expressions of visual pigments and synaptic proteins in

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

KUMAR ABHIRAM JHA, TAPAS C NAG*, SHASHI WADHWA and TARA SANKAR ROY

Department of Anatomy, All India Institute of Medical Sciences,New Delhi 110029, India

*Corresponding author (Email, [email protected])

Light causes damage to the retina, which is one of the supposed factors for age-related macular degeneration inhuman. Some animal species show drastic retinal changes when exposed to intense light (e.g. albino rats). Althoughbirds have a pigmented retina, few reports indicated its susceptibility to light damage. To know how light influences acone-dominated retina (as is the case with human), we examined the effects of moderate light intensity on the retina ofwhite Leghorn chicks (Gallus g. domesticus). The newly hatched chicks were initially acclimatized at 500 lux for 7days in 12 h light: 12 h dark cycles (12L:12D). From posthatch day (PH) 8 until PH 30, they were exposed to 2000 luxat 12L:12D, 18L:6D (prolonged light) and 24L:0D (constant light) conditions. The retinas were processed fortransmission electron microscopy and the level of expressions of rhodopsin, S- and L/M cone opsins, and synapticproteins (Synaptophysin and PSD-95) were determined by immunohistochemistry and Western blotting. Rearing in24L:0D condition caused disorganization of photoreceptor outer segments. Consequently, there were significantlydecreased 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 to24L:0D condition. Our data indicate that the cone-dominated chick retina is affected in constant light condition, withchanges (decreased) in opsin levels. Also, photoreceptor alterations lead to an overall decrease in synaptic proteinexpressions in OPL and IPL and death of degenerated axonal processes in IPL.

[Jha Ka, Nag TC, Wadhwa S and Roy TS 2016 Expressions of visual pigments and synaptic proteins in neonatal chick retina exposed to light ofvariable photoperiods. J. Biosci. 41 667–676]

1. Introduction

Light triggers photoreceptor death upon exposure for pro-longed period. Rodents are used as models to understandthe mechanisms of retinal light damage. They are exposedto intense light for a short period or to light of moderateintensities for long periods. As a consequence, rod cellsdie after light insult (Noell et al. 1966; Wenzel et al.2005). This damaging effect critically depends upon wave-length and intensity of light exposure and its duration(Noell et al. 1966; Penn et al. 1989; Hart et al. 2006;Basha et al. 2014).

On the other hand, information on the cone cell alterationsafter light insult is limited. Cones are important in humanvision and their loss is a problem in certain diseases, espe-cially in patients with retinitis pigmentosa and age-relatedmacular degeneration (AMD; Curcio et al. 1996; Beattyet al. 2000; Nag and Wadhwa 2012). Previous reports em-phasized that depending upon the experimental light condi-tions (e.g., high-intensity light for shorter duration and low-intensity light for longer duration), fluorescent artificial lightcould be harmful to the retina (Penn et al. 1989; Li et al.2001; Wenzel et al. 2005). This type of light is regularlyused in houses and work places where we are continually

http://www.ias.ac.in/jbiosci J. Biosci. 41(4), December 2016, 667–676 * Indian Academy of Sciences 667DOI: 10.1007/s12038-016-9637-6

Keywords. Light damage; opsins; photoreceptors; PSD-95; synaptophysin

Supplementary materials pertaining to this article are available on the Journal of Biosciences Website.

Published online: 28 September 2016

exposed to it for long years and therefore damages our retinain a cumulative way, which is the best understood factor inAMD pathogenesis (Beatty et al. 2000). Light damageexperiments using rodents show loss of photoreceptor nucleiand outer segments, decreased thickness of the retina andalterations in retinal pigment epithelium (RPE; Organisciaket al. 1998; Grimm et al. 2001; Wenzel et al. 2005; Marcet al. 2008). It is imperative to know about the cone vulner-ability in response to light exposure. Furthermore, in today’sscenario, due to increased socio-cultural activities, peopleare exposed to more light than normal home light (350–500 lux) and even to prolonged light phase. It is not clearlyknown how the retina changes upon continual exposure tolight. In this study, we used chicks to understand this, as theypossess about 86% cones in central retina (Morris andShorey 1967). They possess five different types of conephotoreceptors, viz. four types of single cones, and one typeof double cones (Okano et al. 1992). The ellipsoid region ofcones contains oil droplets, which is involved in protectionfrom harmful rays (Hart 2001). Considering vision in humanversus rodents, there are limitations on working with rodentmodels and interpretations of the derived data. First of all,rodents have a rod-dominated retina, with limited cone cellpopulation (e.g., about 0.85% and 3% of total photoreceptorspresent in rats and mice (Szél and Röhlich 1992; Jones andMarc 2005). So, the chick retina is a better model to studycone cells, and of human fovea. Earlier studies indicated thatconstant light exposure at 700 lux induces damage to chickretina (Li et al. 1995). Light intensity of 350–500 lux wasconsidered as normal, above 3500 lux as bright intense light,whereas 10000–30000 lux was considered as high intensitylight (Ashby et al. 2009; Cohen et al. 2011; Read et al.2015). In our previous report, we showed the effects of2000 lux on the morphological organization of chick retinaat different photoperiods, in which 2000 lux was consideredas moderate light intensity (Jha et al. 2015). No informationis available on light-associated synaptic changes consequentto photoreceptor death. In this study, we investigated theeffect of fluorescent light of moderate intensity (2000 lux)at three different photoperiods on the morphological organi-zation of outer and inner plexiform layers (OPL, IPL), andexpression patterns of opsins and synapse-associated pro-teins in chick retina.

2. Materials and methods

2.1 Egg rearing and animal housing

Fertilized eggs of white leghorn chicken (Gallus g.domesticus) were obtained from a local poultry farmand reared in an egg incubator under a normal photope-riod of 12 h light: 12 h dark (12L:12D),70% humidityand 37°C temperature. Egg hatching took place at 21

days of incubation period. The newly hatched chickswere reared in mesh wired cages in 12L:12D photoperiodfor initial seven days. The average ambient illuminationat the cage level was 500 lux (laboratory lightning con-dition), as measured by a digital lux meter, MextechLX1010B, India), with lights switched on at 8 AM andswitched off at 8 PM. The source of illumination wasprovided by fluorescent cool white light, set two feethigh above from the floor of the experimental cage. Thecages were placed at a controlled temperature (33±0.5°C)for initial 7 days. The animals were provided with waterand chick pellet ad libitum. After a week, at posthatchday (PH) 8, chicks were transferred in an experimentallarge wire mesh cage (2×2×2 ft, length×width×height,23±1°C temperature,) and reared upto PH 30. The entireexperiments were performed in accordance with ARVOstatement for the use of animals in Ophthalmic and Vi-sion Research and approved by institutional animal ethicscommittee (approval number: IAEC 583/2012).

2.2 Experimental design and light exposure protocol

The chicks were separated into three groups (Total No ofchicks: 24, 8 chicks/group): Experiment group I: chicks wereplaced in normal 12L:12D photoperiod. Experiment group II(longer photoperiod): chicks were reared in 18L:6D photo-period. Experiment group III (constant light group): chickswere exposed to 24L:0D photoperiod.

The experimental paradigm was described earlier by Jhaet al. (2015). Briefly, the cage illumination was standardizedby using fluorescent light tubes (18 watts × 5 tubes; wave-length range: 400–700 nm; temperature: 6500 K) fittedabove the cage. The distance between two tubes was about100 cm and each tube had separate buttons for switching onand off. The lights were placed two feet above the cage.Emitted light intensity was calculated at the center (2000lux) and four peripheral corners (approx 1950 lux) of thecage at floor level by a lux meter.

2.3 Tissue preparation

Chicks at PH 30 were euthanized, their eyes removed andthe dorsal part marked with India ink. The cornea wasexcised, the lens and vitreous were removed and the eyecupsfixed in 4% paraformaldehyde (4 retina/group) in 0.1 Mphosphate buffer (PB) for 4 h at 4°C. They were cryopro-tected in 15–30% sucrose, embedded in OCT in a cryostat(Microm-HM 525, Thermo scientific, UK) at −20°C, sec-tioned at 14 μm thickness in a dorsal to ventral axis. Sectionswere mounted on to gelatinized slides and stored at −20°Cfor future use.

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2.4 Assessment of retinal changes by transmissionelectron microscopy

After removal of the cornea, lens and vitreous, eyes fromdifferent groups (four retina /group) were fixed in 2% para-formaldehyde and 2.5% glutaraldehyde in 0.1M PB (pH 7.3)for 4 h at 4°C. After wash, samples were osmicated, dehy-drated and embedded in Araldite CY212. Sections (70–80nm) were stained with uranyl acetate and alkaline lead citrateand observed under a Tecnai G2-20 transmission electronmicroscope (FEI Company, The Netherlands). Digitalimages were acquired at magnification range 3500–10000Xusing TIA software. During observation, a number of degen-erated axons were found in IPL, which may be the processesof bipolar, amacrine and ganglion cells. Darkly stained areain ultrathin sections indicated degenerative axons and similarfeatures were also shown in another study in IPL of the retina(Wang et al. 2002). These were counted in a total of 40digitized images obtained from twenty grids, prepared fromfour different retinas in each experimental group (area ofeach image: 20×10 μm =200 μm2). The data are representedas the average number of dead axons /200 μm2 of IPL in allthree groups (supplementary table 1).

2.5 Immunohistochemistry

Cryosections were quenched in 0.3% H2O2, and blocked in10% normal serum in PBS for 2h to block non-specificbinding sites. Retinal sections were then incubated in pri-mary antibodies against rhodopsin (dilution: 5 μg/mL,mouse monoclonal, catalog number: ab98887, Abcam,UK), S-opsin (dilutions: 1–3 μg/mL, rabbit polyclonal,catalog number: ab81017, Abcam, UK), L/M opsin (dilu-tion: 1–3 μg/mL, rabbit polyclonal, catalog number:AB5405, Millipore, MA, USA), synaptophysin (dilution:5 μg/mL; mouse monoclonal, catalog number: MAB5258,Millipore, MA, USA) and PSD-95 (dilution: 5 μg/mL;mouse monoclonal, catalog number: MAB 1596, Millipore,MA, USA) for overnight duration at 4°C. After wash,sections were incubated in FITC- and rhodamine-conjugated secondary antibodies (dilution: 5 μg/mL, Vec-tor Laboratories, CA, USA) for 4h, washed, mounted inVectashield (Vector Laboratories, CA, USA) and examinedunder a fluorescence microscope (Leica DM6000B). Forintensity measurement of opsin expression, images weredigitized at 40x magnification using identical illuminationand microscope setting and intensity was quantified byImageJ software (NIH, USA) as pixel intensities. Imageswere acquired from six adjacent retinal locations in fivealternative sections per eye from three animals and intensitymeasured along 150 μm distance. Pixel intensity was mea-sured in 189000 μm2 area (13500 μm × 14 μm), where13500 μm was the length of retinal tissue (150 μm × 6= 900

μm/retinal section, 900 μm × 5 sections= 4500 μm × 3eyes=13500 μm), 14 μm was the section thickness, andaveraged per group (supplementary tables 2–4).

2.6 Western blotting and quantitation

Western blotting for rhodopsin, S-opsin, synaptophysin andPSD-95 was performed to analyze the quantitative changes intheir expressions among the three groups (N=24, 8 retinae/group). The retina was homogenized in ice-cold protein ex-traction RIPA buffer (3M Tris-HCl, 5M NaCl, 0.01% NonidetP-40, 0.1% glycerol, 100 mM sodium vanadate and 0.05%protease inhibitor cocktail (Sigma-Aldrich Corporation, MO,USA), incubated at 4°C for 1 h and centrifuged at 12,000 rpmfor 30 min at 4°C. The supernatant was collected after centri-fugation and stored at −80°C. Protein concentrations of thesamples were estimated by Bradford’s method. An amount of25 μg of protein was loaded in each lane, along with proteinmarkers (molecular weight range: 10–250 kDa, Bio-Rad,Hercules, CA, USA) in 10–12% SDS polyacrylamide gelsand electrophoresed at 80 Volts. The separated proteins werethen transferred onto nitrocellulose membranes at 65 volts for50 min. The membranes were treated first in 3% non-fat milkand incubated with primary antibodies against rhodopsin(Dilution: 1:1000), S-opsin (1:1000), synaptophysin(1:1000), PSD-95 (1:1000, mouse monoclonal, catalog num-ber: MAB 1596, Millipore, MA, USA), and α-tubulin (1:5000,mouse monoclonal, Sigma-Aldrich Corporation, MO, USA)for 12 h at 4°C. The blots were then incubated in anti-mouseand anti-rabbit secondary antibodies (dilution: 1:200) followedby incubation in avidin-biotin-peroxidase complex for 2 h. Theblots were visualized by treating the membranes in diamino-benzidine tetrahydrochloride hydrate and H2O2. Proteinextracts from rat retina were used as a positive control and α-tubulin and β-actin were used as loading controls for proteins.For densitometric analysis, all blots were scanned using Quan-tity 1 software (Bio-Rad, Hercules, CA, USA) and normalizedwith α-tubulin. L/M opsin antibody was previously used inchicken (Cebulla et al. 2012) and synaptophysin and PSD-95antibodies were used in chicks (Sanyal et al. 2013; Kumaret al. 2014). The rhodopsin and S-opsin antibodies are pre-dicted to work with chicken (from manufacturer’s data sheet).

2.7 Statistical analysis

For statistical analysis, all data are represented as mean ±SEM. A one-way parametric ANOVA, followed by post hocBonferroni test was performed to compare the data amongthe three experimental groups. Analyses were carried outusing SPSS 17 (IBM Inc., USA) and p≤0.05 was consideredto be statistically significant.

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3. Results

3.1 Morphological changes in retinal OPL and IPL

TEM observation at the level of OPL revealed normal andintact synaptic ribbons in 12L:12D group (figure 1a) and18L:6D group retinas (figure 1b) respectively, whereas in24L:0D group, short distorted synaptic ribbons were ob-served (figure 1c). Similarly, in IPL, compared to the num-ber in 12L:12D (figure 1d) and 18L:6D (figure 1e) groups,increased number of degenerated axonal profiles in IPL wasfound in 24L:0D group retina (figure 1f). Posthoc analysisshowed a significant increase in the number of dead axonalprofiles in IPL in 24L:0D group as compared to that in othertwo groups (figure 1g; 24L:0D vs 12L:12D, p 0.001;24L:0D vs 18L:6D, p 0.003).

3.2 Opsin expressions

Retinal sections from all three groups were labeled withL/M opsin, rhodopsin and S-opsin (figure 2a-i). In12L:12D and 18L:6D groups, L/M opsin- immunoreactiv-ity (IR) was intense in cone outer segments and ellipsoids,whereas a decreased IR was seen in 24L: 0D condition,compared to that in 12L:12D and 18L:6D groups (figure 2a-c). IR to rhodopsin and S-opsin in rod and cone outersegments, respectively, was moderate in 12L: 12D(figure 2d and g) and 18L: 6D groups (figure 2e and h)but was reduced in 24L:0D group (figure 2f and i).Figure 2j is a histogram showing immunofluorescenceintensity of L/M opsin in all three groups. There was

significant reduction in L/M-opsin expression in 24L:0Dgroup (p≤0.001) compared to that in 18L:6D and 12L:12Dgroups. There was significant reduction in L/M-opsin ex-pression in 24L:0D group (p≤0.001) compared to that in18L:6D and 12L:12D groups. Figure 2k and l show im-munofluorescence intensity of rhodopsin (figure 2k) and s-opsin (figure 2l) in three different experimental groups.There were significant decreases in rhodopsin and s-opsinimmunofluorescence intensity in 24L:0D group as com-pared that in 18L:6D and 12L:12D (*p<0.001; 24L:0D vs12L:12D and #p<0.001; 24L:0D vs 18L:6D for both pro-teins). Western blotting of L/M opsin was not performed,as the antibody was not specific for this technique, assuggested by the manufacturer (catalog number: AB5405,Millipore, USA). Western blots for other two proteins,viz., rhodopsin (bands at 31 kDa) and S-opsin (bands at29 kDa) revealed significant alterations in their expres-sions among the three groups (figure 3a–c). Post hocanalysis with Bonferroni test further confirmed that rho-dopsin expression was significantly lower (p≤0.001) in24L:0D group compared to that in 18L: 6D group and12L:12D groups, whereas the level was same between18L: 6D and 12L:12D groups (p=0.419; figure 3b). Theexpression of s-opsin was significantly lower in 24L:0Dgroup (p≤0.001) compared to that in 18L:6D and 12L:12Dgroups, wherein it remained unaltered (p=0.318,figure 3c). α-tubulin was used as a loading control andshowed bands of equal intensities at 55 kDa in all groups(figure 3a). Immunoblots of rat retinal extracts showedcorresponding bands at the same molecular weight foreach protein confirming the specificity of the antibodiesto each protein (figure 3a).

Figure 1. Transmission electron micrographs showing the effect of light of different photoperiods on OPL (a-c) and IPL (d-f) in PH30chick retina. (a-c) Normal synaptic ribbons in 12L:12D group (a) and 18L:6D group (b), respectively, while in 24L:0D group (c), short,distortated synaptic ribbons are seen (arrowhead). (d-f) Showing increased number of dead axonal processes in 24L:0D group (f,arrowhead) as compared to that in 12L:12D (d) and 18L:6D (e) groups. (g) Histogram showing significant increase in the number of deadaxonal processes in 24L:0D group (24L:0D vs 12L:12D indicated by *, p 0.001; 24L:0D vs 18L:6D indicated by #, p 0.003).

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3.3 Localization of synaptophysin by IHC and Westernblot analysis of synaptophysin and PSD-95

Retinal sections were labeled with synaptophysin and DAPI(figure 4a-d), the latter (figure 4a) indicated the relativeposition of nuclear layers in all figures. Synaptophysin-IR,being present in OPL and IPL, was almost similar in bothlayers of 12L:12D and 18L:6D groups (figure 4b and c), butdramatically reduced in OPL and upregulated in IPL in24L:0D group (figure 4d). PSD-IR was reduced in OPL in24L:0D group retina as relative to other group retinas, butalmost similar expression in OPL in between 18L:6D and12L:12D group retina (figure 5a-c). The specificity of theexpressions of synaptophysin and PSD-95 was confirmed byWestern blotting (figure 4e and 5d) that showed bands at 37-

and 95 kDa, respectively. There were significant alterationsin the expression of synaptophysin (p≤0.001) and PSD-95(p≤0.001) among the three experimental groups. Post hocanalysis with Bonferroni test further confirmed that theexpressions of both proteins were significantly decreased(synaptophysin: p≤0.001, PSD-95: p≤0.001) in 24L:0Dgroup compared to that in 12L:12D and 18L:6D groups. α-tubulin was used as a loading control and showed bands ofequal intensities at 55 kDa in all groups (figure 4e and 5e).

4. Discussion

The present study shows that moderate, constant fluorescentlight (2000 lux) affects the cone-dominated chick retina.

Figure 2. Opsin- IR in the retina in three photoperiodic conditions. L/M opsin, rhodopsin and S-opsin-IR are clearly present in photoreceptorouter segments, respectively in both 12L:12D (a, d and g; arrowheads) and 18L:6D groups (b, e and h; arrowheads), but is weakly present in 24L:0D group (c, f and i; arrowheads). Scale bar represents 50 μm (shown in figure i, applies to all). (j-l) Histogram showing decreased immunoflu-orescence intensity of L/M opsin (j), rhodopsin (k) and s-opsin (l) in 24L:0D group retina as compared to that in 12L:12D and 18L:6D group retinaand 24L:0D groups. For L/M opsin:-*p<0.002 (24L:0D vs 12L:12D) and # p<0.015 (24L:0D vs 18L:6D); rhodopsin:- * p<0.001 (24L:0D vs12L:12D) and # p<0.001 (24L:0D vs 18L:6D); s-opsin:- p<0.001 (24L:0D versus12L:12D) and # p<0.001 (24L:0D versus 18L:6D).



Figure 3. (a-c) Western blots (a) and histograms (b and c) depicting an overall decreased expression of rhodopsin (b) and s-opsin (c) in24L:0D group in comparison to that in 12L:12D and 18L:6D groups. Data (mean ± SEM) are expressed as rhodopsin/α-tubulin (b) and S-opsin/α-tubulin (c) ratio. The extreme right panel of part a shows protein expression in rat retina as a positive control. Asterisk denotesstatistical significance in 24L:0D versus 12L:12D group (p 0.001) and # represents 24L:0D versus 18L:6D group (p 0.001).

Figure 4. (a-d) Fluorescence micrographs showing DAPI staining (a) and synaptophysin IR in the retina in 12L:12D (b), 18L: 6D (c) and24L:0D group (d). (a) DAPI staining showing different retinal layers (gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclearlayer). (b and c) Normal level of IR in outer- and inner plexiform layer (opl, ipl) in 12L:12D and 18L:6D group retina. (d) Decreasedsynaptophysin IR in opl but increased IR in ipl (sublamina a; stars) in 24L:0D compared to that in (b) and (c). (e) Western blots showoverall decreased expression of synaptophysin in 24L:0D group retina in comparison to that in 12L:12D and 18L:6D groups. (f) Histogramsshow decreased expression of synaptophysin in the retinas of 24L:0D group as compared to that in 18L:6D and 12L: 12D groups. Data(mean ± SEM) are expressed as synaptophysin/α-tubulin. Star denotes statistical significance in 24L:0D versus 12L:12D (p 0.001), and #denotes 24L: 0D versus 18L: 6D (p<0.03), as determined by one-way ANOVA.

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Retinal damage was evident by TEM in OPL and IPL. Light-induced damage was accompanied by decreased expressionof opsins and synapse-associated proteins in 24L:0D group,compared to that in two other groups.

Light damage to the retina is centered largely to photoreceptorouter segments and ONL (Noell et al. 1966; Wasowicz et al.2002). In rodents, light exposure causes increased lipid peroxida-tion to rod outer segments and change in b-wave electroretino-gram (Wiegand et al. 1983), and in chick retina, it affects the oildroplet pigmentation (Hart et al. 2006). It also involved in DNAdamage and chromosome condensation, opsin degradation, de-creased expression of haem oxygenase-1 (an anti-oxidative en-zyme) and c-fos protein, calciummetabolism and peroxidation ofpoly-unsaturated fatty acids in photoreceptor outer segments(Organisciak and Vaughan 2010). The degree of light damage iscritically dependent on the intensity, duration, source and animals(Wenzel et al. 2005). Due to advancement of society, light fromfluorescent tubes is regularly used in industries, airports, clubs andother workplaces (Reese 2008) and therefore will affect the retina,which is one of the known factors in AMD (Beatty et al. 2000).Earlier, using moderate light intensity, histological changes of

chick and pigeon retinas (Marshall et al. 1972; Li et al. 1995)and on photoreceptor apoptosis in quail retina were reported(Thomson et al. 2002). Our previous study revealed that constantlight up to 23 days inducedmorphological changes in chick retinaat various levels (Jha et al. 2015). It caused a decreased thicknessof the retina and of outer and inner nuclear layers due to markednuclear shrinkage. As in rodents (Grimm et al. 2001), constantlight damages the photoreceptor outer segments in chicks. Addi-tionally, in chicks, there is damage to cone oil droplets in constantlight condition. However, no remarkable changes were observedin 18L:6D and 12L:12D groups under the same light intensity(2000 lux). This is possibly due to the idea that a dark periodmorethan 4 hours in an LD cycle minimizes the effect of constant lighton the retina (Li et al. 2000). But the exact protective mechanismremains to be verified, as there are no scientific data on howmanyhours chicks sleep or are active at night. First of all, we did notobserve sleep in experimental chicks after exposure to 2000 lux atdifferent photoperiods during day time. With regard to nightsleep/ wakefulness, it is generally believed that even if the animalssleep, due to their possession of a semi-transparent eyelid, theambient light still continues to affect the retina. The only deviation

Figure 5. (a-c) Fluorescence micrographs showing PSD-95 IR in outer plexiform layer (opl) in the retina in 12L:12D (a), 18L: 6D (b) and24L:0D group (c). (b and c) Similar pattern of IR in opl in 12L:12D and 18L:6D group retina. (c) Decreased PSD-95 IR in opl of 24L:0Dgroup retina as compared to that in (b) and (c). (d) Western blots show decreased expression of PSD-95 in 24L:0D group retina incomparison to that in 12L:12D and 18L:6D groups. (e) Histograms show decreased expression of PSD-95 in the retinas of 24L:0D group ascompared to that in 18L:6D and 12L: 12D groups. Data (mean ± SEM) are expressed as PSD-95/α-tubulin ratio. Star denotes statisticalsignificance in 24L:0D versus 12L:12D (p 0.001), and # denotes 24L: 0D versus 18L: 6D (p<0.001), as determined by one-way ANOVA.



is that the light in this situation may not be that damaging as ithappens when the eyes are open.

In this study, we observed alterations of synaptic ribbons inOPL and degenerating axonal processes in IPL in constantlight condition, which in the latter case may be due to thedamage of ganglion cell dendrites in IPL and degeneratedaxons originating from bipolar and amacrine cells, terminatinginto the IPL. Axonal death in GCL has been reported in severalpathological conditions such as in acute optic neuritis(Gabilondo et al. 2015) and Parkinson’s disease (Garcia-Mar-tin et al. 2014). The neuronal types in INL and GCL that aresensitive to light-induced changes remain to be explored.

4.1 Altered opsin expressions after constant light exposure

Damage to photoreceptor outer segments is central to lightinsult (Noell et al. 1966). Opsin containing visual pigmentsare packed in rod- and cones outer segment discs and in-volved in phototransduction. It is reported that opsin expres-sion is mislocalized in inherited retinal degeneration in RGE/RGE chicks, and reduced in retinal detachment models inchicks and tulp-/- mice, and in retinitis pigmentosa (Li et al.1995; Grimm et al. 2001; Montiani-Ferreira et al. 2005;Cebulla et al. 2012). In our study, the decreased levels ofopsins noted in 24L:0D group indicate that light (2000 lux)causes damage to the outer segment discs, perhaps by due tophotobleaching. Morphological alterations in 24L:0D groupaffected the normal rhythms of photostasis, which could beone of the possible explanations of such damage to outersegments. In rat model of retinal degeneration, blue lightcauses damage to the retina when rhodopsin level is high.Consequently, elevated rhodopsin level in dark rearing con-dition makes the retina more susceptible to light damage(Noell et al. 1966; Organisciak and Vaughan 2010). Sincethe chick retina has more cones than rods (Morris andShorey 1967), it would mean lower level of rhodopsin thancone opsins, and the unaltered opsin levels in 12L:12Dcondition are perhaps due to normal rhythms of photopig-ment regeneration, which can protect the retina from damagecaused by exposure to 2000 lux. In prolonged photoperiod(18L:6D) also, we found no significant alterations in opsinexpression, which supports the idea that a 6 h dark periodcan be sufficient to neutralize the bad effect of prolongedlight phase on the retina, as suggested (Li et al. 2000).

4.2 Synaptophysin and PSD-95 expression in differentphotoperiodic conditions

In the retina, phototransduction proceeds through the OPL toIPL. Visual function is hampered, if there are abnormalities inboth layers. In this study, we found constant light-inducedalterations in the expressions of synaptophysin and PSD-95in chick retina. The former is expressed in OPL and IPL of

retina (Hering and Kröger 1996; Nag and Wadhwa 2001).There are few reports on changes in synaptophysin expressionin disorders like diabetes retinopathy, increased intraocularpressure, retinal degeneration and Alzheimer’s disease (Szeet al. 1997; Kihara et al. 2008; Linberg et al. 2002; Sasakiet al. 2010). In our study, the decreased synaptophysin expres-sion in OPL noted in 24L:0D retina compared to that in18L:6D and 12L:12D retinas is in line with other light-induced experiments on murine model (Pérez and Perentes1994). On the contrary, in 24L:0D group retina, there wasincreased synaptophysin expression in IPL, which is due topossible remodeling of second order neurons in INL after lightdamage. Recently, it has been shown that loss of retinal photo-receptors via chemical toxicity results in synaptophysin upre-gulation in bipolar and amacrine cells (Dagar et al. 2014).

In the central nervous system, PSD-95 is a member of themembrane-associated guanylate kinase present at excitatorypostsynaptic sites and linked with scaffold proteins, which isinvolved in structural and functional neural signaling (El-Hus-seini et al. 2000). In the retina, PSD-95 labels the photorecep-tor terminals in OPL and faintly stains the IPL (Koulen et al.1998). In this study, we observed a reduction of PSD-95expression in 24L:0D group as compared to that in 18L:6Dand 12L:12D groups. It may be due to changes in the OPL andIPL, which alters the connection between photoreceptors, bi-polar, amacrine and ganglion cells. Another reason for suchdecreased expression of PSD-95 is due to damage of glutamateproducing cells (photoreceptor and bipolar cells). Our resultsare similar with those shown by other workers in retinaldamage model in diabetes, ischaemia and obesity, in whichthere is a decrease of PSD-95 level in pathological retina (VanGuilder et al. 2008). So, environmental factors such as con-stant light appear to influence the synaptic proteins (reducedexpressions of synaptophysin and PSD-95), but longer photo-periods do not have any such effects on them.

5. Conclusions

The chick retina, which is cone-dominated, is affected bymoderately intense, constant light condition relative to pro-longed- or normal-light rearing situations. Both rods andcones are damaged and this influences the synaptic organi-zation and morphological and functional well-being of cellslying in the INL. As a result, there are decreased expressionsof synaptophysin, PSD-95 and opsins, indicating that lightcan alter the synaptic organization in cone-dominated retina.Detailed investigations, however, are required to understandhow light influences the inner retina.

Acknowledgements

The work was financially supported by Science and Engi-neering Research Board, DST, New Delhi, India (No. AS-

674 KA Jha et al.


27/2012, TCN). KAJ received fellowships from UGC (NewDelhi). The TEM work was carried out at SAIF, AIIMS,New Delhi.

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MS received 09 December 2015; accepted 17 August 2016

Corresponding editor: NEERAJ JAIN

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