Published as doi: 10.1096/fj.06-6133fje.
(The FASEB Journal. 2006;20:2648-2650.)
© 2006 FASEB
An invertebrate-like phototransduction cascade mediates light detection in the chicken retinal ganglion cells
Maria Ana Contin,
Daniela M. Verra and
Mario E. Guido1
CIQUIBIC (CONICET)-Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina
1Correspondence: CIQUIBIC- Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina. E-mail: mguido{at}mail.fcq.unc.edu.ar
SPECIFIC AIMS
Vertebrate retinal ganglion cells (RGCs) are key main components of a nonvisual circuitry that convey photic and circadian information to the brain.
In this work, we investigated the intrinsic photoreceptive capability of chicken RGCs in culture by assessing the effect of light on the synthesis of radioactive melatonin; we characterized these cells as rhabdomeric photoreceptors (RPs) based on homology according to specific markers, and we examined the nature of the phototransduction cascade operating in these retinal cells.
PRINCIPAL FINDINGS
To examine the synthesis of 3H-melatonin in the RGC cultures, cells were exposed to bright light (white cool fluorescence light of 
1500 lux) and maintained for 4 days under a 12:12 h light-dark (LD) cycle. At day 5, cultures were released to constant darkness (DD) and fed 3H-tryptophan for 8 h at different phases during 24 h. Results shown in Fig. 1
A demonstrated that in DD there was a daily variation in the synthesis of 3H-melatonin of RGC cultures with higher levels of 3H-melatonin at zeitgeber times (ZTs) 8 and 10 during the subjective day, while basal levels were found at ZTs 14–22 during the projected night. Levels of 3H-melatonin at ZTs 8 and 10 were significantly greater than those at other times examined.
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Figure 1. Synthesis of 3H-melatonin in RGC cultures from E8 chicken embryos. A) Temporal regulation of 3H-melatonin synthesis in RGC cultures. Cultures were synchronized to a 12:12 h LD cycle for 4 days [L: cool white fluorescence light of 1500 lux turned on at time 0 (ZT 0) and off at 12 h (ZT 12)]. On day 5, cultures remained in DD. Cells were fed 3H-tryptophan (25 µCi/ml) for 8 h at different phases across 24 h. Data are mean ± SE (n=7–9/group) from 3 independent experiments and correspond to the total content of 3H-melatonin extracted from the cells and culture medium (F=4.09, P<0.006 by one-way ANOVA with factor of time). B) Light effect on 3H-melatonin synthesis. RGCs were cultured in 10 ml of B27-DMEM containing vitamin A and synchronized to a 12:12 h LD cycle for 4 days (L: cool white fluorescence light of 1500 lux turned on at ZT 0 and off at ZT 12. On day 5 at time 0, L was turned on and cells were fed 3H-tryptophan (25 µCi/ml) for 4 h at different phases during the subjective day. Controls were exposed to light at time 0 and released to dark during 4 h 3H-tryptophan labeling. Results are sum of 3H-melatonin content isolated from cells and culture medium. Data are mean ± SE (n=7-9/group) from 3 independent experiments. P < 0.02 by ANOVA. C). Effect of all-trans-retinal administration in RGC cultures. Cultures were handled as in B. At time 0, light was turned on and all-trans-retinal (0.1 µM) added at ZT 4 together with 3H-tryptophan. Data are mean ± SE (n=6-9/group) from 3 independent experiments. ** P < 0.0015, ***P < 0.0001 by pairwise comparison.
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Strikingly, when RGC cultures synchronized to a LD cycle were exposed to bright light during the subjective day (ZT8) on day 5, diurnal levels of 3H-melatonin were significantly decreased as compared to controls maintained in the dark after 4 h of labeling with 3H-tryptophan (Fig. 1B
). These findings demonstrated that chicken RGCs are intrinsically photosensitive. Also, since most opsin photopigments use retinal chromophore in both invertebrates and vertebrates, we investigated whether light adsorption can be enhanced by the addition to the culture medium of all-trans-retinal, a precursor for the chromophore 11-cis retinal. To test this, cultures were fed 0.1 µM all-trans-retinal in addition to the basal level of vitamin A (Dulbecco’s modified Eagle’s medium-B27, Gibco, Gaithersburg, MD). Results showed that the inhibitory effect of light on the 3H-melatonin synthesis is further increased by the addition of the chromophore to the culture medium (Fig. 1C
).
To investigate if the invertebrate phototransduction cascade is the pathway acting in intrinsically photosensitive RGCs, we pharmacologically blocked the phosphoinositide (PIP2) breakdown with specific phospholipase C (PLC) antagonists such as 100 µM neomycin (Neo) and 5 µM U73122 {1-[6-(17–3-methoxyestra-1,3,5,10-trien-17-yl)amino] hexyl-1H-pyrrole-2,5-dione} in the RGC cultures. These compounds inhibit InsP3 production, the activation of InsP3 receptors and probably the transient receptor potential (TRP) and TRP-like (TRPL) channels as well. We found that RGC cultures treated with Neo or with U73122 significantly affected the labeling of 3H-melatonin by reversing the suppressive effect of light, when compared to light controls treated with the vehicle (Fig. 2
A). No significant differences were observed between the dark control and the pharmacologically treated cultures in light (Fig. 2A
). To investigate the participation of the TRP and TRPL channels in RGC cultures, we examined the effect of 1 mM lanthanum (La3+), a known TRP channel blocker on the light inhibition of 3H-melatonin synthesis. We found that there is a significant increase in levels of labeled melatonin in cultures exposed to light and treated with La3+ when compared to vehicle-treated controls kept in light (Fig. 2B
). In addition, we assessed the effect of decreasing levels of intracellular Ca2+ by the administration of the Ca2+ chelator BAPTA-AM [1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (10 mM)]. This treatment significantly increased 3H-melatonin levels in light as compared with controls treated with the vehicle only (Fig. 2B
). No significant differences were observed between the dark control and the pharmacologically treated cultures in light (Fig. 2B
). We also investigated both the involvement of cGMP in the process triggered by light in chicken RGCs, and the participation of a phosphodiesterase (PDE). We used the PDE6 activity inhibitor zaprinast (0.1 M). Results shown in Fig. 2C
revealed a significant effect of 0.1 M zaprinast on levels of 3H-melatonin of RGCs maintained in the dark, mimicking the inhibitory effect of light. By contrast, we examined the effect of stimulating the guanylate cyclase (GC) activity by NO donor sodium nitropuriside (SNP; 1 mM) on the synthesis of 3H-melatonin in RGC cultures kept in the dark. SNP significantly decreased levels of 3H-melatonin synthesized in the RGCs in the dark with respect to dark controls (vehicle) (Fig. 2C
).
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Figure 2. Effect of different pharmacological treatments on the light suppression of 3H-melatonin synthesis in RGC cultures from E8 chicken embryos. Cultures were synchronized to a 12:12 h LD cycle for 4 days (L: cool white fluorescence light of 1500 lux turned on at ZT 0 and off at ZT 12). On day 5, L was turned on at time 0 and cells were fed 3H-tryptophan (25 µCi/ml) at ZT 4 together with the different effectors. Dark controls received identical treatments. Data are mean ± SE (n=6–9/group) from 3 independent experiments. A) Effect of PLC inhibitors neomycin (Neo, 100 µM) and U73122 (5 µM). **P < 0.007 as compared to light control treated with the vehicle. B) Effect of the Ca2+ chelator BAPTA-AM (10 mM) and the TRP/TRPL-channel blocker lanthanum (Lan, 1 mM). *P < 0.04, **P < 0.001 as compared to the light control (vehicle). C) Effect of cGMP modulators in controls maintained during 4 h of labeling in dark: the NO donor (SNP) (1 mM) and the PDE6 inhibitor zaprinast (ZAP, 0.1 M). *P < 0.04 for SNP or *P < 0.02 for zaprinast treatments as compared to dark controls (vehicle).
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CONCLUSIONS AND SIGNIFICANCE
Our observations constitute the first demonstration that RGCs in culture are capable of sensing photic information from the environment. RGCs that were previously synchronized to LD cycles exhibited a daily variation in the synthesis of melatonin under DD with maximum levels during the subjective day and lower values at the subjective night. Strikingly, when light was turned on, cultured cells respond to the photic input by substantially suppressing levels of recently synthesized 3H-melatonin. The inhibitory effect of light was further enhanced by increasing amounts of all-trans-retinal, the ubiquitous chromophore utilized all through the phylogenetic tree. These results indicate that RGCs in culture somehow detect light by using a vitamin A-based chromophore. In addition, RGCs showed mRNA expression for developmental factors (Pax6 and Brn-3), photopigment (Opn4), clock genes (Cry1 and Cry2), and the Gq G-protein involved in the invertebrate photocascade. These observations support the idea that chicken RGCs may have evolved from an ancient RP precursor cell.
We postulate that the cascade of phototransduction operating in the chicken RGCs involves 1) a distinct G-protein isoform, Gq, that activates a PLC that hydrolyzes PIP2 to diacylglycerol (DAG) and InsP3; and 2) the participation of Ca2+-permeable light-sensitive channels such as TRP and TRPL channels and/or the InsP3-sensitive Ca2+ stores. Moreover, the signal triggered by light implies the increase in cGMP levels and potentially the activity of a PDE controlling levels of cGMP. Thus, RGCs respond to light by triggering neurochemical events similar to those of the invertebrate phototransduction cascade (see Fig. 3
).
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Figure 3. Schematic diagram shows nonvisual phototransduction in chicken retina. It begins when light activates the photopigment causing photoisomerization of 11-cis retinal to all-trans-retinal. This leads to activation of G protein Gq, which in turn, activates a PLC. PLC hydrolyzes PIP2 to IP3 and DAG leading to opening of Ca2+-permeable light-sensitive TRP and TRP-like channels, and/or Ca2+ release from InsP3-sensitive stores. Light signal implies increase in cGMP levels by GC activation and probably regulation of PDE activity. All this produces the cell membrane depolarization. 3H-melatonin is synthesized from 3H-tryptophan in a pathway involving AA-NAT and HIOMT enzymes. To study the photocascade in RGC cultures, specific inhibitors of signaling pathway components were used to suppress light inhibition of melatonin synthesis. PLC antagonists (neomycin [1] and U73122 [2]), a TRP channel blocker (lanthanum [3]) and a Ca+2 chelator (BAPTA-AM [4]) produce a significant increase of 3H-melatonin levels in light as compared with controls. In addition, when GC was activated (SNP [5]) or PDE inhibited (zaprinast [6]), synthesis of 3H-melatonin in dark-kept cultures was significantly decreased resembling light effect. Chicken RGCs detect light via an invertebrate-like phototransduction cascade; however, it is still unknown how photocascade is linked to biosynthesis of melatonin in RGCs.
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The physiological significance for the presence of RPs in the eye may be related to the requirement for light detection at early developmental stages before any sign of formal vision occurs, which can be essential for the embryo to temporally regulate vital developmental programs and physiology in response to the environment.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6133fje
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Copyright © 2006 by The Federation of American Societies for Experimental Biology.
