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Ivan Anastassov, Weiwei Wang, Felice Dunn; Establishment of convergence at rod bipolar cell dendrites in postnatal mouse retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2976.
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© ARVO (1962-2015); The Authors (2016-present)
Responses to dim stimuli start their efferent journey through the primary rod bipolar pathway where input from multiple rods is pooled into individual rod bipolar cells (RBC) at the first visual synapse. Convergence of rods is important for retinal function and depends on the development of a proper complement of dendritic tips and proteins in RBCs. It is yet unknown how the dendrites of RBCs develop and contact the right number of rods.
Retinas from the Grm6-TdTomato mouse line were taken at P8, 14, 21, 30 and 82. In this line, tdTom is expressed under the Grm6 promoter in individual RBCs, but expression is only observed in a small number of RBCs allowing for easy resolution and identification of individual cells. Grm6-Tom retinas were flat-mounted and stained with antibodies against mGluR6, TRPM1, PKCa, Ribeye and Ctbp2. DiI injections were made in retinas of GustGFP mice where RBCs express GFP. Tissue was imaged with conventional and super-res confocal microscopy. Images were processed with ImageJ, cells were masked with Amira and analysis was performed in Prism and Matlab.
Dendritic tip number increased from P8 (15.88±4.4; mean±sd) to P30 (56.31±3.77) and reached an asymptote at P82 (58.33±10.95) with a saturating exponential fit constant t=21.75 days. Preliminary data from super res microscopy shows a higher dendritic tip number of 77±8.49/RBC at P82. RBC-associated mGluR6 puncta increased with age; at P8 we observed 23.56±3.75 puncta, reaching an asymptote at P82 with 52±8.68 puncta, t=9.79 days. Dendritic tip presence of mGluR6 and TRPM1 was examined at all ages; at P8, less than 50% of identified tips had both proteins present, this number gradually increased to ~75% of tips by P82, t=7.41 days. However, examination of individual protein presence within tips without bias to either protein, revealed that mGluR6 appeared with t=4.04 days compared to t=7.54 days for TRPM1. Furthermore, we encountered tips missing either protein at all examined ages.
RBCs target the appropriate number of rods during early postnatal development, as judged by our fixed tissue data and number of dendritic tips associated with each cell. At eye opening (P14), a scotopic ERG b-wave is present but only ~58% of morphologically identified tips have both mGluR6 and TRPM1. We propose that mGluR6 appears in RBC tips prior to TRPM1, suggesting that each protein is trafficked to tips independently.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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