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Jun Zhang, Jeffrey Diamond; Synaptic localization of AMPAR subunits in A17 amacrine cells at the rod bipolar cell ribbon synapse. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1479.
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© ARVO (1962-2015); The Authors (2016-present)
Physiological studies have suggested that calcium-permeable (GluA2-lacking) AMPA receptors (AMPARs) trigger GABA release from A17 amacrine cells at ribbon synapses in rod bipolar cells (RBCs). The goal of the present study is to determine the precise location of AMPARs on A17 dendrites at RBC ribbon synapse using postembedding immunogold EM.
Immunogold labeling was performed on retinal tissue taken from P18 Sprague-Dawley rats. ~70 nm-thick ultrasections were collected on nickel grids. Grids were incubated overnight with primary antibodies against AMAPR subunits (GluA1, 1:100; GluA2, 1:100, and GluA3, 1:100, all from Millipore) and then with 10 nm gold-conjugated secondary antibodies in TBS-Triton. After being counterstained with 5% uranyl acetate and 0.3% lead citrate, grids were viewed on a JEOL1200 EM and images were digitalized. A total of 27 RBC dyads were analyzed. The distance between the centers of the each gold particle and of the RBC ribbon synapse was measured. Two-tailed Student’s t-test was used for statistical analysis.
Immunogold labeling for GluA3 was found both synaptically and perisynaptically on A17 dendrites at RBC ribbon synapse. 50% (79) of the particles were located within 200nm of the center of the ribbon synapse. The remaining particles were visible along the dendritic plasma membrane: 40% were located 200 to 400 nm from the center of the ribbon synapse; 10% were located more than 500 nm away. Neither GluA1 nor GluA2 immunoreactivity was evident on A17 dendrites.
A17 dendrites do not appear to express GluA2, consistent with previous physiological results. The evidence for GluA3 subunit localization suggests that GABA release from A17 cells in the rat retina most likely depend on GluA3-mediated calcium signal.
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