Inner Segments Of Photoreceptor Cells Research Articles

Long chain acyl-CoA synthetase 6 facilitates the local distribution of di-docosahexaenoic acid- and ultra-long-chain-PUFA-containing phospholipids in the retina to support normal visual function in mice.

Docosahexaenoic acid (DHA) and ultra-long-chain polyunsaturated fatty acids (ULC-PUFAs) are uniquely enriched in membrane phospholipids of retinal photoreceptors. Several studies have shown that di-DHA- and ULC-PUFA-containing phospholipids in photoreceptors have an important role in maintaining normal visual function; however, the molecular mechanisms underlying the synthesis and enrichment of these unique lipids in the retina, and their specific roles in retinal function remain unclear. Long-chain acyl-coenzyme A (CoA) synthetase 6 (ACSL6) preferentially converts DHA into DHA-CoA, which is a substrate during DHA-containing lipid biosynthesis. Here, we report that Acsl6 mRNA is expressed in the inner segment of photoreceptor cells and the retinal pigment epithelial cells, and genetic deletion of ACSL6 resulted in the selective depletion of di-DHA- and ULC-PUFA-containing phospholipids, but not mono-DHA-containing phospholipids in the retina. MALDI mass spectrometry imaging (MALDI-MSI) revealed the selective distribution of di-DHA- and ULC-PUFA-containing phospholipids in the photoreceptor outer segment (OS). Electroretinogram of Acsl6-/- mice exhibited photoreceptor cell-derived visual impairment, whereas the expression levels and localization of opsin proteins were unchanged. Acsl6-/- mice exhibited an age-dependent progressive decrease of the thickness of the outer nuclear layers, whereas the inner nuclear layers and OSs were normal. These results demonstrate that ACSL6 facilitates the local enrichment of di-DHA- and ULC-PUFA-containing phospholipids in the retina, which supports normal visual function and retinal homeostasis.

Presence of calpain-5 in mitochondria

Calpains are Ca2+-dependent cysteine proteases that are widely distributed in animal tissues and modulate a variety of cellular processes. There are 15 members of the calpain family in mammals. In animal cells, there are three types of calpains, viz., calpain-1, calpain-2, and calpain-10 in the mitochondria. The three types of calpains have been shown to play significant roles in pathophysiological conditions, including in apoptosis- and necrosis-like cell death. One of the severe retinal diseases, autosomal dominant neovascular inflammatory vitreoretinopathy, is known to be induced by mutations of the calpain-5 gene. However, the distribution of calpain-5 in the retina has not been elucidated. Therefore, in the present study, we determined the localization of calpain-5 in the porcine retina. We detected calpain-5 in the inner segment of photoreceptor cells using immunohistochemistry. With immunoelectron microscopy, calpain-5 was localized in the mitochondria of photoreceptor cells. Western blot analyses showed that calpain-5 was present in each mitochondrial subfraction. Furthermore, we showed that the molecular weight of mitochondrial calpain-5 was slightly smaller than cytosolic one. Our results demonstrated that a novel mitochondrial calpian, calpain-5, was localized in the mitochondria of retinal photoreceptor cells.

Unique cell biological profiles of retinal disease-causing missense mutations in the polarity protein Crumbs

ABSTRACT Mutations in human crumbs 1 ( CRB1 ) are a major cause of retinal diseases that lead to blindness. CRB1 is a transmembrane protein found in the inner segment of photoreceptor cells (PRCs) and the apical membrane of Muller glia. The function of the extracellular region of CRB1 is poorly understood, although more than 80 disease-causing missense mutations have been mapped to it. We have recreated four of these mutations, affecting different extracellular domains, in Drosophila Crumbs (Crb). Crb regulates epithelial polarity and growth, and contributes to PRC differentiation and survival. The mutant Crb isoforms showed a remarkable diversity in protein abundance, subcellular distribution and ability to rescue the lack of endogenous Crb, elicit a gain-of-function phenotype or promote PRC degeneration. Interestingly, although expression of mutant isoforms led to a substantial rescue of the developmental defects seen in crb mutants, they accelerated PRC degeneration compared to that seen in retinas that lacked Crb, indicating that the function of Crb in cellular differentiation and cell survival depends on distinct molecular pathways. Several Crb mutant proteins accumulated abnormally in the rhabdomere and affected rhodopsin trafficking, suggesting that abnormal rhodopsin physiology contributes to Crb/CRB1-associated retinal degeneration.

Docosahexaenoic acid preserves visual function by maintaining correct disc morphology in retinal photoreceptor cells

Docosahexaenoic acid (DHA) has essential roles in photoreceptor cells in the retina and is therefore crucial to healthy vision. Although the influence of dietary DHA on visual acuity is well known and the retina has an abundance of DHA-containing phospholipids (PL-DHA), the mechanisms associated with DHA's effects on visual function are unknown. We previously identified lysophosphatidic acid acyltransferase 3 (LPAAT3) as a PL-DHA biosynthetic enzyme. Here, using comprehensive phospholipid analyses and imaging mass spectroscopy, we found that LPAAT3 is expressed in the inner segment of photoreceptor cells and that PL-DHA disappears from the outer segment in the LPAAT3-knock-out mice. Dynamic light-scattering analysis of liposomes and molecular dynamics simulations revealed that the physical characteristics of DHA reduced membrane-bending rigidity. Following loss of PL-DHA, LPAAT3-knock-out mice exhibited abnormalities in the retinal layers, such as incomplete elongation of the outer segment and decreased thickness of the outer nuclear layers and impaired visual function, as well as disordered disc morphology in photoreceptor cells. Our results indicate that PL-DHA contributes to visual function by maintaining the disc shape in photoreceptor cells and that this is a function of DHA in the retina. This study thus provides the reason why DHA is required for visual acuity and may help inform approaches for overcoming retinal disorders associated with DHA deficiency or dysfunction.

Unique cell biological profiles of retinal disease-causing missense mutations in the polarity protein Crumbs.

Mutations in human crumbs 1 (CRB1) are a major cause of retinal diseases that lead to blindness. CRB1 is a transmembrane protein found in the inner segment of photoreceptor cells (PRCs) and the apical membrane of Müller glia. The function of the extracellular region of CRB1 is poorly understood, although more than 80 disease-causing missense mutations have been mapped to it. We have recreated four of these mutations, affecting different extracellular domains, in Drosophila Crumbs (Crb). Crb regulates epithelial polarity and growth, and contributes to PRC differentiation and survival. The mutant Crb isoforms showed a remarkable diversity in protein abundance, subcellular distribution and ability to rescue the lack of endogenous Crb, elicit a gain-of-function phenotype or promote PRC degeneration. Interestingly, although expression of mutant isoforms led to a substantial rescue of the developmental defects seen in crb mutants, they accelerated PRC degeneration compared to that seen in retinas that lacked Crb, indicating that the function of Crb in cellular differentiation and cell survival depends on distinct molecular pathways. Several Crb mutant proteins accumulated abnormally in the rhabdomere and affected rhodopsin trafficking, suggesting that abnormal rhodopsin physiology contributes to Crb/CRB1-associated retinal degeneration.

The protein tyrosine phosphatase interacting protein 51 (PTPIP51) is required for the differentiation of photoreceptors

Proliferation and differentiation of retinal progenitor cells (RPCs) are tightly controlled by extrinsic cues and distinct combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we investigated the role of the protein tyrosine phosphatase interacting protein 51 (PTPIP51) in the differentiation of RPCs. The expression pattern of PTPIP51 was analyzed by immunostaining during post-natal retinal development in the rat. Ex vivo electroporation has been used to silence or misexpress PTPIP51 in post-natal retinal explants, and the retinal phenotype was investigated after 3–7days in vitro (div). PTPIP51 expression in the retina started postnatally and was maintained throughout adulthood, especially in retinal ganglion cells and in the inner segment of photoreceptor cells. Silencing of Ptpip51 expression in postnatal retina failed to modify the commitment of late RPCs in the different lineages but severely impaired the final differentiation of photoreceptors, observed by a decrease in the fraction of Rhodopsin-positive cells after 7div. By contrast, misexpression of PTPIP51 in early or late RPCs failed to modify the differentiation of the RPCs. Our data demonstrate that PTPIP51 is implicated in the differentiation process of immature photoreceptors. Because PTPIP51 is specifically localized in the inner segment, PTPIP51 may contribute to the complex stage of maturation of the apical segment of these cells.

The GAFa domain of phosphodiesterase‐6 contains a rod outer segment localization signal

Photoreceptor phosphodiesterase-6 (PDE6) is a peripheral membrane protein synthesized in the inner segment of photoreceptor cells. Newly synthesized PDE6 is transported to the outer segment (OS) where it serves as a key effector enzyme in the phototransduction cascade. Proper localization of PDE6 in photoreceptors is critically important to the function and survival of photoreceptor cells. The mechanism of PDE6 transport to the OS remains largely unknown. In this study, we investigated potential OS targeting signals of PDE6 by constructing cGMP-binding, cGMP-specific phosphodiesterase-5/PDE6 chimeric proteins and analyzing their localization in rods of transgenic Xenopus laevis. We found that efficient OS localization of chimeric isoprenylated PDE enzymes required the presence of a targeting motif within the PDE6 GAFa domain. Furthermore, the GAFa-dependent localization signal was sufficient to target GAFa fusion protein to the OS. Our results support the idea that effective trafficking of the peripheral membrane proteins to the OS of photoreceptor cells requires a sorting/targeting motif in addition to a membrane-binding signal.

Immunohistochemical Localization of Glycogen Synthase and GSK3β: Control of Glycogen Content in Retina

Glycogen has an important role in energy handling in several brain regions. In the brain, glycogen is localized in astrocytes and its role in several normal and pathological processes has been described, whereas in the retina, glycogen metabolism has been scarcely investigated. The enzyme glycogen phosphorylase has been located in retinal Müller cells; however the cellular location of glycogen synthase (GS) and its regulatory partner, glycogen synthase kinase 3β (GSK3β), has not been investigated. Our aim was to localize these enzymes in the rat retina by immunofluorescence techniques. We found both GS and GSK3β in Müller cells in the synaptic layers, and within the inner segments of photoreceptor cells. The presence of these enzymes in Müller cells suggests that glycogen could be regulated within the retina as in other tissues. Indeed, we showed that glycogen content in the whole retina in vitro was increased by high glucose concentrations, glutamate, and insulin. In contrast, retina glycogen levels were not modified by norepinephrine nor by depolarization with high KCl concentrations. Insulin also induced an increase in glycogen content in cultured Müller cells. The effect of insulin in both, whole retina and cultured Müller cells was blocked by inhibitors of phosphatidyl-inositol 3-kinase, strongly suggesting that glycogen content in retina is modulated by the insulin signaling pathway. The expression of GS and GSK3β in the synaptic layers and photoreceptor cells suggests an important role of GSK3β regulating glycogen synthase in neurons, which opens multiple feasible roles of insulin within the retina.

Observation on retinal ultrastructures of the human fetus at the gestational age of 9 months

Objective To observe the retinal ultrastructure of the human fetal at the age of 9 months, and to investigate the clinical significance of the observation on retinal neuron development during the prenatal period. Methods Four human fetal eyes of 2 fetus at the gestational age of 9 months, including 1 at 35 and the other at 36 weeks, were obtained after termination of pregnancy due to trauma. The gestational ages of the fetus were estimated according to both last menstrual period (LMP) of the pregnant women and the weight/crown-heel length of fetus at the delivery. From each eyeball, 4 pieces of retina at the posterior pole were obtained and observed after specimens handling according to the procedure of routine electron microscopy. Eight pieces of retina which were randomly selected from total of 16 pieces of retina in each group were processed and observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Permissions from pregnant women and family members were guaranteed. Results At the gestational age of 9 months, the outer nuclear layer of fetal retina contained 5 to 6 layers of photoreceptor cells (PRC), and spherical-like membrane structures were found outside of the outer limiting membrane (OLM). Among many tight-aligned inner segments of PRCs there was zonula adherens of OLM, mitochondrias at inner side of OLM, and cilium at outer side of OLM. Outer segment of PRCs were short and contained a few irregularly arranged disc membrane. Some PRC had a multi-shaped nucleus in which equal amount of euehromatin and heteroehromatin. There were only few and thin axon branches from photoreceptor cells, and very few axons contacted with inner nuclear layer (INL) and no typical synapse was found. The INL contained 4 to 5 layers of cell bodies, in which many cellular nuclear had uneven density of euchromatin and heterochromatin; some were lobulated nucleus with clear karyotheca. In inner plexiform layer (IPL), the nerve cells had small branches, and only little connection among the synapses and few synapse structures were found. Although not many retinal ganglion cells (RGC) existed, RGC had both intact cell membrane and some rough endoplasmic reticulum (RER). The karyotheca of RGC had double-layers structures, and the nucleus was mainly consisted of euchromatin. Internal limiting membrane (ILM) had double-layer membrane structures, and the well-arranged nerve fiber layer was located at the outer side of ILM, with some micropores on the surface. Conclusions At the gestational age of 9 months, all layers of the human retinal has been formed, but some cell structure and cell connections are not yet mature, suggesting that at this time of period, human retina is still at an important stage of developing and remodeling. Key words: Retina/growth & development; Retina/ultrastructure; Microscopy; electron; transmission

Expression of erythropoietin and erythropoietin receptor in detached retina in rat model

Objective To observe the expression of erythropoietin (EPO) and EPO receptor (EPOR) in detached retina in rat model.Methods Fourty-eight male SD rats were randomly divided into control group and retinal detachment (RD) groups (1 hour,3,6,12,24,48,and 72 hours group) with 6 rats (12 eyes) in each group.1.4% hyaluronic acid was slowly injected into the subretinal space to induce the detachment of the upper retina to set up the RD model.The expression levels of mRNA and protein of EPO and EPOR were measured by RT-PCR and western-blotting analysis.Meanwhile,the locations of EPO and EPOR in retina were checked by immunohistochemistry.Results Both of the mRNA and protein levels of EPO and EPOR increased after RD,and reached the peak at the 48th hour after RD.The mRNA levels of EPO and EPOR were significantly higher in the 6 and 12 hours group than that in the control group(P< 0.05).The protein levels of EPO and EPOR were significantly higher in 3 hours group than that in the control group(P<0.05).Immunohistochemistry indicated weak expression of EPO from ganglion cell layer to inner and outer segment of photoreceptor cells,and strong expression in the corresponding location was found 48 hours after RD.Expression of EPOR from ganglion cell layer to inner segment of photoreceptor cells in the normal retina was detected,and strong expression in the corresponding location was found 48 hours after RD.Conclusion The expression of EPO and EPOR in retina increases gradually after RD,and reaches the peak at the 48th hour;most of the layers of neural retina can express EPO and EPOR. Key words: Erythropoietin,recombinant; Receptors,erythropoietin; Retinal detachment/etiology; Animal experimentation

Experimental autoimmune uveoretinitis initiated by non‐phagocytic destruction of inner segments of photoreceptor cells by Mac‐1+ mononuclear cells

EAU in mice is a model of human posterior uveitis. EAU is a Th1-dependent disease that has been assumed to target the neural retina and related tissues; however, in situ effector cells and the target have not yet been clearly demonstrated. In the present study, we induced EAU in B10R mice by immunizing them with human interphotoreceptor retinoid-binding protein peptide 161-180. Histological examinations revealed that EAU occurred approximately 11 days after the immunization and reached a peak on day 14. Retinae from normal or EAU mice were treated with proteases to obtain mono-dispersed cells. The mono-dispersed cells thus obtained were separated into three to four fractions by discontinuous Percoll density-gradient (e.g. PBS/40/60) centrifugation. In normal mice, 94% of the total cells were recovered in two fractions (i.e. PBS/40 and pellet); and these fractions mainly contained inner and outer segments and cell bodies of photoreceptor cells and RPE cells, respectively. In EAU mice, additional cells (i.e. inflammatory cells) were obtained at the 40/60 interface. Electron microscopic examination showed that tissue damage during EAU was initiated by non-phagocytic destruction of inner segments by Mac-1(+) mononuclear cells on day 11, followed by phagocytic activity of macrophages against outer segments and RPE cells on day 14. In vitro culturing of normal retinal cells with EAU infiltrates suggested the involvement of TNF-alpha and NO in the tissue damage. These results indicate that EAU was initiated by non-phagocytic destruction of inner segments of photoreceptor cells by Mac-1(+) mononuclear cells.

Differential localization of rat Eag1 and Eag2 potassium channels in the retina

Despite of their wide expression in the brain, the precise neurophysiological role of rat Eag1 (rEag1) and Eag2 (rEag2) K + channels remains elusive. Our previous studies in hippocampal pyramidal neurons demonstrate a somatodendritic localization of rEag1 and rEag2 channels, suggesting that the two channel isoforms may contribute to setting the membrane excitability of somas and dendrites. Here, we aim to further characterize the cellular and subcellular localization patterns of rEag1 and rEag2 proteins by studying their laminar distribution in the retina. Confocal microscopic analyses of immunofluorescence data revealed that rEag1 and rEag2 K + channels exhibit distinct cellular expression pattern in the retina. rEag1 immunoreactivity was most prominent in the outer half of the inner plexiform layer, whereas strong rEag2 immunostain was found in the outer and inner segments of photoreceptor cells, the outer plexiform layer, and the inner nuclear layer. These results suggest that rEag1 and rEag2 K + channels may play a significant role in the transmission of electrical signals along the retinal neuronal circuits. We also performed double-labeling experiments to demonstrate that rEag1 and rEag2 are predominantly expressed in the somatodendritic compartment of retinal neurons. In addition, we presented evidence suggesting that rEag1 channels may be expressed in the GABAergic amacrine cell. Finally, based on their different immunostaining patterns over the inner region of the retina, we propose that compared to rEag2, rEag1 expression encompasses a significantly broader range of the somatodendritic compartment of the retinal ganglion cell.

The Proteome of the Mouse Photoreceptor Sensory Cilium Complex

Primary cilia play critical roles in many aspects of biology. Specialized versions of primary cilia are involved in many aspects of sensation. The single photoreceptor sensory cilium (PSC) or outer segment elaborated by each rod and cone photoreceptor cell of the retina is a classic example. Mutations in genes that encode cilia components are common causes of disease, including retinal degenerations. The protein components of mammalian primary and sensory cilia have not been defined previously. Here we report a detailed proteomics analysis of the mouse PSC complex. The PSC complex comprises the outer segment and its cytoskeleton, including the axoneme, basal body, and ciliary rootlet, which extends into the inner segment of photoreceptor cells. The PSC complex proteome contains 1968 proteins represented by three or more unique peptides, including approximately 1500 proteins not detected in cilia from lower organisms. This includes 105 hypothetical proteins and 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders, increasing their priority as candidate genes. The PSC complex proteome also contains many cilia proteins not identified previously in photoreceptors, including 13 proteins produced by genes that harbor mutations that cause cilia disease and seven intraflagellar transport proteins. Analyses of PSC complexes from rootletin knock-out mice, which lack ciliary rootlets, confirmed that 1185 of the identified PSC complex proteins are derived from the outer segment. The mass spectrometry data, benchmarked by 15 well characterized outer segment proteins, were used to quantify the copy number of each protein in a mouse rod outer segment. These results reveal mammalian cilia to be several times more complex than the cilia of unicellular organisms and open novel avenues for studies of how cilia are built and maintained and how these processes are disrupted in human disease.

Divergent Distribution in Vascular and Avascular Mammalian Retinae Links Neuroglobin to Cellular Respiration

The visual function of the vertebrate retina relies on sufficient supply with oxygen. Neuroglobin is a respiratory protein thought to play an essential role in oxygen homeostasis of neuronal cells. For further understanding of its function, we compared the distribution of neuroglobin and mitochondria in both vascular and avascular mammalian retinae. In the vascular retinae of mouse and rat, oxygen is supplied by the outer choroidal, deep retinal, and inner capillaries. We show that in this type of retina, mitochondria are concentrated in the inner segments of photoreceptor cells, the outer and the inner plexiform layers, and the ganglion cell layer. These are the same regions in which oxygen consumption takes place and in which neuroglobin is present at high levels. In the avascular retina of guinea pig the deep retinal and inner capillaries are absent. Therefore, only the inner segments of the photoreceptors adjacent to choroidal capillaries display an oxidative metabolism. We demonstrate that in the retina of guinea pigs both neuroglobin and mitochondria are restricted to this layer. Our results clearly demonstrate an association of neuroglobin and mitochondria, thus supporting the hypothesis that neuroglobin is a respiratory protein that supplies oxygen to the respiratory chain.

Interaction of transducin-α with LGN, a G-protein modulator expressed in photoreceptor cells

LGN and activator of G-protein signaling 3 (AGS3) belong to the class of G-protein modulators containing G-protein regulatory motifs (GPR proteins). Evidence for the functions of these molecules has only started to emerge. Immunostaining of mouse retina cross-sections and serial tangential sectioning of the retina combined with immunoblot analysis revealed that LGN is expressed in the inner segments of photoreceptor cells. Double immunolabeling demonstrated that, following light-dependent translocation from the outer segments, the α-subunit of the visual G-protein transducin (Gtα) colocalizes with LGN in the basal part of the inner segments. LGN and Gtα coprecipitate from the retinal extracts, supporting the notion of the interaction between the proteins. Furthermore, the GPR domain of LGN potently inhibits receptor-mediated guanine nucleotide exchange and steady-state GTPase activity of transducin. The localization and interaction with Gtα suggest LGN roles in modulation of transducin translocation and other photoreceptor cell functions.

Orexins and Their Receptors in the Human Retina

Objectives: Orexins A and B are neuropeptides involved in the regulation of feeding behavior, energy homeostasis and arousal. In the human retina, however, immunohistochemical localization of orexins and their receptors, OX-R1 and OX-R2, has not been ascertained. Methods: We localized orexins A and B, OX-R1 and OX-R2 in the human retina using immunohistochemistry. Retinae from 2 Alzheimer’s disease (AD) patients provided preliminary evidence for possible orexin alterations. Results: Orexin A, orexin B and OX-R1 were localized in ganglion and amacrine cells, cellular processes in the inner and outer plexiform layer and in the inner segments of photoreceptor cells. There was no OX-R2 immunoreactivity in the retina. The staining intensity for both orexins was decreased in the AD patients. Conclusion: This immunohistochemical study provides the first evidence for the distribution of orexin A, orexin B and OX-R1 in the human retina. The localization pattern suggests a modulatory role for orexins in the interactions of those retinal cells which transmit light information to the suprachiasmatic nuclei, and thus may be involved in circadian rhythm entrainment.

Expression pattern of sigma receptor 1 mRNA and protein in mammalian retina

Sigma receptors are nonopiate and nonphencyclidine binding sites that are thought to be neuroprotective due to modulation of N-methyl- d-aspartate (NMDA) receptors. Sigma receptor 1 expression has been demonstrated in numerous tissues including brain. Recently, studies using binding assays have demonstrated sigma receptor 1 in neural retina, however these studies did not demonstrate in which retinal cell type(s) sigma receptor 1 was present nor did they establish unequivocally the molecular identity of the receptor. The present study was designed to address these issues. Reverse transcription–polymerase chain reaction (RT–PCR) analysis amplified sigma receptor 1 in neural retina, RPE–choroid complex, and lens isolated from mice. A similar RT–PCR product was amplified also in three cultured cell lines, rat Müller cells, rat ganglion cells and human ARPE-19 cells. In situ hybridization analysis revealed abundant sigma receptor 1 expression in ganglion cells, cells of the inner nuclear layer, inner segments of photoreceptor cells and retinal pigment epithelial (RPE) cells. Immunohistochemical studies detected the sigma receptor 1 protein in retinal ganglion, photoreceptor, RPE cells and surrounding the soma of cells in the inner nuclear layer. These data provide the first cellular localization of sigma receptor 1 in neural retina and establish the molecular identity of sigma receptor 1 in retinal cells. The demonstration that sigma receptor 1 is present in ganglion cells is particularly noteworthy given the well-documented susceptibility of these cells to glutamate toxicity. Our findings suggest that retinal ganglion cells may be amenable to the neuroprotective effects of sigma ligands under conditions of neurotoxicity such as occurs in diabetes.

Circadian Clock Regulation of pH in the Rabbit Retina

Although it is generally accepted that the acid-base ratio of tissue, as represented by the pH, is strictly regulated to maintain normal function, recent studies in the mammalian nervous system have shown that neuronal activity can result in significant shifts in pH. In the mammalian retina, many cellular phenomena, including neuronal activity, are regulated by a circadian clock. We thus investigated whether a clock regulates retinal pH, using pH-sensitive microelectrodes to measure the extracellular pH (pH(o)) of the in vitro rabbit retina in the subjective day and night, that is, under conditions of constant darkness. These measurements demonstrated that a circadian clock regulates the pH(o) of the rabbit retina so that the pH(o) is lower at night than in the day. This day/night difference in retinal pH(o) was observed when the rabbits were maintained on a normal light/dark cycle and after they were maintained on a light/dark cycle that was phase-delayed by 9 hr. Continuous recordings of retinal pH(o) around subjective dusk indicated that the change from daytime to nighttime pH(o) is relatively fast and suggested that the clock that regulates pH(o) is located in the retina. The lowest pH(o) recorded in the retina in both the day and night was in the vicinity of the inner segments of photoreceptor cells, supporting the idea that photoreceptors serve as the primary source of protons. The circadian-induced shift in pH(o) was several times greater than light-induced pH(o) changes. These findings suggest that a circadian clock in the mammalian retina regulates retinal pH.

Retina-derived fetuin (RDF): Analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot

PURPOSE. This study was undertaken to determine the pres-ence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light micros-copy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. METHODS. Retinal tissues and sections from embryonic, early postnatal and adult normal rats and retinal pigment epithe-lial (RPE) cells from postnatal rats were immunostained for fetuin with a polyclonal fetuin antibody and a peroxidase conjugated-secondary antibody using immunocytochemistry and Western blot analyses. The cDNA generated from RNA isolated from early postnatal rat retinas and RPE was probed with primers for rat fetuin, amplified by PCR and the PCR products were analyzed by Southern blot. RESULTS. Fetuin (RDF) was immunolocalized to cells of the neuroepithelium in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, in-ner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve pro-cesses were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicat-ing that this protein may be developmentally regulated. Using RT-PCR, the message for rat fetuin was demonstrated in the retina and RPE of normal postnatal rats. Southern blot con-firmed that the PCR product from the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin. CONCLUSIONS. Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cys-teine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.

Dominant expression of rat prostanoid DP receptor mRNA in leptomeninges, inner segments of photoreceptor cells, iris epithelium, and ciliary processes.

Prostaglandin (PG) D2 is one of the major prostanoids in the mammalian brain and eye tissues. Its function is mediated by the prostanoid DP receptor, which is specific for PGD2 among the various prostanoids. In this study, we cloned the full-length cDNA for the rat DP receptor and used it for detection of DP receptor mRNA in various rat tissues. Northern blotting and RT-PCR analyses revealed that this DP receptor was expressed most intensely in the eye tissues, moderately in the leptomeninges and oviduct, and weakly in the epididymis. The tissue distribution profile of the mRNA for the rat DP receptor is overlapped with those of hematopoietic and lipocalin-type PGD synthases. Among rat eye tissues, the expression was the highest in the iris. In situ hybridization and in situ RT-PCR revealed DP receptor mRNA to be localized in the epithelium of the iris and ciliary body and in photoreceptor cells of the retina, suggesting the involvement of the receptor in the physiological regulation of intraocular pressure and the vision process. In the brain, DP receptor mRNA was dominantly expressed in the leptomeninges and was not detected in the brain parenchyma including the ventral rostral forebrain, the surface area of which is reportedly involved in sleep induction by PGD2.