Circadian Modulation of c-Fos Expression Occurs only in the SCN and not in Other Visual Projection Areas in the Rat

Brief photic stimuli at different circadian times induce differential expression of c-Fos in the suprachiasmatic nuclei (SCN). Whether circadian modulation of light-induced c-Fos expression occurs in other visual projection areas is not known. We addressed this question by estimating the immunohistochemical expression of c-Fos induced by 60 min light pulses at three different circadian times. The areas studied were the SCN, the ventral lateral geniculate nucleus, the intergeniculate leaflet, the ventral tegmental area, the superior colliculus and a non-visual control, the paraventricular thalamic nucleus (PVT). Light pulses induced an increase in the number of c-Fos immunoreactive cells in the SCN as a function of the circadian time. Remaining visual structures showed a light-induced increase in c-Fos expression but this was not dependent on the circadian time. The non-visual control area (PVT) did not respond to light pulses. Since no circadian modulation was found in the intergeniculate leaflet, which rec eives collateral projections from the same retinal ganglion cells that project to the SCN, nor in other primary visual projection areas, the present findings suggest that the circadian modulation of light-induced c-Fos expression in the SCN depends mainly on the functional properties of its intrinsic neurons.     

Circadian Modulation of c-Fos Expression Occurs only in the SCN and not in Other Visual Projection Areas in the Rat

Brief photic stimuli at different circadian times induce differential expression of c-Fos in the suprachiasmatic nuclei (SCN). Whether circadian modulation of light-induced c-Fos expression occurs in other visual projection areas is not known. We addressed this question by estimating the immunohistochemical expression of c-Fos induced by 60 min light pulses at three different circadian times. The areas studied were the SCN, the ventral lateral geniculate nucleus, the intergeniculate leaflet, the ventral tegmental area, the superior colliculus and a non-visual control, the paraventricular thalamic nucleus (PVT). Light pulses induced an increase in the number of c-Fos immunoreactive cells in the SCN as a function of the circadian time. Remaining visual structures showed a light-induced increase in c-Fos expression but this was not dependent on the circadian time. The non-visual control area (PVT) did not respond to light pulses. Since no circadian modulation was found in the intergeniculate leaflet, which rec eives collateral projections from the same retinal ganglion cells that project to the SCN, nor in other primary visual projection areas, the present findings suggest that the circadian modulation of light-induced c-Fos expression in the SCN depends mainly on the functional properties of its intrinsic neurons.     

Calcium imaging reveals a network of intrinsically light-sensitive inner-retinal neurons

BACKGROUND: Mice lacking rod and cone photoreceptors (rd/rd cl) are still able to regulate a range of responses to light, including circadian photoentrainment, the pupillary light reflex, and suppression of pineal melatonin by light. These data are consistent with the presence of a novel inner-retinal photoreceptor mediating non-image-forming irradiance detection. RESULTS: We have examined the nature and extent of intrinsic light sensitivity in rd/rd cl retinae by monitoring the effect of light stimulation (470 nm) on intracellular Ca(2+) via FURA-2 imaging. Using this approach, which does not rely on pharmacological or surgical isolation of ganglion cells from the rod and cone photoreceptors, we identified a population of light-sensitive neurons in the ganglion cell layer (GCL). Retinal illumination induced an increase of intracellular Ca(2+) in approximately 2.7% of the neurons. The light-evoked Ca(2+) fluxes were dependent on the intensity and duration of the light stimulus. The light-responsive units formed an extensive network that could be uncoupled by application of the gap junction blocker carbenoxolone. Three types of light-evoked Ca(2+) influx were observed: sustained, transient, and repetitive, which are suggestive of distinct functional classes of GCL photoreceptors. CONCLUSIONS: Collectively, our data reveal a heterogeneous syncytium of intrinsically photosensitive neurons in the GCL coupled to a secondary population of light-driven cells, in the absence of rod and cone inputs.     

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR)

The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as “visual cycle” involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.     

Lysophosphatidic acid-induced Ca(2+) mobilization in the neural retina of chick embryo

Lysophosphatidic acid (LPA) plays various roles in the regulation of cell growth as a lipid mediator. We studied the effect of LPA on intracellular Ca(2+) concentration ([Ca2+]i) with Fura-2 in the neural retina of chick embryo during neurogenesis. Bath application of LPA (1-100 microM) to the embryonic day 3 (E3) chick retina caused an increase in [Ca2+](i) in a dose-dependent manner, with an EC(50) value of 9.2 microM. The Ca(2+) rise was also evoked in a Ca(2+)-free medium, suggesting that release of Ca(2+) from intracellular Ca(2+) stores (Ca(2+) mobilization) was induced by LPA. U-73122, a blocker of phospholipase C (PLC), inhibited the Ca(2+) rise to LPA. Pertussis toxin partially inhibited the Ca(2+) rise to LPA, indicating that G(i)/G(o) protein was at least partially involved in the LPA response. The developmental profile of the LPA response was studied from E3 to E13. The Ca(2+) rise to LPA declined drastically from E3 to E7, in parallel with decrease in mitotic activity of retinal progenitor cells. The signal transduction pathway and developmental profile of the Ca(2+) response to LPA were the same as those of the Ca(2+) response to adenosine triphosphate (ATP), which enhances the proliferation of retinal progenitor cells. The coapplication of LPA with ATP resulted in enhancement of Ca(2+) rise in the E3 chick retina. Our results show that LPA induces Ca(2+) mobilization in the embryonic chick retina during neurogenesis.     

Edible Seaweed, Eisenia bicyclis, Protects Retinal Ganglion Cells Death Caused by Oxidative Stress

The purpose of the present study was to determine whether edible seaweed, Eisenia bicyclis, is effective in blunting the negative influence of N-methyl-D-aspartate (NMDA) on rat retinas and of oxidative stress-induced transformed retinal ganglion cell (RGC-5 cell line) death. The ethanol extract of E. bicyclis (EEEB) significantly attenuated the negative insult of L: -buthionine-(S,R)-sulfoximine plus glutamate on RGC-5 cells. Treatment of the RGC-5 cells with EEEB reduced the reactive oxygen species and recovered the reduced glutathione level caused by various radical species such as H(2)O(2), OH·, or O(2)·(-). Moreover, EEEB inhibited lipid peroxidation on rat brain homogenates caused by sodium nitroprusside. Applying NMDA to the retina affected the thickness of the inner plexiform layer (IPL) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) produced a positive effect on ganglion cells. Importantly, EEEB protected the thinning of IPL and increased TUNEL positive cells in the ganglion cell layer (GCL). Five phlorotannin derivatives were isolated using chromatographic methods and liquid chromatography-mass spectroscopy analysis which has been known as an antioxidant. In conclusion, EEEB has a neuroprotective effect in vitro and in vivo. Furthermore, the major constituents of this extract, phlorotannins, could possibly be active compounds due to their antioxidative potency.     

Mesotrypsin, a brain trypsin, activates selectively proteinase-activated receptor-1, but not proteinase-activated receptor-2, in rat astrocytes

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca(2+) mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca(2+) responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC(50)) value of 25 nm. The maximal effect of mesotrypsin on Ca(2+) mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (Ala-pFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH(2), synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca(2+) response. Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain.     

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine

Purpose

RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.

Methods

661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.

Results

Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.

Conclusions

661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.     

Stimulation of Prostaglandin EP2 Receptors on RGC-5 Cells in Culture Blunts the Negative Effect of Serum Withdrawal

Reduced neurotrophic support is one possible cause for retinal ganglion cells dying in glaucoma. Experiments were designed to investigate the effect of EP2 receptor agonist butaprost on transformed retinal ganglion (RGC-5) cells where reduced neurotrophic support was simulated by serum withdrawal. Cultures were analysed for cell viability, flow cytometry, reactive oxygen species and apoptosis. Western blot and immunohistochemistry were used to provide information for the occurrence of PGE₂ receptor-types. We demonstrated the existence of all four types of PGE₂ receptors in RGC-5 cells and exposure of cultures to butaprost resulted in an elevation of cAMP. Serum deprivation induced RGC-5 cell death was significantly attenuated by butaprost as well as by rolipram and forskolin where intracellular cAMP levels were increased. These data are of value in relation to the possible use of EP2 receptor agonists to reduce both elevated intraocular pressure and retinal ganglion cell death as occurs in glaucoma.     

Expression of p97/VCP and ubiquitin during postnatal development of the degenerating rat retina

In this study, we aimed to investigate the distribution pattern of ubiquitin and p97/VCP in the rat retina during postnatal development. Eyeballs from 1-, 4-, 10-, 36- and 72-week-old rats were examined by immunohistochemistry, and protein colocalization was determined by immunofluorescence microscopy. In the 1-week-old rat retina, p97/VCP was strongly expressed in the neuroblast layer, however no ubiquitin immunoreactivity was observed. p97/VCP immunoreactivity was present in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS) of the photoreceptor layer, and retinal pigment epithelium in the 4- and 10-week-old rat retinas. p97/VCP immunoreactivity increased significantly in the 10-week-old rat retinas. Ubiquitin was barely seen in the 4-week-old rat retinas, and ubiquitin expression was weak in the GCL and the IPL of the 10-week-old rat retinas. In the 36- and 72-week-old rats, the presence of ubiquitin was remarkable in the IS, INL, IPL and GCL, however, p97/VCP immunoreactivity was significantly decreased. Colocalization of ubiquitin and p97/VCP was also observed in the INL, IS, GCL and ONL of 36- and 72-week-old rat retinas. Our results indicate that p97/VCP immunoreactivity in the retina significantly decreases after rats reach 10 weeks of age, whereas ubiquitin immunoreactivity increases with aging. These results suggest that an altered expression pattern of p97/VCP and ubiquitin in the developing rat retina may associate with age-related retinal degeneration.     

Nicotinamide attenuates retinal ischemia and light insults to neurones

The aim of the present studies was to determine whether nicotinamide is effective in blunting the negative influence of ischemia/reperfusion to the rat retina in situ and of light to transformed retinal ganglion cells (RGC-5 cells) in culture. Ischemia was delivered to the retina of one eye of rats by raising the intraocular pressure. Nicotinamide was administered intraperitoneally just before ischemia and into the vitreous immediately after the insult. Electroretinograms (ERGs) of both eyes were recorded before and 5 days after ischemia. Seven days after ischemia, retinas were analysed for the localization of various antigens. Retinal and optic nerve extracts were also prepared for analysis of specific proteins and mRNAs. Also, RGC-5 cells in culture were given a light insult (1000 lux, 48 and 96 h) and evidence for reduced viability and apoptosis determined by a variety of procedures. Nicotinamide was added to some cultures to see whether it reversed the negative effect of light. Ischemia/reperfusion to the retina affected the localization of Thy-1, neuronal nitric oxide synthase (NOS) and choline acetyltransferase (ChAT), the a- and b-wave amplitudes of the ERG, the content of various retinal and optic nerve proteins and mRNAs. Significantly, nicotinamide statistically blunted many of the effects induced by ischemia/reperfusion which included the activation of poly-ADP-ribose polymerase (PARP). Light-induced apoptosis of RGC-5 cells in culture was attenuated by nicotinamide and the PARP inhibitor NU1025. The presented data show that nicotinamide attenuates injury to the retina and RGC-5 cells in culture caused by ischemia/reperfusion and by light, respectively. Evidence is provided to suggest that nicotinamide acts as a PARP inhibitor and possibly an antioxidant.     

Melanopsin and mechanisms of non-visual ocular photoreception

In addition to rods and cones, the mammalian eye contains a third class of photoreceptor, the intrinsically photosensitive retinal ganglion cell (ipRGC). ipRGCs are heterogeneous irradiance-encoding neurons that primarily project to non-visual areas of the brain. Characteristics of ipRGC light responses differ significantly from those of rod and cone responses, including depolarization to light, slow on- and off-latencies, and relatively low light sensitivity. All ipRGCs use melanopsin (Opn4) as their photopigment. Melanopsin resembles invertebrate rhabdomeric photopigments more than vertebrate ciliary pigments and uses a G(q) signaling pathway, in contrast to the G(t) pathway used by rods and cones. ipRGCs can recycle chromophore in the absence of the retinal pigment epithelium and are highly resistant to vitamin A depletion. This suggests that melanopsin employs a bistable sequential photon absorption mechanism typical of rhabdomeric opsins.     

The flavonoid baicalin counteracts ischemic and oxidative insults to retinal cells and lipid peroxidation to brain membranes

The purpose of the present study was to determine whether the flavonoid, baicalin is effective at blunting the negative influence of ischemia/reperfusion to the rat retina in situ and of various insults to a transformed retinal ganglion cells (RGC-5 cells) in culture. Baicalin was administered intraperitoneally just before and after an ischemic insult to retina of one eye of a rat. Ischemia was delivered by raising the intraocular pressure above the systolic blood pressure for 50min. Seven days after ischemia, retinas were analysed for the localisation of various antigens. Retinal extracts were also analysed for various mRNAs. Moreover, the content of specific proteins was deduced in retinal and optic nerve extracts. Also, RGC-5 cells in culture were given one of three different insults, light (1000lx for 2 days), hydrogen peroxide (200microM H(2)O(2) for 24h) or serum deprivation (48h) where cell survival and reactive oxygen species (ROS) formation was assayed. Moreover, a lipid peroxidation assay was used to compare the antioxidant capacity of baicalin with the flavonoid, epigallocatechin gallate (EGCG). Ischemia/reperfusion to the retina affected the localisation of Thy-1 and choline acetyltransferase (ChAT) and the content of various proteins (optic nerve and retina) and mRNAs (retina). Importantly, baicalin statistically blunted most of the effects induced by ischemia/reperfusion. Only the increase in caspase-8 and caspase-3 mRNAs caused by ischemia/reperfusion were unaffected by baicalin treatment. Baicalin also attenuated significantly the negative insult of light, hydrogen peroxide and serum withdrawal to RGC-5 cells. In the lipid peroxidation studies, baicalin was also found to be equally effective as EGCG to act as an antioxidant. Significantly, the negative insult of serum withdrawal on RGC-5 cell survival was blunted by baicalin but not by EGCG revealing the different properties of the two flavonoids.     

The Wnt Signaling Pathway Protects Retinal Ganglion Cell 5 (RGC-5) Cells from Elevated Pressure

The Wnt pathway is an essential signaling cascade that regulates survival and differentiation in the retina. We recently demonstrated that retinal ganglion cells (RGCs) have constitutively active Wnt signaling in vivo. However, the role of Wnt in RGC viability or function is unknown. In this study, we investigated whether Wnt protects the retinal ganglion cell line RGC-5 from elevated pressure, oxidative stress, and hypoxia injuries. Expression of RGC marker genes in the RGC-5 cultures was confirmed by immunocytochemistry and PCR. We demonstrated that the Wnt3a ligand significantly reduced pressure-induced caspase activity in RGC-5 cells (n = 5, P = 0.03) and decreased the number of TUNEL-positive cells (n = 5, P = 0.0014). Notably, Wnt3a-dependent protection was reversed by the Wnt signaling inhibitor Dkk1. In contrast, Wnt3a did not protect RGC-5 cells from oxidative stress or hypoxia. Furthermore, Wnt3a significantly increased growth factor expression in the presence of elevated pressure but not in the presence of oxidative stress and hypoxia. These results indicate that Wnt3a induces injury-specific survival pathways in RGC-5 cells, potentially by upregulating neuroprotective growth factors. Therefore, activation of the Wnt pathway by Wnt3a could be investigated further as a tool to develop novel molecular therapeutic strategies for the prevention of RGC death in retinal disease.     

Regulation of cGMP synthesis in photoreceptors: role in signal transduction and congenital diseases of the retina

Calcium feedback in vertebrate photoreceptors regulates synthesis of cGMP, a second messenger in phototransduction. The decrease in the free intracellular Ca(2+) concentrations caused by illumination stimulates two isoforms of retinal membrane guanylyl cyclase (RetGC) via Ca(2+)-sensor proteins and thus contributes to photoreceptor recovery and light adaptation. Unlike other members of the membrane guanylyl cyclase family, retinal guanylyl cyclases do not have identified extracellular peptide ligands. Recoverin-like proteins, GCAP-1 and GCAP-2, interact with the intracellular portion of the cyclases and stimulate its activity through dimerization of the cyclase subunits. Several mutations that affect the function of photoreceptor guanylyl cyclase and the activator protein have been linked to various forms of congenital human retinal diseases, such as Leber congenital amaurosis, cone and cone-rod dystrophy.     

Light Exposue Activates Retina Ganglion Cell Lysophosphatidic Acid Acyl Transferase and Phosphatidic Acid Phosphatase by a c-fos-Dependent Mechanism

We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in ganglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in vitro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light; no increase was observed in preparations obtained from light-exposed animals treated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.     

Differential expression of melanopsin isoforms Opn4L and Opn4S during postnatal development of the mouse retina

Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development.     

Endothelin receptors in light-induced retinal degeneration

Excessive light exposure leads to retinal degeneration in albino animals and exacerbates the rate of photoreceptor apoptosis in several retinal diseases. In previous studies we have described the presence of endothelin-1 (ET-1) and its receptors (ET-A and ET-B) in different sites of the mouse retina, including the retinal pigment epithelium, the outer plexiform layer (OPL), astrocytes, the ganglion cell layer (GCL), and vascular endothelia. After light-induced degeneration of photoreceptors, endothelinergic structures disappear from the OPL, but ET-1 and ET-B immunoreactivities increase in astrocytes. Here, we present novel observations about the course of light-induced retinal degeneration in BALB-c mice exposed to 1500 lux during 4 days with or without treatment with tezosentan, a mixed endothelinergic antagonist. Retinal whole mounts were immunostained with anticleaved caspase-3 (CC-3) serum to identify apoptotic photoreceptor cells within the outer nuclear layer (ONL). Glial activation was measured as glial fibrillary acidic protein (GFAP) immunoreactivity in retinal whole mounts and in Western blots from retinal extracts. Tezosentan treatment significantly reduced both the number of CC3-immunoreactive cells and GFAP levels, suggesting that inhibition of endothelinergic receptors could play a role in photoreceptor survival. Using confocal double immunofluorescence, we have observed that ET-A seems to be localized in bipolar cell dendrites, whereas ET-B is localized in horizontal cells. Our observations suggest the existence of an endothelinergic mechanism modulating synaptic transmission in the OPL. This mechanism could perhaps explain the effects of tezosentan treatment on photoreceptor survival.