Varoqueaux and N. Brose), GABACρ receptor subunit (rabbit polyclonal, 1:500, kindly provided by R. Enz), GABAAα1 receptor subunit (polyclonal guinea-pig, 1:5,000, kindly provided by J.M. Fritschy), GABAAα1 receptor subunit (polyclonal rabbit, 1:2,000, Selleckchem Cisplatin kindly provided by J.M. Fritschy), GABAAα2 receptor subunit (polyclonal guinea-pig, 1:2,000, kindly provided by J.M. Fritschy), and GABAAα3 receptor subunit (polyclonal guinea-pig, 1:3,000, kindly provided by J.M. Fritschy).

Secondary antibodies utilized were either anti-isotypic Alexa Fluor conjugates (1:1,000, Invitrogen, Carlsbad, CA, USA) or DyLight conjugates (1:1,000, Jackson ImmunoResearch, West Grove, PA, USA). Image stacks were acquired on an Olympus FV1000 laser scanning confocal microscope. Fixed tissue was imaged using a 1.35 NA 60× oil immersion objective Bortezomib clinical trial at a voxel size of 0.069, 0.069, 0.3 μm or 0.051, 0.051, 0.3 μm (x, y, z) for images used in quantification. To visualize RBC-amacrine cell contacts at different developmental time points, we crossed the GAD67-GFP and grm6-tdtomato mouse lines and imaged the retinas using a custom-built two-photon microscope and an Olympus 60× water objective. Each optical plane was averaged three to four times. Raw image stacks were processed

using MetaMorph (Molecular Devices, Sunnyvale, CA, USA) and Amira (Visualization Sciences Group, Burlington, MA, USA). For volume estimation, the PKC signal of

RBC boutons was masked in three dimensions using Non-specific serine/threonine protein kinase the “label field” function of Amira. Subsequently, this PKC mask was multiplied with the GAD or GABA receptor channel to isolate signal specifically within the RBC boutons. Next, a constant threshold for image stacks from the same retina (including WT-KO region comparison across the retina), was applied to detect the volume occupied by the signal using the “label voxel” function of Amira. The percent of signal volume as compared to PKC mask volume was estimated using the “tissue statistics” function of Amira. The specificity of GABA receptor antibodies utilized in this study was tested by performing colocalization analysis and assessing the extent of random associations (Soto et al., 2011). In vertical retinal sections, we found 87% of GABAAα3 receptor clusters apposed to the presynaptic marker, VIAAT. Retinas were fixed by eyecup immersion in 2% paraformaldehye/2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 3 hr. The tissue was then washed in buffer, fixed further in 1% osmium tetroxide in cacodylate buffer for 1 hour prior to en bloc staining with 1% uranyl acetate. The tissue was then dehydrated in graded ethanol series, embedded in araldite resin, sectioned, and stained with 5% lead citrate.