Using electron microscopy, immunohistochemistry,

and electrophysiology, we assessed whether perturbed neurotransmission affects the structural formation and maintenance of GABA receptor subtypes on RBC axon terminals. Because RBC terminals also receive some glycinergic inhibition (Eggers et al., 2007, Wässle et al., 2009), we further analyzed whether an upregulation of glycine receptors occurred to compensate for the reduction in GABAergic transmission. Furthermore, we asked whether the loss of GABAergic inhibition causes alterations to RBC output beyond that expected solely from disinhibition in the GABA-deficient circuit. Because GABA release from A17 amacrine cells normally requires RBC drive due to the reciprocal synaptic arrangement between these two cell types, we determined whether lack of GABAergic transmission also caused developmental changes in the glutamatergic synapses of A17 amacrine cells. GABAergic inhibition onto axon terminals buy MK-8776 of RBCs is mediated by a variety of amacrine cells, including A17 cells that provide reciprocal feedback inhibition (Figure 1A). GABA is synthesized in amacrine cells by two isoforms of glutamate decarboxylase, GAD67 and GAD65 (Haverkamp and Wässle, 2000; Vardi and Auerbach, 1995). We found that in the sublamina of the inner

plexiform layer (IPL) where RBC axonal terminals stratify (sublamina 5, ON-layer), GAD67 immunoreactivity was more abundant compared to GAD65 (Figure 1B). Quantification buy Ceritinib of the immunolabeling (see

Experimental Procedures) showed that dendritic processes positive for GAD67 exhibited greater volume overlap with protein kinase C (PKC) immunoreactive RBC axonal boutons, compared to GAD65 containing processes (Figure 1C), suggesting that GAD67-positive amacrine cell processes provide the majority of GABAergic inhibition onto RBC axon terminals. GPX6 To visualize amacrine cell contacts onto RBC terminals during development, we utilized the GAD67-GFP transgenic line ( Chattopadhyaya et al., 2004), where a fraction of GAD67-positive amacrine cells ( Figure S1A available online) express GFP. Amacrine cells typically synapse onto bipolar cell axons at enlarged varicosities ( Dowling and Boycott, 1966). Using the GAD67-GFP transgenic line, we visualized large varicosities of GFP-labeled amacrine processes contacting RBC boutons at sites immunopositive for GAD67, but not GAD65 ( Figure S1B). We further determined when GABAergic contacts develop on RBCs, labeled by expression of the fluorescent protein tdtomato under the grm6 promoter ( Figure 1D). Appositions between GAD67-GFP positive processes and RBC axonal boutons were already apparent at postnatal day (P) 10 ( Figure 1D), several days after axonal differentiation in the bipolar cells ( Morgan et al., 2006). At P15, large varicosities were clearly present at sites of contact between amacrine cell dendrites and RBC boutons, which remained evident at P21.