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Abstract The retina is the light sensing tissue of the eye which contains multiple layers of cells required for the detection and transmission of a visual signal. Loss of the light-sensing photoreceptors leads to defects in visual function and blindness. Previously, we found that mosaic deletion of Kcnj13, and subsequent loss of the potassium channel Kir7.1, in mice leads to photoreceptor degeneration and recapitulates the human retinal disease phenotype (Zhong et al., 2015). Kcnj13 expression in the retinal pigment epithelium (RPE) is essential for normal retinal electrophysiology, function, and survival. Mice with homozygous loss of Kcnj13 die at postnatal day 1 (P1), requiring a tissue-specific approach to study retinal degeneration phenotypes in adult mice. We used the CRISPR-Cas9 system to generate a floxed, conditional loss-of-function (cKO) Kcnj13 flox allele to study the pathogenesis of Kcnj13 deficiency in the retina. To investigate if the Kcnj13 is required in the RPE for photoreceptor function and survival, we used Best1-cre , which is specifically expressed in the RPE. We observed complete loss of Kcnj13 expression in Cre-positive RPE cells. Furthermore, our findings show that widespread loss of Kcnj13 in the RPE leads to severe and progressive thinning of the outer nuclear layer and a reduced response to light. Finally, to detect Best1-cre expression in the RPE of live animals without sacrificing the animal for histology, we generated a Cre-reporter-containing Kcnj13 cKO mouse line (cKOR: Kcnj13 flox/flox ; Best1-cre; Ai9) which can be rapidly screened using retinal fluorescence microscopy. These findings provide new tools for studying the roles of Kcnj13 in retinal homeostasis. Highlights • RPE-specific loss of Kcnj13 results in photoreceptor dysfunction and death. • Visual defects in RPE-specific loss of Kcnj13 animals coincide with progressive retinal degeneration. • RPE-specific Cre expression can be evaluated in living Kcnj13 cKOR mice using retinal fluorescence microscopy. [ABSTRACT FROM AUTHOR]

Abstract The aim of this study was to compare the quality of electroretinogram (ERG) recordings using a custom built active electrode with attached amplifier versus a standard (passive) ERG electrode. Scotopic and photopic ERG responses were recorded from five adult albino rabbits using a custom built active electrode on one eye and a passive electrode on the other. For the active electrode, the ERG-jet electrode (Universo S.A., La Chaux-De-Fonds, Switzerland) was used as the transducer with the cable cut short and soldered directly to the input of a customized amplifier. The passive electrode was a standard ERG jet electrode. The signal to noise ratio and reproducibility of ERGs were compared. The noise was significantly lower in the active electrode compared to the passive electrode (p = 0.009) resulting in signals being recorded at lower stimulation strengths with the active electrode. The scotopic a-wave was significantly larger in the active electrode at all supra-threshold stimulation intensities (p < 0.05) and the scotopic b-wave amplitudes were also higher in the active electrode at all supra-threshold stimulation intensities but was only statistically significant between −3.25 and −1 log cd.s.m−2 (p < 0.05). The photopic a- and b-wave amplitudes were also higher in the active electrode and statistically significant between −0.75 and 0.48 log cd.s.m−2 for the a-wave and −1.25 to −1 log cd.s.m−2 for the b-wave (p < 0.05). The intra-observer repeatability, inter-sessions reproducibility and reliability of the signals were better in the active electrode as evidenced by lower coefficient of variation (CV) and coefficient of repeatability (CR) with high intra-class correlation coefficient (ICC) of the a- and b-wave parameters of the active electrode. These findings suggest that the custom built active ERG electrode produces less noise than the passive electrode, allowing responses to be recorded at lower stimulation strengths. It produces greater signal amplitudes and improved reproducibility and is therefore a better device for investigating retinal function. Highlights • The use of an active electrode effectively eliminates line noise in ERG recordings. • The improved signal to noise ratio allows signals to be recorded at lower stimulation strengths. • ERGs have improved reproducibility with an active electrode design. • Higher amplitude responses can be seen in an active electrode due to reduced load on the transducer. [ABSTRACT FROM AUTHOR]

The mammalian retina consists of multiple cell layers including photoreceptor cells, which are light sensing neurons that play essential functions in the visual process. Previously, we identified mutations in SPATA7, encoding spermatogenesis associated protein 7, in families with Leber Congenital Amaurosis (LCA) and juvenile Retinitis Pigmentosa (RP), and showed that Spata7 null mice recapitulate the human disease phenotype of retinal degeneration. SPATA7 is expressed in the connecting cilium of photoreceptor (PR) cells in the mouse retina, as well as in retinal pigment epithelium (RPE) cells, but the functional role of Spata7 in the RPE remains unknown. To investigate whether Spata7 is required in PRs, the RPE, or both, we conditionally knocked out Spata7 in photoreceptors and RPE cells using Crx-Cre and Best1-Cr e transgenic mouse lines, respectively. In Spata7 photoreceptor-specific conditional (cKO) mice, both rod and cone photoreceptor dysfunction and degeneration is observed, characterized by progressive thinning of the outer nuclear layer and reduced response to light; however, RPE-specific deletion of Spata7 does not impair retinal function or cell survival. Furthermore, our findings show that both Rhodopsin and RPGRIP1 are mislocalized in the Spata7 Flox/- ; Crx-Cre cKO mice, suggesting that loss of Spata7 in photoreceptors alone can result in altered trafficking of these proteins in the connecting cilium. Together, our findings suggest that loss of Spata7 in photoreceptors alone is sufficient to cause photoreceptor degeneration, but its function in the RPE is not required for photoreceptor survival; therefore, loss of Spata7 in photoreceptors alters both rod and cone function and survival, consistent with the clinical phenotypes observed in LCA and RP patients with mutations in SPATA7 . [ABSTRACT FROM AUTHOR]

Abstract: Homocysteine is an amino acid required for the metabolism of methionine. Excess homocysteine is implicated in cardiovascular and neurological disease and new data suggest a role in various retinopathies. Mice lacking cystathionine-beta-synthase (cbs −/−) have an excess of retinal homocysteine and develop anatomical abnormalities in multiple retinal layers, including photoreceptors and ganglion cells; heterozygous (cbs +/−) mice demonstrate ganglion cell loss and mitochondrial abnormalities in the optic nerve. The purpose of the present study was to determine whether elevated homocysteine, due to absent or diminished cbs, alters visual function. We examined cbs −/− (3 weeks) and cbs +/− mice (5, 10, 15, 30 weeks) and results were compared to those obtained from wild type (WT) littermates. Conventional dark- and light-adapted ERGs were recorded, along with dc-ERG to assess retinal pigment epithelial (RPE) function. The visual evoked potential (VEP) was used to assess transmission to the visual cortex. The amplitudes of the major ERG components were reduced in cbs −/− mice at age 3 weeks and VEPs were delayed markedly. These findings are consistent with the early retinal disruption observed anatomically in these mice. In comparison, at 3 weeks of age, responses of cbs +/− mice did not differ significantly from those of WT mice. Functional abnormalities were not observed in cbs +/− mice until 15 weeks of age, at which time amplitude reductions were noted for the ERG a- and b-wave and the light peak component, but not for other components generated by the RPE. VEP implicit times were delayed in cbs +/− mice at 15 and 30 weeks, while VEP amplitudes were unaffected. The later onset of functional defects in cbs +/− mice is consistent with a slow loss of ganglion cells reported previously in the heterozygous mutant. Light peak abnormalities indicate that RPE function is also compromised in older cbs +/− mice. The data suggest that severe elevations of homocysteine are associated with marked alterations of retinal function while modest homocysteine elevation is reflected in milder and delayed alterations of retinal function. The work lays the foundation to explore the role of homocysteine in retinal diseases such as glaucoma and optic neuropathy. [Copyright &y& Elsevier]

Abstract: In recent years, retinal research has benefited from major advances in optical imaging approaches. Investigations of the structural and functional organization of the vertebrate retina using live preparations have been facilitated by improvements in cell labeling methods, and by microscopy techniques that permit high-resolution of cells in vitro and in vivo. In particular, the generation of transgenic animals with fluorescently labeled retinal cells has permitted real-time visualization of cell generation, migration, differentiation and growth in the developing retina. Neuronal activity can also be examined by optical imaging using activity reporters directed to specific retinal cell types. Optical techniques such as multiphoton microscopy and total internal reflection fluorescence microscopy (TIRFM) have helped unravel the physiological properties and function of retinal cells. Here, we focus on the latest cell labeling methods that have proven highly useful in many aspects of retinal research. We also highlight several examples of how newly developed imaging technology itself has facilitated investigations that have advanced our understanding of retinal circuits and their development. [Copyright &y& Elsevier]