Wild-type Retinas Research Articles

Attenuation of EphrinB2 Reverse Signaling Decreases Vascularized Area and Preretinal Vascular Tuft Formation in the Murine Model of Oxygen-Induced Retinopathy

EphB4 and ephrinB2 are known key regulators of retinal vascular development, but due to their capacity for bidirectional signaling, delineation of their individual roles in this process remains unclear. To better dissect out individual contributions, a model of proliferative retinopathy in mice with attenuated ephrinB2 reverse signaling was studied. It was hypothesized that endothelial ephrinB2 reverse signaling regulates hypoxia-induced capillary sprouting, as well as the pathologic formation of neovascular tufts in postnatal retinal microvascular networks. Genetically manipulated mice with attenuated ephrinB2 reverse signaling (ephrinB2(lacZ/+)), along with wild-type (WT) controls, were exposed to oxygen-induced retinopathy (OIR), a postnatal model of proliferative retinopathy. At peak disease (postnatal day 18), microvascular networks were analyzed to examine intraretinal revascularization, capillary sprouting, and pathologic neovascularization responses. EphB4 and phosphorylated ephrinB protein expression patterns along retinal microvessels were also assessed. EphrinB2(lacZ/+) mice exhibited reduced hypoxia-induced revascularization (P ≤ 0.04) and reduced formation of neovascular tufts (P < 0.001), as compared with WT controls. Corresponding to the observed inhibition of retinal angiogenesis, ephrinB2(lacZ/+) retinas displayed an increased number of blind-ended capillary sprout tips (P < 0.02) and endothelial filopodial processes (P = 0.001). In WT and ephrinB2(lacZ/+) OIR-exposed retinas, ephrinB was confined to endothelial cells, with expression detected along angiogenic vascular processes including neovascular tufts and blind-ended capillary sprouts. EphrinB2 reverse signaling is a regulator of key processes during retinal vascularization and controls pathologic retinal angiogenesis through direct effects on capillary sprouting and endothelial filopodia formation.

Vision-guided ocular growth in a mutant chicken model with diminished visual acuity

Visual experience is known to guide ocular growth. We tested the hypothesis that vision-guided ocular growth is disrupted in a model system with diminished visual acuity. We examine whether ocular elongation is influenced by form-deprivation (FD) and lens-imposed defocus in the Retinopathy, Globe Enlarged (RGE) chicken. Young RGE chicks have poor visual acuity, without significant retinal pathology, resulting from a mutation in guanine nucleotide-binding protein β3 (GNB3), also known as transducin β3 or Gβ3. The mutation in GNB3 destabilizes the protein and causes a loss of Gβ3 from photoreceptors and ON-bipolar cells (Ritchey et al., 2010). FD increased ocular elongation in RGE eyes in a manner similar to that seen in wild-type (WT) eyes. By comparison, the excessive ocular elongation that results from hyperopic defocus was increased, whereas myopic defocus failed to significantly decrease ocular elongation in RGE eyes. Brief daily periods of unrestricted vision interrupting FD prevented ocular elongation in RGE chicks in a manner similar to that seen in WT chicks. Glucagonergic amacrine cells differentially expressed the immediate early gene Egr1 in response to growth-guiding stimuli in RGE retinas, but the defocus-dependent up-regulation of Egr1 was lesser in RGE retinas compared to that of WT retinas. We conclude that high visual acuity, and the retinal signaling mediated by Gβ3, is not required for emmetropization and the excessive ocular elongation caused by FD and hyperopic defocus. However, the loss of acuity and Gβ3 from RGE retinas causes enhanced responses to hyperopic defocus and diminished responses to myopic defocus.

Pattern of Expression of p53, Its Family Members, and Regulators during Early Ocular Development and in the Post-Mitotic Retina

Because of its role in cell cycle regulation and apoptosis, p53 may be involved in maintaining the post-mitotic state of the adult eye. To shed light on the role of p53 in retinal development and maintenance, this study investigated the pattern of expression of p53, its family members, and its regulators during the development of the mouse eye. Relative quantitative real-time PCR (qRT-PCR) was used to determine the steady-state levels of target transcripts in RNA extracted from wild-type mouse whole eyes or retinas between embryonic day (E) 15 and post-natal day (P) 30. Immunoblotting was used to compare the steady-state levels of the protein to that of the transcript. Transcript and protein levels for p53 in the eye were highest at E17 and E18, respectively. However, both p53 transcript and protein levels dropped precipitously thereafter, and no protein was detected on immunoblots after P3. Expression patterns of p63, p73, Mdm2, Mdm4, and Yy1 did not follow that of p53. Immunohistochemistry analysis of the developing eye showed that both p53 and Mdm2 are abundantly expressed at E18 in all layers of the retinal neuroblast. Downregulation of p53 in the post-mitotic retina suggests that, although p53 may be involved in ocular and retinal development, it may play a minimal role in healthy adult retinal function.

AMPA receptor-dependent, light-evoked d-serine release acts on retinal ganglion cell NMDA receptors

NMDA receptor (NMDAR) activation requires coincident binding of the excitatory neurotransmitter glutamate and a coagonist, either glycine or D-serine. Changes in NMDAR currents during neural transmission are typically attributed to glutamate release against a steady background of coagonist, excluding the possibility of coagonist release. AMPA receptor (AMPAR) stimulation evokes D-serine release, but it is unknown whether this is a physiological phenomenon capable of influencing synaptic responses. In this study, we utilized the intact retina to determine whether light-evoked synaptic activity in retinal ganglion cells (RGCs) is shaped by a dynamic pool of coagonist. The application of AMPAR antagonist abolished light-evoked NMDAR currents, which were rescued by adding coagonist to the bath. When NMDA was globally applied to RGCs via bath or picospritzing, the coagonist occupancy was also dependent on AMPARs but to a lesser extent than that observed during light responses, suggesting a difference in extrasynaptic coagonist regulation. By saturating the glutamate binding site of NMDARs, we were able to detect released coagonist reaching RGCs during light-evoked responses. Mutant mice lacking the d-serine-synthesizing enzyme serine racemase were deficient in coagonist release. Coagonist release in wild-type retinas was notably greater in ON than in OFF responses and depended on AMPARs. These findings suggest activity-dependent modulation of coagonist availability, particularly D-serine, and may add an extra dimension to NMDAR coincidence detection in the retina.

Endoplasmic Reticulum Stress-associated Cone Photoreceptor Degeneration in Cyclic Nucleotide-gated Channel Deficiency

Cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 account for >70% of all known cases of achromatopsia. Cones degenerate in achromatopsia patients and in CNGA3(-/-) and CNGB3(-/-) mice. This work investigates the molecular basis of cone degeneration in CNG channel deficiency. As cones comprise only 2-3% of the total photoreceptor population in the wild-type mouse retina, we generated mouse lines with CNG channel deficiency on a cone-dominant background, i.e. CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) mice. The retinal phenotype and potential cell death pathways were examined by functional, biochemical, and immunohistochemical approaches. CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) mice showed impaired cone function, opsin mislocalization, and cone degeneration similar to that in the single knock-out mice. The endoplasmic reticulum stress marker proteins, including Grp78/Bip, phospho-eIF2α, phospho-IP(3)R, and CCAAT/enhancer-binding protein homologous protein, were elevated significantly in CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) retinas, compared with the age-matched (postnatal 30 days) Nrl(-/-) controls. Along with these, up-regulation of the cysteine protease calpains and cleavage of caspase-12 and caspase-7 were found in the channel-deficient retinas, suggesting an endoplasmic reticulum stress-associated apoptosis. In addition, we observed a nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G in CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) retinas, implying a mitochondrial insult in the endoplasmic reticulum stress-activated cell death process. Taken together, our findings suggest a crucial role of endoplasmic reticulum stress in cone degeneration associated with CNG channel deficiency.

Activation of ganglion cells in wild-type and P23H rat retinas with a small subretinal electrode

Electronic retinal prostheses are being developed for people who become blind due to loss of photoreceptors from the disease retinitis pigmentosa. Previously, we reported on the responses of RGCs in the P23H rat (a model of retinitis pigmentosa) and the Sprague-Dawley (SD) rat to stimulation with a 400-μm diameter electrode (Jensen and Rizzo, 2011). With recent clinical trials now utilizing smaller (50–200 μm) electrodes, I sought to investigate the electrically evoked responses of RGCs in P23H and SD rat retinas with a smaller (125-μm diameter) electrode. Here, I report on the electrically evoked spike activity from RGCs that arose from stimulation of the retinal neural network. With biphasic current pulses of 1 ms per phase, the thresholds for activation of SD rat RGCs ranged from 0.52 to 2.8 μA; thresholds of P23H rat RGCs ranged from 1.2 to 7.8 μA. Median thresholds of RGCs were 1.4 μA in SD rats and 2.5 μA in P23H rats. These thresholds measurements were obtained with the recording electrode placed over the stimulating electrode. I also examined how thresholds of RGCs change as a function of distance (100–500 μm) from the center of the stimulating electrode. The median threshold currents of RGCs were much higher in P23H rats for all distances. What was striking was that the thresholds for activation of RGCs in P23H rat retinas rose much more rapidly. When the recording electrode was only 100–200 μm from the center of the stimulating electrode, the median threshold current of P23H rat RGCs rose by 460%. In contrast, the median threshold current of SD rat RGCs increased only 29%. I also investigated the contribution of photoreceptors to the electrically evoked responses of ON-center RGCs in SD rat retinas by examining the change in RGC thresholds when photoreceptor input to ON bipolar cells was blocked with the mGluR6 antagonist CPPG. At 500–600 μm, CPPG suppressed the light-evoked responses of the RGCs and at the same time increased the amount of current needed to generate an electrically evoked response. Similar to what was observed with SD rat RGCs, CPPG suppressed the light-evoked responses of ON-center P23H rat RGCs. However, the stimulation thresholds were not significantly altered. In conclusion, the data show that the threshold currents for indirect stimulation of both SD and P23H rat RGCs with a 125-μm diameter electrode are much lower than what we found previously with a 400-μm diameter electrode. To achieve high resolution vision with a multielectrode array, the spread of activation of RGCs needs to be limited. Our findings indicate that the spread of activation of RGCs is more confined in the degenerate retina. Lastly, my findings indicate that photoreceptors contribute to the lower stimulation thresholds of RGCs in normal, healthy retinas.

BK Channels Mediate Pathway-Specific Modulation of Visual Signals in theIn VivoMouse Retina

The modulatory role of large-conductance Ca(2+)-activated K(+) (BK) channels in the nervous system has been extensively studied. In the retina, it has been shown that BK channels play a pivotal role in modulating feedback from A17 amacrine cells to rod bipolar cells (RBCs). Here, we used electroretinography to examine the functional role of BK channels for rod and cone vision in the retina in vivo using a genetically engineered mouse lacking functional BK channels (Bk(-/-)). Under dark-adapted and light-adapted conditions, the lack of BK channels had no effect on photoreceptor activity, suggesting that these ion channels do not modulate photoreceptor responses. At the bipolar cell level, the ERG signals attributed to RBCs in Bk(-/-) mice were not different from those in wild-type mice at low scotopic stimulus intensities. However, at high scotopic and low mesopic stimulus intensities, close to RBC saturation, a significant reduction of ERG signals reflecting RBC activity was present in the Bk(-/-) retina. At higher mesopic stimulus intensities activating both RBCs and cone bipolar cells (CBCs), no difference in ERG signals between Bk(-/-) and wild-type mice was found. In photopic stimulus paradigms, activity of ON- and OFF-CBCs in Bk(-/-) and wild-type retinae was indistinguishable. These findings demonstrate that BK channels modulate visual responses in vivo at the bipolar cell level at intermediate stimulus conditions.

Loss of Llgl1 in retinal neuroepithelia reveals links between apical domain size, Notch activity and neurogenesis

To gain insights into the cellular mechanisms of neurogenesis, we analyzed retinal neuroepithelia deficient for Llgl1, a protein implicated in apicobasal cell polarity, asymmetric cell division, cell shape and cell cycle exit. We found that vertebrate retinal neuroepithelia deficient for Llgl1 retained overt apicobasal polarity, but had expanded apical domains. Llgl1 retinal progenitors also had increased Notch activity and reduced rates of neurogenesis. Blocking Notch function by depleting Rbpj restored normal neurogenesis. Experimental expansion of the apical domain, through inhibition of Shroom3, also increased Notch activity and reduced neurogenesis. Significantly, in wild-type retina, neurogenic retinal progenitors had smaller apical domains compared with proliferative neuroepithelia. As nuclear position during interkinetic nuclear migration (IKNM) has been previously linked with cell cycle exit, we analyzed this phenomenon in cells depleted of Llgl1. We found that although IKNM was normal, the relationship between nuclear position and neurogenesis was shifted away from the apical surface, consistent with increased pro-proliferative and/or anti-neurogenic signals associated with the apical domain. These data, in conjunction with other findings, suggest that, in retinal neuroepithelia, the size of the apical domain modulates the strength of polarized signals that influence neurogenesis.

Adequate Time Window and Environmental Factors Supporting Retinal Graft Cell Survival in rd Mice.

Postnatal photoreceptor cells can be integrated into the wild-type adult retina in mice, and retinal transplantation is now one therapeutic option for retinal degenerative diseases when photoreceptor degeneration is the primary cause of the disease. The aim of this study was to specify the optimal time window during the course of retinal degeneration and to modulate the host and/or graft environment for a successful transplantation. We first studied the background features of the mice with phosphodiesterase 6b (PDE6b) gene mutation (rd; C3H/Hej) and found that the infiltration of microglia and glial fibrillary acidic protein (GFAP) expression once increased at the peak of rod death (∼2-3 weeks of age) but then reduced for a following period until gliosis began to take place with enhanced GFAP expression (∼8 weeks of age). The postnatal retinal cells (p4-p7) were successfully transplanted during this period with neurite extension into the host retina. In later transplantations (6 or 8 weeks of age), graft cells survived better in the presence of chondroitinase ABC (ChABC), which digests chondroitin sulfate proteoglycan (CSPG), an essential component of gliosis. In contrast, in earlier transplantations (4 weeks of age), graft cells survived better in the presence of valproic acid (VPA), a neural differentiating reagent, or glatiramer acetate, an immune modulator. These suggest that, immediately after the outer nuclear layer (ONL) degeneration, an inflammatory reaction may be easily induced but the host neurons may be more able to accept donor cells in the presence of neural differentiating factor. These results will help optimize transplantation conditions when we consider clinical application.

Comparative intrinsic optical signal imaging of wild-type and mutant mouse retinas

Functional measurement is important for retinal study and disease diagnosis. Transient intrinsic optical signal (IOS) response, tightly correlated with functional stimulation, has been previously detected in normal retinas. In this paper, comparative IOS imaging of wild-type (WT) and rod-degenerated mutant mouse retinas is reported. Both 2-month and 1-year-old mice were measured. In 2-month-old mutant mice, time course and peak value of the stimulus-evoked IOS were significantly delayed (relative to stimulus onset) and reduced, respectively, compared to age matched WT mice. In 1-year-old mutant mice, stimulus-evoked IOS was totally absent. However, enhanced spontaneous IOS responses, which might reflect inner neural remodeling in diseased retina, were observed in both 2-month and 1-year-old mutant retinas. Our experiments demonstrate the potential of using IOS imaging for noninvasive and high resolution identification of disease-associated retinal dysfunctions. Moreover, high spatiotemporal resolution IOS imaging may also lead to advanced understanding of disease-associated neural remodeling in the retina.

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin α-subunit knockout (Tα(-/-)) mice which lack rod signaling(8).

CHM/REP1 cDNA delivery by lentiviral vectors provides functional expression of the transgene in the retinal pigment epithelium of choroideremia mice

Choroideremia (CHM) is a progressive X-linked degeneration of three ocular layers: photoreceptors, retinal pigment epithelium (RPE) and choroid, caused by the loss of Rab Escort Protein-1 (REP1). As a recessive monogenic disorder, CHM is potentially curable by gene addition therapy. The present study aimed to evaluate the potential use of lentiviral vectors carrying CHM/REP1 cDNA transgene for CHM treatment. We generated lentiviral vectors carrying either CHM/REP1 cDNA or EGFP transgene under the control of the elongation factor-1α promoter (EF-1α) or its shortened version EFS. We transduced human (HT1080) and dog (D17) cells, CHM patient's fibroblasts and mouse primary RPE cells in vitro, as well as wild-type and CHM mouse retinas in vivo by subretinal injections. Transgene expression was confirmed by immunoblotting, fluorescence-activated cell sorting, immunofluorescence and confocal microscopy. CHM/REP1 transgene functionality was assessed by an in vitro prenylation assay. Lentiviral vectors with CHM/REP1 and EGFP transgenes efficiently transduced HT1080, D17 and CHM fibroblast cells; CHM/REP1 transgene lead to an increase in prenylation activity. Subretinal injections of lentiviral vectors into mouse retinas resulted in efficient transduction of the RPE (30-35% of total RPE cells transduced after a 1-µl injection), long-term expression for at least 6 months and a decrease in amount of unprenylated Rabs in the CHM RPE. Transduction of neuroretinal cells was restricted to the injection site. Lentiviral CHM/REP1 cDNA transgene rescues the prenylation defect in CHM mouse RPE and thus could be used to restore REP1 activity in the RPE of CHM patients.

Glutathione Peroxidase 4 Is Required for Maturation of Photoreceptor Cells

Oxidative stress is implicated in the pathologies of photoreceptor cells, and the protective role of antioxidant enzymes for photoreceptor cells have been well understood. However, their essentiality has remained unknown. In this study we generated photoreceptor-specific conditional knock-out (CKO) mice of glutathione peroxidase 4 (GPx4) and showed the critical role of GPx4 for photoreceptor cells. In the wild-type retina the dominant GPx4 expression was in the mitochondria, indicating the mitochondrial variant was the major GPx4 in the retina. In the GPx4-CKO mice, although photoreceptor cells developed and differentiated into rod and cone cells by P12, they rapidly underwent drastic degeneration and completely disappeared by P21. The photoreceptor cell death in the GPx4-CKO mice was associated with the nuclear translocation of apoptosis-inducing factor (AIF) and TUNEL-positive cells. Photoreceptor cells before undergoing apoptosis (P11) exhibited decreased mitochondrial biomass, decreased number of connecting cilia, as well as disorganized structure of outer segments. These findings indicate that GPx4 is a critical antioxidant enzyme for the maturation and survival of photoreceptor cells.

Protective role of somatostatin receptor 2 against retinal degeneration in response to hypoxia

In mouse retinal explants, octreotide, a somatostatin [somatotropin release-inhibiting factor (SRIF)] receptor 2 (sst(2)) agonist, prevents the hypoxia-induced vascular endothelial growth factor upregulation. In mice with oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity, either sst(2) overexpression or octreotide have been found to limit hypoxia-induced angiogenic processes. Here, we investigated whether sst(2) influences retinal degeneration in response to hypoxia in wild-type (WT), sst(1)- and sst(2)-knockout (KO) mice. In retinal explants, we determined the role of sst(2) on apoptotic signals. In control condition, caspase-3 activity and the Bax/Bcl-2 ratio were lower in sst(1)-KO than in WT, but higher in sst(2)-KO than in WT retinas. In all strains, a comparable increase in caspase-3 activity and the Bax/Bcl-2 ratio was observed after hypoxia. The hypoxia-induced increase in apoptotic signals was recovered by octreotide in both WT and sst(1)-KO retinas. To investigate the role of sst(2) on retinal function, we recorded electroretinogram (ERG) in response to light flashes in OIR mice. ERG responses did not differ between WT and KO mice with the exception of oscillatory potentials (OPs) which, in sst(1)-KO mice, displayed much larger amplitude. In all strains, hypoxia drastically reduced a-, b-waves and OPs. In both WT and sst(1)-KO mice, octreotide recovered a- and b-waves, but did not recover OPs in sst(1)-KO mice. Neither apoptotic signals nor ERG was affected by octreotide in sst(2)-KO mice. These results show that sst(2) may protect retinal cells from hypoxia, thus implementing the background to establish potential pharmacological targets based on sst(2) pharmacology.

Retinal Function and Structure in the Hypotransferrinemic Mouse

The iron carrier transferrin is expressed at remarkably high levels in normal retinas and is upregulated during retinal degeneration. The authors characterized the consequences of genetically reduced retinal transferrin production on retinal structure and function. Hypotransferrinemic (HPX⁻/⁻) mice treated with weekly intraperitoneal salvage transferrin injections were examined at 1 and 2 months of age. HPX⁻/⁻, HPX⁺/⁻, and wild-type (WT) mice were evaluated by electroretinography, ophthalmoscopy, and histology. Retinal iron content and transferrin levels were measured. RNA levels of genes involved in iron homeostasis and antioxidative response were determined by quantitative PCR. Oxidative injury was assessed by immunostaining for 4-hydroxy-2-nonenal (HNE). At 2 months, dark-adapted, mixed rod-cone response b-wave amplitudes were significantly lower in HPX⁻/⁻ mice than in WT mice (340 ± 112 μV vs. 624 ± 134 μV [mean ± SEM]; P = 0.002). Oscillatory potentials were significantly suppressed in HPX mice, and ophthalmoscopy demonstrated marked retinal pallor. Quantitative immunostaining revealed a 39% reduction of transferrin content in HPX⁻/⁻ compared with WT retinas (P = 0.01). mRNA levels of Tf, Tf receptor, and ceruloplasmin were decreased, whereas mRNA for antioxidant genes were elevated in HPX⁻/⁻ retinas. HNE staining was reduced in mice carrying the mutant HPX allele. Histologic examination demonstrated preserved retinal structure, and retinal iron content was similar across the strains. Despite the lack of wild-type retinal transferrin production and low levels of retinal transferrin protein, the retinal morphology and retinal iron content in HPX⁻/⁻ mice treated by systemic salvage transferrin injections are normal until age 2 months. However, retinal function and gene expression of some of the iron-associated genes are significantly altered.

Whole-Exome Sequencing Identifies Mutations in GPR179 Leading to Autosomal-Recessive Complete Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs(∗)57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.

Profiling Retinal Biochemistry in the MPDZ Mutant Retinal Dysplasia and Degeneration Chick: A Model of Human RP and LCA

Raman microscopy, a rapid nondestructive technique that profiles the composition of biological samples, was used to characterize retinal biochemistry in the retinal dysplasia and degeneration (rdd) and wild-type (wt) chick retina during retinogenesis and at hatching. Embryonic day (E)13 and posthatch day (P)1 rdd and wt retinal cross-sections (n = 3 of each line at each age) were profiled using 633 helium-neon laser excitation. The biochemical composition was determined using computational analysis of the Raman spectra. In parallel histology, TUNEL and glial fibrillary acidic protein (GFAP) immunostaining were used to visualize retinal dysfunction. Principal component (PC) analysis of the Raman spectra identified 50 major biochemical profiles, but only PCs that made significant contributions to variation within rdd and wt retina were mapped. These significant PCs were shown to arise from DNA, various fatty acids, melanin, and a number of proteins. Distinct patterns of GFAP immunostaining and a larger population of TUNEL-positive nuclei were observed in the rdd versus wt retina. This study has demonstrated that Raman microscopy can discriminate between major retinal biomolecules, thus providing an unbiased account of how their composition varies due to the impact of the MPDZ null mutation in the rdd chick relative to expression in the normal wt retina.

Vascular endothelial growth factor in the ischemic retina and its regulation by somatostatin

In a retinal ischemic ex vivo model, we have reported protective effects of somatostatin (SRIF) receptor 2 (sst(2) ). As an ischemic condition not only causes cell death but also induces a vascular response, we asked whether vascular endothelial growth factor (VEGF) is altered in this model and whether its expression, release or localization are affected by sst(2) activation. Ex vivo retinas of wild-type (WT) and sst(1) KO mice (which over-express sst(2) ) were incubated in ischemic conditions with SRIF, octreotide (OCT) or a VEGF trap. Ischemia in WT retinas caused increase of VEGF release and decrease of VEGF mRNA. Both effects were counteracted by SRIF or OCT. VEGF immunoreactivity was in retinal neurons and scarcely in vessels. Ischemia caused a significant shift of VEGF immunoreactivity from neurons to vessels. The increase of vascular VEGF was reduced in sst(1) KO retinas and in WT retinas treated with SRIF or OCT. VEGF trap also limited this increase, demonstrating that vascular VEGF was of extracellular origin. Together, the data show a VEGF response to ischemia, in which VEGF released by damaged neurons reaches the retinal capillaries. The activation of sst(2) protects neurons from ischemic damage, thereby limiting VEGF release and the VEGF response.

Electrically Evoked Responses Of Retinal Ganglion Cells In Wild Type and Rd10 Mouse Retinas.

Electrically Evoked Responses Of Retinal Ganglion Cells In Wild Type and Rd10 Mouse Retinas.

Evolutionary relationships and diversification of barhl genes within retinal cell lineages

BackgroundBasic helix-loop-helix and homeodomain transcription factors have been shown to specify all different neuronal cell subtypes composing the vertebrate retina. The appearance of gene paralogs of such retina-specific transcription factors in lower vertebrates, with differently evolved function and/or conserved non-coding elements, might provide an important source for the generation of neuronal diversity within the vertebrate retinal architecture. In line with this hypothesis, we investigated the evolution of the homeobox Barhl family of transcription factors, barhl1 and barhl2, in the teleost and tetrapod lineages. In tetrapod barhl2, but not barhl1, is expressed in the retina and is important for amacrine cell specification. Zebrafish has three barhl paralogs: barhl1.1, barhl1.2 and barhl2, but their precise spatio-temporal retinal expression, as well as their function is yet unknown.ResultsHere we performed a meticulous expression pattern comparison of all known barhl fish paralogs and described a novel barhl paralog in medaka. Our detailed analysis of zebrafish barhl gene expression in wild type and mutant retinas revealed that only barhl1.2 and barhl2 are present in the retina. We also showed that these two paralogs are expressed in distinct neuronal lineages and are differently regulated by Atoh7, a key retinal-specific transcription factor. Finally, we found that the two retained medaka fish barhl paralogs, barhl1 and barhl2, are both expressed in the retina, in a pattern reminiscent of zebrafish barhl1.2 and barhl2 respectively. By performing phylogenetic and synteny analysis, we provide evidence that barhl retinal expression domain is an ancestral feature, probably lost in tetrapods due to functional redundancy.ConclusionsFunctional differences among retained paralogs of key retina-specific transcription factors between teleosts and tetrapods might provide important clues for understanding their potential impact on the generation of retinal neuronal diversity. Intriguingly, within teleosts, retention of zebrafish barhl1.2 and its medaka ortholog barhl1 appears to correlate with the acquisition of distinct signalling mechanisms by the two genes within distinct retinal cell lineages. Our findings provide a starting point for the study of barhl gene evolution in relation to the generation of cell diversity in the vertebrate retina.