible light from a cold light source with 140,000 lux for 30 min. Control animals of both strains were not irradiated. The animals had their pupils dilated to prevent light absorbance by iris pigmentation. 22 h after irradiation, the rats were sacrificed and their eyes enucleated. Posterior segments, containing retina and choroid, were prepared for light and electron microscopy. Twenty different sections of specified and equal areas were examined in every eye. In albino rats severe retinal damage was observed after light exposure, rod outer segments (ROS) were shortened and the thickness of the outer nuclear layer (ONL) was significantly diminished. Choriocapillaris blood vessels were obstructed. In wide areas the retinal pigment epithelium (RPE) was absent in albino rats after irradiation. In contrast, LE rats presented much less cell damage in the RPE and retina after bright light exposure, although intra-individual differences were observed. The thickness of the ONL was almost unchanged compared to controls. ROS were shortened in LE rats, but the effect was considerably less than that seen in the albinos. Only minimal changes were found in choroidal blood vessels of pigmented rats. The RPE showed certain toxic damage, but cells were not destroyed as in the non-pigmented animals. The number of melanin granules in the RPE of LE rats was reduced after irradiation. Ocular melanin protects the retina and choroid of pigmented eyes against light-induced cell toxicity. Physical protection of iris melanin, as possible in eyes with non-dilated pupils, does not seem to play a major role in our setup. Biochemical mechanisms, like reducing oxidative intracellular stress, are more likely to be responsible for melanin-related light protection in eyes with dilated lens aperture.     

Intensity-dependent phase-adjustments in the locomotor activity rhythm of the nocturnal field mouse Mus booduga

The locomotor activity rhythm of the nocturnal field mouse Mus booduga was monitored under constant darkness (DD) and free-running periods (tau) were estimated. Following a free-run of about 15 days in DD, the animals were exposed to periodic light pulses (LPs) of various intensities (1 lux, 10 lux, 50 lux, 100 lux, and 1,000 lux) and 15 minutes duration for 65 days at intervals of 24 hours to investigate the influence of intensity of light on the phase-angle-difference (psi) between the onset of locomotor activity and the time of LP administration. The experimentally observed values of psi and tau for a LP of 1,000 lux intensity used for 15 minutes every 24 hr, showed a sigmoid shaped relationship with tau. This relationship was similar to that predicted based on the nonparametric model of entrainment, which uses the tau and the LP phase response curve (PRC) constructed using LP of similar duration and intensity. The functional nature of the relationship between psi and tau was not found to change significantly with increasing intensities of LP used to entrain the locomotor activity rhythm. However, psi was significantly modulated by the intensity of LP. These results suggest that the periodic sensitivity of the circadian pacemaker underlying the locomotor activity rhythm in the nocturnal field mouse M. booduga to LPs plays an important role in maintaining a characteristic psi with the zeitgeber and the psi changes in a light intensity-dependent manner.     

Alpha-phenyl-N-tert-butylnitrone (PBN) prevents light-induced degeneration of the retina by inhibiting RPE65 protein isomerohydrolase activity

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.     

CYCLIC NUCLEOTIDE LEVELS IN NORMAL AND BIOLOGICALLY FRACTIONATED MOUSE RETINA: EFFECTS OF LIGHT AND DARK ADAPTATION

Abstract— The content of cyclic AMP and cyclic GMP was measured in whole eyes and in normal retinas from C57BL(6)J mice, in receptorless retinas from congenic mice homozygous for the receptor dystrophy gene (rd/rd), and in retinas from mice treated postnatally with monosodium glutamate. Normal retinas contain approx 320 μg of protein: dystrophic (rd/rd) retinas contain approx 110μg of protein, lack rods but possess some surviving cone somata and terminals: glutamate-modified retinas contain approx 200 μg of protein and have both a reduced area and thickness with a marked deficiency of ganglion cells and amacrine cells. In normal mice, more than 90% of the cyclic GMP, but only 607, of the cyclic AMP of the whole eye was in the retina. In normal dark-adapted retinas isolated under dim red light cyclic AMP and cyclic GMP content was 4.1 and 20.2pmol/retina, respectively. The content of both cyclic AMP and cyclic GMP was 40% less, 2.5 and 11.5pmol/retina, respectively, in light-adapted retinas. In dark-adapted retinas isolated under infra-red light, cyclic AMP content was 40%, higher than that in retinas isolated under dim red light; cyclic GMP content was the same under these two conditions. Receptorless retinas contained approx 50% as much cyclic AMP and only 1-2% as much cyclic GMP as normal retinas. Although glutamate-modified retinas also had approx 50% as much cyclic AMP, they contained 60-85%, as much cyclic GMP as normal retinas. Light decreased by 30-50% levels of both cyclic AMP and cyclic GMP in glutamate-modified retinas, but only reduced cyclic nucleotide levels in receptorless retinas by 20%.
These data indicate that 95% or more of the cyclic GMP is in photoreceptor cells, whereas cyclic AMP is more evenly distributed throughout the retina. In addition, both cyclic AMP and cyclic GMP levels are influenced by light- and dark-adaptation.     

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.     

Extended exposure to continuous low intensity light abolishes the photosensitivity of retinal dopamine neurons

Male Sprague-Dawley rats were divided into 2 groups. One group (experimental) was housed for 6 months in continuous low intensity light while the other (control) was exposed to standard 14 hr light: 10 hr dark cyclic lighting conditions for the entire time. For both the control and experimental groups the light intensity was 350-700 Lux. After 6 months, the experimental rats were returned to cyclic lighting. At one week and again at 2 months the light aversion behavior of all rats was tested in a light/dark test box. The experimental rats chose the dark side of the box only 58% of the time while control animals preferred the dark 79% of the time. Since rats normally are nocturnal and avoid light, these results suggest that the experimental rats may have permanently lost a functionally significant portion of the ability to detect light. After the second behavioral test all rats were dark adapted and 15 hr later the effect of short term (30 or 60 min) exposure to light on DA turnover in one retina from each rat was assessed. The other retina from each rat was fixed and examined histologically. Light significantly enhanced the alpha methyl-p-tyrosine induced decline of DA in the retinas of the control rats but exerted no similar effect in the experimental animals. The retinal DA contents of the experimental rats were substantially depleted. Histological examination suggested that the outer nuclear layers of the experimental retinas were more severely damaged than those from rats exposed to continuous light for 4 months but still contained a few pycnotic photoreceptor nuclei and nearly normal looking inner neural layers. These results indicate that extended exposure to light eventually abolishes light aversion behavior and at this time there is also a loss of the photosensitivity of the dopaminergic amacrine neurons.     

Effect of different light intensities on growth,reproduction, amino acid synthesis,fat and sugar contents in ulva fasciata delile

Various physiological processes of Ulva fasciata were investigated in the laboratory under light intensities of 1500, 2500 and 3500 lux respectively.It was shown that there is a strong correlation between light intensity and growth rate, which increased with the increase in light intensity up till 2500 lux. Light intensities above 3000 lux resulted in bleaching of the algal thalli.In no instance there was any discharge of swarmers in total darkness nor at very reduced light intensities of about 100 lux.Zoospores were always negatively phototactic, while gametes were positively phototactic, appearing always on the well-illuminated sides of the culture bottles.The maximum yield of total nitrogen, dry weight, and amino acid content coincides with the optimum light intensity. Under such conditions leucine, valine, -alanine and glutamic acids are found in abundance, while phenyl alanine, -aminobutyric and glycine are moderately represented.The amount of total fat content increases with the increse in light intensity up till 3500 lux. This might refer to a strong correlation between the rate of photosynthesis and the fat synthesis.It was found that fructose and raffinose were present in negligible amounts under reduced light intensities (1500 lux), while sucrose was found in rather higher quantities. The quantity of glucose is higher than that of fructose and raffinose but much less than that of sucrose under the same light intensity.Alexandria UniversityKuwait UniversityKuwait University     

The effect of light intensity on the breeding and development of rats and golden hamsters

The growth, gestation and lactation of rats and hamsters subjected to different light intensities (15-2,000 lux) have been investigated. In the male rat, adrenal, testis and kidney weights were higher with increased light intensity, and there was an optimal light intensity (250 lux) for number of litters born, and a different optimum (60 lux) for the number of young per litter. In the hamster there were no differences related to light intensity, apart from an increase in the incidence of anoestrus where light intensities were below 60 lux.     

Effect of Light Intensity on the Phase and Period Response Curves in the Nocturnal Field Mouse Mus booduga

The effect of light intensity on the phase response curve (PRC) and the period response curve (τRC) of the nocturnal field mouse Mus booduga was studied. PRCs and τRCs were constructed by exposing animals free-running in constant darkness (DD), to fluorescent light pulses (LPs) of 100 lux and 1000 lux intensities for 15min duration. The waveform of the PRCs and τRCs evoked by high light intensity (1000 lux) stimuli was significantly different compared to those constructed using low light intensity (100 lux). Moreover, a weak but significant correlation was observed between phase shifts and period changes when light stimuli of 1000 lux intensity were used; however, the phase shifts and period changes in the 100 lux PRC and τRC were not correlated. This suggests that the intensity of light stimuli affects both phase and period responses in the locomotor activity rhythm of the nocturnal field mouse M. booduga. These results indicate that complex mechanisms are involved in entrainment of circadian clocks, even in nocturnal rodents, in which PRC, τRC, and dose responses play a significant role.     

Effect of light periodicity and intensity on the growth and survival of Heterobranchus longifilis Val. 1840 (Teleostei: Clariidae) larvae after 14 days of rearing

Heterobranchus longifilis Val. 1840 larvae were reared under two light intensities, 30 lux and 915 lux, and at varying photoperiods. Results show that maximum survival (82.5 ± 6.5% respectively) at 30 lux was obtained at continuous illumination [24 h light (L)], while the minimum (65 ± 21.2%) was at the 6 h L : 18 h dark (D) treatment. Survival at 24 h D averaged 71.3 ± 6.3%, with no significant difference (P < 0.05) in growth of larvae. Maximum larval survival at 915 lux was 87.5 ± 17.7% at the 18 h L treatment. Growth was not significantly different (P < 0.05) in the treatments. Comparison of the two light intensities showed that survival was better at a photoperiod above 12 h irrespective of intensity, while growth was significantly better at the 915 lux intensity.     

A QTL on distal Chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors

C57BL/6J-c^2J (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J × BALB/c)F₁× c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c. When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases. Received: 14 December 1999 / Accepted: 18 February 2000     

The fruit fly Drosophila melanogaster favors dim light and times its activity peaks to early dawn and late dusk

The light preferences of fruit flies were tested by 2 different means. First, flies were allowed to choose between different illuminations, and their favorite resting, grooming, and feeding places were determined with an infrared-sensitive camera. Second, the activity levels of the animals during their main daily activity period were determined photoelectrically (via infrared light beams) under different light intensities. Both methods revealed that the flies prefer dim light. They rested, groomed, and fed preferentially in places with a light intensity between 5 and 10 lux, and they showed the highest activity level when the light intensity during the day was kept at 10 lux. Furthermore, when dawn and dusk were simulated by logarithmically increasing/decreasing the light intensity during a 1.5-h interval, the flies' activity maxima occurred at about 7.5 lux during early dawn and late dusk. The results suggest that fruit flies time their clocks by early dawn and late dusk and avoid bright light during the day.     

Use of liquid chromatography with electrochemistry to measure effects of varying intensities of white light on DOPA accumulation in rat retinas

Exposure of dark-adapted rats to light enhances the activity of the retinal dopamine (DA) neurons. The purpose of this study was to determine if the response of these neurons to light varies with different intensities of light. The accumulation of dihydroxyphenylalanine (DOPA) after inhibition of L-aromatic amino acid decarboxylase with NSD-1015 was used as a measure of the $\text{in vivo}$ activity of these DA neurons. Retinal DOPA accumulation was significantly increased in dark-adapted rats that had been exposed to light for only 5 min. The activation of the retinal DA neurons by cool white fluorescent lighting was dependent upon the light intensity. Light intensities of 0.1 and 0.5 lux did not stimulate the retinal DA neurons. There was a significant, but submaximal, activation of the neurons by 5.0 lux, and intensities of 32.2 lux or more maximally stimulated the neurons. The method involving liquid chromatography (LC) with electrochemistry (EC) which was used in these experiments to measure retinal DOPA and DA concentrations is also described in detail.