Systemic but not intraocular Epo Gene Transfer Protects the Retina from Light-and Genetic-Induced Degeneration

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Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse

PURPOSE. To test the hypothesis that Nrl Ϫ/Ϫ photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS. The photoreceptor layer of fixed retinal tissue of 4-to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single-and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the awave of the ERG. RESULTS. Outer segments of Nrl Ϫ/Ϫ photoreceptors (ϳ7 m) are shorter than those of wild-type (WT) rods (ϳ25 m) and cones (ϳ15 m); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl Ϫ/Ϫ photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl -/-a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the max of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was ϳ50 ms, more than two times faster than that of rods. CONCLUSIONS. Many morphological, molecular, and electrophysiological features of the Nrl Ϫ/Ϫ photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease. (Invest Ophthalmol Vis Sci

PDE9A is expressed in the inner retina and contributes to the normal shape of the photopic ERG waveform

The ubiquitous second messenger cGMP is synthesized by guanylyl cyclase and hydrolyzed by phosphodiesterase (PDE). cGMP mediates numerous signaling pathways in multiple tissues. In the retina, cGMP regulates signaling in nearly every cell class including photoreceptors, bipolar cells, amacrine cells, and ganglion cells. In order to understand the specific role of cGMP and its regulating enzymes in different cell types, it is first necessary to localize these components and dissect their influence on the circuits. Here we tested the contribution of PDE9A to retinal processing by recording the electroretinograms (ERG) of PDE9A ™/™ (KO) mice and by localizing the enzyme. We found that while the scotopic ERG of KO was the same as that of wild type (WT) in both amplitude and kinetics, the photopic ERG was greatly affected. The greatest effect was on the recovery of the b-wave; the falling phase and the b-wave duration were significantly longer in the KO mice for all photopic stimuli (UV, green, or saturating white flashes). The rising phase was slower in KO than in WT for UV and green stimuli. For certain stimuli, amplitudes of both the a- and b-waves were smaller than in WT. Using Lac-Z expression in KO retinas as a reporter for PDE9A expression pattern, we found that PDE9A is localized to GABA-positive and GABA-negative amacrine cells, and likely also to certain types of ganglion cells. Our results indicate that PDE9A, by controlling the level of cGMP, modulates inhibitory processes within the cone pathway. We speculate that these circuits involve NO/cGMP signaling pathways

The Light Response of ON Bipolar Neurons Requires G o

s by means of metabotropic glutamate receptor 6 (mGluR6) (Nakajima et al., 1993; Nomura et al., 1994). Initially, mGluR6 was thought to trigger a cascade resembling that for phototransduction (Nawy and Jahr, 1990; Shiells and Falk, 1990). However, the main components of the phototransduction cascade (transducin, phosphodiesterase, and a cGMP-gated cation channel) were not found in the ON bipolar dendrites (Wassle et al., 1992; Vardi et al., 1993). Instead, a different trimeric G-protein was found: G o (Vardi et al., 1993; Vardi, 1998). G o has been suggested as the second step in the ON bipolar cascade because of the following: (1) it colocalizes with mGluR6 in the dendrites (Vardi, 1998; Vardi et al., 2000); (2) agonist-bound mGluR6 can activate its # subunit (G# o ) in vitro (Weng et al., 1998); and (3) G# o dialyzed into ON bipolar cells reduces the response to glutamate (Nawy, 1999). However, doubt persists regarding the role of G o because, rather than coupling mGluR6 to the eff

Functionally rodless mice: transgenic models for the investigation of cone function in retinal disease and therapy. Vision Res. 2002 Feb;42(4):401-15. IF: 2.0

Two genetically engineered strains of mice were used to characterize murine cone function electroretinographically, without interference of rod-driven responses: (1) mice with a deletion of the gene for the rod transducin alpha-subunit (transducin alpha-/-), and (2) mice with rod arrestin deleted (arrestin -/-). In the first three months of age, both strains have a normal complement of rods and normal rod structure, but transducin alpha-/- mice have no rod-driven responses to light, while rod-driven activity of arrestin -/- mice can be suppressed by a single intense flash for hours. In response to intense flashes the electroretinograms of these strains of mice showed a readily identifiable, pure-cone a-wave of approximately 10 microV saturating amplitude. A 530 nm background that saturates rod responses of wild type mice was found to desensitize the b-wave responses of mice of both transgenic lines, whether the b-waves were driven by photons captured by M- or UV-cone pigments. The desensitizing effect of the 530 nm background on UV-pigment driven responses provides new evidence in support of the hypothesis of functional co-expression of the M-pigment in cones expressing primarily the UV-pigment

Phospholipase C β4 is involved in modulating the visual response in mice

Expression of G protein-regulated phospholipase C (PLC) β4 in the retina, lateral geniculate nucleus, and superior colliculus implies that PLC β4 may play a role in the mammalian visual process. A mouse line that lacks PLC β4 was generated and the physiological significance of PLC β4 in murine visual function was investigated. Behavioral tests using a shuttle box demonstrated that the mice lacking PLC β4 were impaired in their visual processing abilities, whereas they showed no deficit in their auditory abilities. In addition, the PLC β4-null mice showed 4-fold reduction in the maximal amplitude of the rod a- and b-wave components of their electroretinograms relative to their littermate controls. However, recording from single rod photoreceptors did not reveal any significant differences between the PLC β4-null and wild-type littermates, nor were there any apparent differences in retinas examined with light microscopy. While the behavioral and electroretinographic results indicate that PLC β4 plays a significant role in mammalian visual signal processing, isolated rod recording shows little or no apparent deficit, suggesting that the effect of PLC β4 deficiency on the rod signaling pathway occurs at some stage after the initial phototransduction cascade and may require cell–cell interactions between rods and other retinal cells

Differential function of Gγ13 in rod bipolar and ON cone bipolar cells

Heterotrimeric G-proteins (comprising Gα and Gβγ subunits) are critical for coupling of metabotropic receptors to their downstream effectors. In the retina, glutamate released from photoreceptors in the dark activates metabotropic glutamate receptor 6 (mGluR6) receptors in ON bipolar cells; this leads to activation of Go , closure of transient receptor potential melastatin 1 channels and hyperpolarization of these cells. Go comprises Gαo , Gβ3 and a Gγ. The best Gγ candidate is Gγ13, although functional data to support this are lacking. Thus, we tested Gγ13 function by generating Gng13(-/-) knockout (KO) mice, recording electroretinograms (ERG) and performing immunocytochemical staining. The amplitude of scotopic ERG b-waves in KO mice was lower than in wild-type (WT) mice. Furthermore, in both KO and WT mice, the ERG b-wave decreased with age; this decrease was much more pronounced in KO mice. By contrast, the photopic ERG b-waves in KO mice were hardly affected at any age. In KO mice retinas, immunostaining for Gβ3 and for the GTPase activating proteins RGS7, RGS11, R9AP and Gβ5 decreased significantly in rod bipolar cells but not in ON cone bipolar cells. Staining for Gαo and certain other cascade elements decreased only slightly. Analysis of our ON bipolar cDNA library showed that these cells express mRNAs for Gγ5, Gγ10 and Gγ11. Quantitative RT-PCR of retinal cDNA showed greater values for these transcripts in retinas of KO mice, although the difference was not significant. Our results suggest that Gγ13 contributes to mGluR6 signalling in rod bipolar cells more than in ON cone bipolar cells, and that this contribution includes both coupling the receptor and maintaining a stable localization of the mGluR6-related cascade elements