Ectopic synaptic ribbons in dendrites of mouse retinal ON- and OFF-bipolar cells

The ectopic distribution of synaptic ribbons in dendrites of mouse retinal bipolar cells was examined by using genetic ablation of metabotropic glutamate receptor subtype 6 (mGluR6), electron microscopy, and immunocytochemistry. Ectopic ribbons were observed in dendrites of rod and ON-cone bipolar cells in the mGluR6-deficient mouse but not in those of wild-type mice. The number of rod spherules facing the ectopic ribbons in mGluR6-deficient rod bipolar dendrites increased gradually during early growth and reached a plateau level of about 20% at 12 weeks. These ectopic ribbons were immunopositive for RIBEYE, a ribbon-specific protein, but the associated vesicles were immunonegative for synaptophysin, a synaptic-vesicle-specific protein. The presence of ectopic ribbons was correlated with an increase in the roundness of the invaginating dendrites of the rod bipolar cells. We further confirmed ectopic ribbons in dendrites of OFF-cone bipolar cells in wild-type retinas. Of the four types of OFF-cone bipolar cells (T1–T4), only the T2-type, which had a greater number of synaptic ribbons at the axon terminal and a thicker axon cylinder than the other types, had ectopic ribbons. Light-adapted experiments revealed that, in wild-type mice under enhanced-light adaptation (considered similar to the mGluR6-deficient state), the roundness in the invaginating dendrites and axon terminals of rod bipolar cells increased, but no ectopic ribbons were detected. Based on these findings and known mechanisms for neurotransmitter release and protein trafficking, the possible mechanisms underlying the ectopic ribbons are discussed on the basis of intracellular transport for the replenishment of synaptic proteins

A Battery of Cell- and Structure-specific Markers for the Adult Porcine Retina

The pig is becoming an increasingly used non-primate model in experimental studies of human retinal diseases and disorders. The anatomy, size, and vasculature of the porcine eye and retina closely resemble their human counterparts, which allows for application of standard instrumentation and diagnostics used in the clinic. Despite many reports that demonstrate immunohistochemistry as a useful method for exploring neuropathological changes in the mammalian central nervous system, including the pig, the porcine retina has been sparsely described. Hence, to facilitate further immunohistochemical analysis of the porcine retina, we report on the successful use of a battery of antibodies for staining of paraformaldehyde-fixed cryosectioned retina. The following antibodies were evaluated for neuronal cells and structures: recoverin (cones and rods), Rho4D2 (rods), transducin-γ (cones), ROM-1 (photoreceptor outer segments), calbindin (horizontal cells), PKC-α (bipolar cells), parvalbumin (amacrine and displaced amacrine cells), and NeuN (ganglion cells and displaced amacrines). For detecting synaptic connections in fiber layers, we used an antibody against synaptobrevin. For detecting retinal pigment epithelium, we studied antibodies against cytokeratin and RPE65, respectively. The glial cell markers used were bFGF (Müller cells and displaced amacrine cells), GFAP (Müller cells and astrocytes), and vimentin (Müller cells). Each staining effect was evaluated with regard to its specificity, sensitivity, and reproducibility in the identification of individual cells, specific cell structures, and fiber layers, respectively. The markers parvalbumin and ROM-1 were tested here for the first time for the porcine retina. All antibodies tested resulted in specific staining of high quality. In conclusion, all immunohistochemical protocols presented here will be applicable in fixed, cryosectioned pig retina. (J Histochem Cytochem 58:377–389, 2010

Union makes strength: a worldwide collaborative genetic and clinical study to provide a comprehensive survey of RD3 mutations and delineate the associated phenotype

Contains fulltext : 117915.pdf (publisher's version ) (Open Access)Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration (RD), and the most common cause of incurable blindness diagnosed in children. It is occasionally the presenting symptom of multisystemic ciliopathies which diagnosis will require a specific care of patients. Nineteen LCA genes are currently identified and three of them account for both non-syndromic and syndromic forms of the disease. RD3 (LCA12) was implicated as a LCA gene based on the identification of homozygous truncating mutations in two LCA families despite the screening of large cohorts of patients. Here we provide a comprehensive survey of RD3 mutations and of their clinical expression through the screening of a cohort of 852 patients originating worldwide affected with LCA or early-onset and severe RD. We identified three RD3 mutations in seven unrelated consanguineous LCA families - i.e., a 2 bp deletion and two nonsense mutations - predicted to cause complete loss of function. Five families originating from the Southern Shores of the Mediterranean segregated a similar mutation (c.112C>T, p.R38*) suggesting that this change may have resulted from an ancient founder effect. Considering the low frequency of RD3 carriers, the recurrence risk for LCA in non-consanguineous unions is negligible for both heterozygote and homozygote RD3 individuals. The LCA12 phenotype in our patients is highly similar to those of patients with mutant photoreceptor-specific guanylate cyclase (GUCY2D/LCA1). This observation is consistent with the report of the role of RD3 in trafficking of GUCYs and gives further support to a common mechanism of photoreceptor degeneration in LCA12 and LCA1, i.e., inability to increase cytoplasmic cGMP concentration in outer segments and thus to recover the dark-state. Similar to LCA1, LCA12 patients have no extraocular symptoms despite complete inactivation of both RD3 alleles, supporting the view that extraocular investigations in LCA infants with RD3 mutations should be avoided