The mean charge of AMPA-evoked currents was not different in wild-type and grm6-TeNT RBCs at both P11–P13 and P30 ( Figure S4). To distinguish GABAA and GABAC components of the evoked response, AMPA puffs were repeated in the presence of (1,2,5,6-tetrahydropyridine-4yl) methyphospinic
acid (TPMPA) (GABAC receptor antagonist) or SR95531 (GABAA receptor antagonist) ( Figure 3C). Quantification of mean charge and amplitude (data not shown) of the evoked response (total, GABAA, or GABAC) showed no significant differences between RBCs in wild-type and grm6-TeNT retinas at P11–P13 ( Figure 3D) and at P30 ( Figure 3E). Together, these results suggest that functional GABAergic synapses are formed normally and maintained even when the bipolar cells fail to transmit effectively to amacrine cells. To assess the functional Roxadustat consequences of reduced GABA synthesis on the formation and maintenance of inhibitory synapses onto RBC axon terminals, we performed whole-cell recordings of these neurons in the GAD1KO retina. As expected, spontaneous inhibitory postsynaptic currents
(sIPSCs) were rare in GAD1KO at both ages examined ( Figures 4A and 4B). This indicates that deletion of GAD67 was not accompanied by a compensatory upregulation of the other GABA synthesizing enzyme, GAD65. Because synaptic release of GABA in these mutants is greatly impaired, we puffed GABA onto GAD1KO RBC axon terminals and recorded the evoked chloride currents in order to assess I-BET151 order whether any postsynaptic changes occurred in GAD1KO RBCs. Interestingly, RBC responses to GABA application in GAD1KO were unchanged at P11–P13 but were dramatically reduced at P30 (example recordings in Figure 4C). Both mean amplitude and charge of the evoked
responses decreased by P30 ( Figure 4D). Moreover, the rise time was slower for evoked responses in P30 GAD1KO RBCs, whereas their decay time was faster, Dichloromethane dehalogenase as compared to control ( Figure 4E). Thus, GABAergic transmission plays an important role in the maintenance, although not in the initial formation, of functional GABAergic synapses on RBC axon terminals. In wild-type animals, RBCs receive little glycinergic input (Eggers et al., 2007). Indeed, immunolabeling for the α1 or α2 subunit of the glycine receptor (Ivanova and Müller, 2006; Wässle et al., 2009) showed little glycine receptor expression on RBC axon terminals (Figures S5A and S5B). However, a severe reduction of GABAergic transmission in GAD1KO may be compensated for by an upregulation of glycine receptor-driven input onto RBC axon terminals. We investigated this possibility by quantifying the expression of glycine receptors containing α1 or α2 subunits on RBC axon terminals in GAD1KO retina. But, we did not find any upregulation of these glycine receptor subtypes in the RBC terminals of GAD1KO ( Figure S6).