Daniela M. Verra

An invertebrate-like phototransduction cascade mediates light detection in the chicken retinal ganglion cells

Prebilaterian animals perceived ambient light through nonvisual rhabdomeric photoreceptors (RPs), which evolved as support of the chordate visual system. In vertebrates, the identity of nonvisual photoreceptors and the phototransduction cascade involved in nonimage forming tasks remain uncertain. We investigated whether chicken retinal ganglion cells (RGCs) could be nonvisual photoreceptors and the nature of the photocascade involved. We found that primary cultures of chicken embryonic RGCs express such RP markers as transcription factors Pax6 and Brn3, photopigment melanopsin, and G‐protein q but not markers for ciliary photoreceptors (α‐transducin and Crx). To investigate the photoreceptive capability of RGCs, we assessed the direct effect of light on 3H‐melatonin synthesis in RGC cultures synchronized to 12:12 h light‐dark cycles. In constant dark, RGCs displayed a daily variation in 3H‐melatonin levels peaking at subjective day, which was significantly inhibited by light. This light effect was further increased by the chromophore all‐trans‐retinal and suppressed by specific inhibitors of the invertebrate photocascade involving phosphoinositide hydrolysis (100 μM neomycin; 5 μM U73122) and Ca2+ mobilization (10 mM BAPTA; 1 mM lanthanum). The results demonstrate that chicken RGCs are intrinsically photosensitive RPs operating via an invertebrate‐like phototransduction cascade, which may be responsible for early detection of light before vision occurs.—Contin, M. A., Verra, D. M., Guido, M. E. An invertebrate‐like phototransduction cascade mediates light detection in the chicken retinal ganglion cells FASEB J. 20, E2249–E2257 (2006)

Early onset and differential temporospatial expression of melanopsin isoforms in the developing chicken retina.

PURPOSE Retinal ganglion cells (RGCs) expressing the photopigment melanopsin (Opn4) display intrinsic photosensitivity. In this study, the presence of nonvisual phototransduction cascade components in the developing chicken retina and primary RGCs cultures was investigated, focusing on the two Opn4 genes: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. METHODS Retinas were dissected at different embryonic (E) and postnatal (P) days, and primary RGC cultures were obtained at E8 and kept for 1 hour to 5 days. Samples were processed for RT-PCR and immunochemistry. RESULTS Embryonic retinas expressed the master eye gene Pax6, the prospective RGC specification gene Brn3, and components of the nonvisual phototransduction cascade, such as Opn4m and the G protein q (Gq) mRNAs at very early stages (E4-E5). By contrast, expression of photoreceptor cell markers (CRX, red-opsin, rhodopsin, and α-transducin) was observed from E7 to E12. Opn4m protein was visualized in the whole retina as early as E4 and remained elevated from E6 to the postnatal days, whereas Opn4x was weakly detected at E8 and highly expressed after E11. RGC cultures expressed Gq mRNA, as well as both Opn4 mRNAs and proteins. Opn4m was restricted exclusively to the GC layer at all ages, whereas Opn4x was limited to the forming GC layer and optic nerve at E8, but by E15, its expression was mostly in Prox1(+) horizontal cells. CONCLUSIONS The early expression onset of nonvisual phototransduction molecules could confer premature photosensitivity to RGCs, while the appearance of Opn4x expression in horizontal cells suggests the identification of a novel type of photosensitive cell in birds.

Light activation of the phosphoinositide cycle in intrinsically photosensitive chicken retinal ganglion cells.

PURPOSE In vertebrates, intrinsically photosensitive retinal ganglion cells (ipRGCs) acting as nonvisual photoreceptors transmit environmental illumination information to the brain, regulating diverse non-image-forming tasks. The phototransduction cascade in chicken ipRGCs has been shown to resemble that of rhabdomeric photoreceptors and involves phospholipase C (PLC) activation. The current work was an investigation of the participation of the phosphoinositide (PIP) cycle in this mechanism and of whether changes in activities of inositol 1,4,5-trisphosphate (IP(3)) and PIP kinase are triggered by light. METHODS Primary cultures of Thy-1 immunopurified chicken embryonic RGCs were exposed to bright light pulses or kept in the dark, to assess intracellular Ca(2+) mobilization by Fluo-3 AM fluorescence microscopy, IP(3) levels, and enzymatic activities of diacylglycerol, phosphatidylinositol, and phosphatidylinositol phosphate kinases (DAGK, PIK, and PIPK, respectively), by radioactive assays. The presence of different melanopsins (Opn4m and Opn4x) and other photopigments was determined by RT-PCR and immunochemistry. RESULTS Cultured RGCs expressing different nonvisual photopigments displayed a significant and rapid increase in IP(3) levels (1.3-fold) and Ca(2+) mobilization by light, which was reversed by administration of the PLC inhibitor U73122 (5 μM). Brief light pulses also caused a very rapid and transient activation of DAGK, PIK, and PIPK compared with that in the dark control. CONCLUSIONS The results indicate for the first time that light stimulation of chicken RGC cultures activates the PIP cycle, causing an increase in intracellular levels of IP(3), changes in levels of phosphatidic acid, PIP, and PIP(2); and mobilization of Ca(2+).

Early Appearance of Nonvisual and Circadian Markers in the Developing Inner Retinal Cells of Chicken

The retina is a key component of the vertebrate circadian system; it is responsible for detecting and transmitting the environmental illumination conditions (day/night cycles) to the brain that synchronize the circadian clock located in the suprachiasmatic nucleus (SCN). For this, retinal ganglion cells (RGCs) project to the SCN and other nonvisual areas. In the chicken, intrinsically photosensitive RGCs (ipRGCs) expressing the photopigment melanopsin (Opn4) transmit photic information and regulate diverse nonvisual tasks. In nonmammalian vertebrates, two genes encode Opn4: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. RGCs express both Opn4 genes but are not the only inner retinal cells expressing Opn4x: horizontal cells (HCs) also do so. Here, we further characterize primary cultures of both populations of inner retinal cells (RGCs and HCs) expressing Opn4x. The expression of this nonvisual photopigment, as well as that for different circadian markers such as the clock genes Bmal1, Clock, Per2, and Cry1, and the key melatonin synthesizing enzyme, arylalkylamine N-acetyltransferase (AA-NAT), appears very early in development in both cell populations. The results clearly suggest that nonvisual Opn4 photoreceptors and endogenous clocks converge all together in these inner retinal cells at early developmental stages.