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R. E. Marc, K. Rapp, J. M. Frederick, S. Karan, W. Baehr; Extreme Remodeling by Cone Bipolar Cells in Retinal Degenerations. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2500.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal remodeling alters bipolar cell (BC) connectivity in retinal degenerations. Previous studies focused on remodeling secondary to rod-specific degenerations. Here we analyzed guanyl cyclase (GC1 and GC2) double knockout (DKO) mice (Baehr et al. Invest Ophthalmol Vis Sci 2006;47: E-Abstract 4318) to explore the effects of primary cone loss/cone dysfunction on cone BCs.
Adult wt and 6 month-old GC-DKO mice were euthanized and the eyes enucleated. Libraries of BCs were generated by 45 min of retinal dissociation in L-15 medium (Invitrogen) + 30 units/ml papain (Worthington). After four L-15 washes, each retina was triturated by pipet into nine 35mm dishes containing 2 ml L-15 and a concanavalin A (Sigma) coated coverslip. After 1 hr, cells were fixed in 4% formaldehyde + 0.1 M phosphate buffer (PB), 3% sucrose and 1 mM Ca (pH 7.4), and stored at 4 deg C. Rod BCs were visualized with anti-PKCalpha IgGs (Sigma) and OFF cone BCs with anti-neurokinin 3 receptor (NK-3R) IgGs (Novus Biologicals). The cells were washed 3 x 10 min in PB, incubated overnight with primary antibodies (1:1000) in PB + 1% Triton X-100 (PBX), washed 3 times with PB, probed with Cy3 conjugated secondary IgGs in PBX for 1 hr, and washed. Coverslips were mounted with DIFTAGS medium (Immunon) and cells captured by Z-stack confocal imaging. Confocal libraries were isosurface rendered with Imaris 5.0.1 (Bitplane AG).
Imaging of dissociated wt retinas revealed diverse BC morphs, with numerous, classic PKCalpha+ rod BCs (5-6 um flask shaped somas, 20-25 um axons, bushy dendrites) and NK3-R+ OFF cone BCs (spherical 7 um somas, 12-17 um axons, sparse dendrites). Each retina produced hundreds of each class. GC-DKO retinas produced few normal BCs and most of those were PKCalpha+, consistent with the presence of 2-3 layers of rod nuclei at sacrifice. However, virtually no normal cone BCs were found in the mutant retinas. Most NK3-R+ cells were spherical, larger than normal, and lacked distinctive dendrites. Many possessed thin axons and several NK3-R+ cells were successfully retrieved with multiple axons.
Previous work has documented loss of BC dendrites in rod-selective models of degeneration. As cone transduction is non-functional in the GC-DKO mouse, remodeling of OFF cone BCs is likely to be linked directly to impaired cone synaptic signaling. Cone BCs lose their dendrites and exhibit evidence of supernumerary axon formation, consistent with the formation of microneuromas in phase 3 remodeling. This argues that retinal degenerations resemble temporal lobe epilepsy where supernumerary axon sprouting is a prime defect.
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