Mario E. Guido

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)

Retinal Ganglion Cells Are Autonomous Circadian Oscillators Synthesizing N-Acetylserotonin during the Day

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.

c-Fos associates with the endoplasmic reticulum and activates phospholipid metabolism

c‐Fos, a transcription factor that constitutes DNA‐binding AP‐1 complexes, regulates gene expression that promotes long‐lasting cellular changes. We show that, in addition to its transcription factor activity, c‐Fos regulates the metabolism of phospholipids cytoplasmically by an AP‐1‐independent activity. Two waves of c‐Fos expression that promote subsequent waves of stimulation of 32P‐orthophosphate incorporation into phospholipids are evidenced in quiescent cultured fibroblasts induced to re‐enter the cell cycle. The first wave of c‐Fos expression peaks at 7.5 min and returns to control levels by 15 min. The second wave starts by 30 min and remains elevated at 120 min. In the first wave, the lipids that incorporate 32P are predominantly second‐messenger polyphosphoinositides (PIP, PIP2, PIP3); whereas in the second wave, membrane‐biogenesis‐related lipids (PI, PE, PA), become radioactive. Both waves of phospholipid activation depend on c‐Fos expression. It is interesting that a peptide that blocks AP‐1 nuclear import does not affect phospholipid activation. Immunocytochemical examination showed c‐Fos immunoreactivity associated to the endoplasmic reticulum. We conclude that c‐Fos, rapidly induced upon cell stimulation, associates to the endoplasmic reticulum where it first regulates the synthesis/ replenishment of phospholipids required for signal transduction pathways and subsequently regulates enzymes involved in the genesis of new membrane necessary for cell growth.

Immediate Early Gene Expression Within the Visual System: Light and Circadian Regulation in the Retina and the Suprachiasmatic Nucleus

Immediate early genes are a family of genes that share the characteristic of having their expression rapidly and transiently induced upon stimulation of neuronal and non-neuronal cells. In this review, first a short description of the IEGs is given, then it is discussed the stimulus-induced and circadian-induced variations in the expression of IEGs in the visual system, mainly in the retina and the suprachiasmatic nucleus. The possible physiological consequences of these variations in IEG expression are also considered. Finally, we refer to two aspects of our recent studies and those of other laboratories involving light-driven IEG expression. The first is the finding that in the chick retina, the expression of c-fos is differentially modulated in the different cell types and that c-fos regulates the synthesis of the quantitatively most important lipids of all cells, the phospholipids, by a non-genomic mechanism. The second is the occurrence of differential waves of IEG expression in the mammalian suprachiasmatic nucleus regarding light induction or spontaneous oscillations.

The metabolism of phospholipids oscillates rhythmically in cultures of fibroblasts and is regulated by the clock protein PERIOD 1

The mammalian circadian timing system is composed of countless cell oscillators distributed throughout the body and central pacemakers regulating temporal physiology and behavior. Peripheral clocks display circadian rhythms in gene expression both in vivo and in culture. We examined the biosynthesis of phospholipids as well as the expression of the clock gene period 1 (Per1) and its potential involvement in the regulation of the phospholipid metabolism in cultured quiescent NIH 3T3 cells synchronized by a 2 h serum shock. A 30 min pulse of radiolabeled precursor was given at phases ranging from 0.5 to 62 h after serum treatment. We observed a daily rhythm in the phospholipid labeling that persisted at least for two cycles, with levels significantly decreasing 29 and 58 h after treatment. Per1 expression exhibited a rapid and transient induction and a daily rhythmicity in antiphase to the lipid labeling. After Per1 expression knockdown, the rhythm of phospholipid labeling was lost. Furthermore, in cultures of CLOCK mutant fibroblasts—cells with a clock mechanism impairment—PER1 was equally expressed at all times examined and the phospholipid labeling did not oscillate. The results demonstrate that the biosynthesis of phospholipids oscillates daily in cultured fibroblasts by an endogenous clock mechanism involving Per1 expression.

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.

Synthesis of retinal ganglion cell phospholipids is under control of an endogenous circadian clock: Daily variations in phospholipid‐synthesizing enzyme activities

Retinal ganglion cells (RGCs) are major components of the vertebrate circadian system. They send information to the brain, synchronizing the entire organism to the light‐dark cycles. We recently reported that chicken RGCs display daily variations in the biosynthesis of glycerophospholipids in constant darkness (DD). It was unclear whether this rhythmicity was driven by this population itself or by other retinal cells. Here we show that RGCs present circadian oscillations in the labeling of [32P]phospholipids both in vivo in constant light (LL) and in cultures of immunopurified embryonic cells. In vivo, there was greater [32P]orthophosphate incorporation into total phospholipids during the subjective day. Phosphatidylinositol (PI) was the most 32P‐labeled lipid at all times examined, displaying maximal levels during the subjective day and dusk. In addition, a significant daily variation was found in the activity of distinct enzymes of the pathway of phospholipid biosynthesis and degradation, such as lysophospholipid acyltransferases (AT II), phosphatidate phosphohydrolase (PAP), and diacylglycerol lipase (DGL) in cell preparations obtained in DD, exhibiting differential but coordinated temporal profiles. Furthermore, cultures of immunopurified RGCs synchronized by medium exchange displayed a circadian fluctuation in the phospholipid labeling. The results demonstrate that chicken RGCs contain circadian oscillators capable of generating metabolic oscillations in the biosynthesis of phospholipids autonomously.

Expression of Novel Opsins and Intrinsic Light Responses in the Mammalian Retinal Ganglion Cell Line RGC-5. Presence of OPN5 in the Rat Retina

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca2+mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca2+ levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca2+ mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.

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+).

A nonmammalian vertebrate model of blindness reveals functional photoreceptors in the inner retina

In mammals, photoreceptors located in the inner retina convey photic information to the brain, regulating diverse non‐image‐forming tasks such as pupillary light reflexes and photic synchronization (entrainment) of daily activity rhythms. In nonmammalian vertebrates, the retina, deep brain photoreceptors, and pineal organ may be photoreceptive. Here we investigated light perception in the absence of functional cone and rod photoreceptors using GUCY1* chickens, birds carrying a null mutation that causes blindness at hatch. They showed light responses in both the pupillary light reflex and the entrainment of feeding rhythms to a 12:12 h light‐dark cycle. Light responses persisted even when the extraretinal photoperception was abolished, but they were lost after enucleation;this strongly indicates the essential role played by the inner retina. A sensitivity spectrum study for the pupillary reflex that combined pupil responses to different monochromatic lights of various intensities demonstrated that a single opsin/vitamin A‐based photopigment peaking at 484 nm drives photic responses;the best fit (lowest sum of squares, R2=0.9622) was attained with an opsin:vitamin A2 template. The results are the first characterization of functional inner retinal photoreceptors participating in the regulation of non‐image‐forming activities in nonmammalian vertebrates.—Valdez, D. J., Nieto, P. S., Garbarino‐Pico, E., Avalle, L. B., Díaz‐Fajreldines, H., Schurrer, C., Cheng, K. M., Guido, M. E. A nonmammalian vertebrate model of blindness reveals functional photoreceptors in the inner retina. FASEB J. 23, 1186–1195 (2009)