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Jun Zhang, Jingsheng Tuo, Defen Shen, Wei Li, Chi-Chao Chan; Neurite Outgrowth From Horizontal And Bipolar Cells In Ccl2/Cx3cr1 Deficient Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1765.
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© ARVO (1962-2015); The Authors (2016-present)
Our recent study showed aberrant photoreceptor terminals and a significant decrease of cone synapses in Ccl2-/-/Cx3cr1-/- (DKO) mice that develop focal retinal degeneration mimicking age-related macular degeneration (AMD) (Zhang, ARVO #1436, 2010). To study the responses of horizontal cells and rod bipolar cells, the postsynaptic targets of photoreceptors, to the degeneration of presynaptic photoreceptor terminals, we evaluated these two postsynaptic processes using immunofluorescence (IF) and electron microscopy (EM).
DKO and wide type (WT) mice (at 1, 6 and 12 month) were used for the study. The eyecups were fixed with routine protocols for IF and EM, respectively. IF on retinal sections were performed by incubating overnight in a mixture of antibodies of monoclonal anti-mouse CtBP2 (1:100) with either polyclonal anti-rabbit PKC (1:10000) or polyclonal anti-rabbit calbindin D28 (1:500) at 4°C. Sections were then incubated in a mixture of anti-mouse IgG-FITC (1:100) and anti-rabbit IgG-Cy3 (1:400). IF was visualized with a confocal microscope. For EM, retinal strips were further postfixed in 1% OsO4, dehydrated in alcohol and embedded in resin for 72 h at 60°C. Ultrasections were collected in 200 mesh grids and then counterstained with 5% uranyl acetate and 0.3% lead citrate. Ultrasections were viewed on a JEOL 1010EM.
In WT mice of different ages, both calbindin labeled horizontal processes and PKC labeled rod bipolar dendrites exclusively ramify within the outer plexiform layer (OPL). In DKO mice, however, a few horizontal processes and rod bipolar dendrites extended aberrantly into the outer nuclear layer (ONL) at age of one month. These aberrant processes increased in numbers with aging. Double labeling of WT retina showed close associations of CtBP2 labeled synaptic ribbons with either horizontal processes or rod bipolar dendrites, which were localized in the OPL across all ages. In DKO mice, however, such associations were also found in the ONL, in addition to the OPL, in each age group. EM examination further confirmed that aberant processes received synaptic inputs from photoreceptor terminals.
Both IF and EM evidences demonstrated that horizontal and bipolar processes sprout to form ectopic synapses in the ONL of DKO mice, suggesting a neuronal remodeling in the retinal lesions of the AMD mouse model.
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