Glutamate Transporters EAAT2 and EAAT5 Differentially Shape Synaptic Transmission from Rod Bipolar Cell Terminals

Co-expression of EAAT2 and EAAT5 in mouse retinal RBs is confirmed by scRT-PCR analysis. A, scRT-PCR analyses of EAAT2 and EAAT5 mRNA expression in a single RB cell from a P17 mouse retina and the other RB from an adult mouse retina. Co-expression of EAAT2 and EAAT5 could be seen in both RBs. A ladder with DNA fragments between 100 and 1000 bp is shown on the left. B, The percentages of EAAT2 and EAAT5 expression in individual RBs from both P17 and adult mice. See also Table 2. C, Schematic diagrams illustrating the molecular heterogeneity for EAAT2 and EAAT5 expression in individual RBs from P17 and adult mice. The percentages of either subtype and a combination of EAAT2 and EAAT5 are shown, and the exact cell numbers are given in parentheses. See also Table 3.

EAAT2 is located near ribbons in the axon terminals of RBs. A, Confocal images showing immunofluorescence triple labeling of EAAT2 (green), RIBEYE (magenta), and PKCα (blue) in a frozen mouse retinal section. EAAT2 was expressed strongly in the OPL, and moderately in the INL, IPL, and GCL. Note that, in the INL, EAAT2 was expressed in the somata of some cone bipolar cells but not RB cells labeled by PKCα (asterisks). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 20 μm. B, Magnification of the images in the dashed line frames of A. EAAT2 was expressed in RB axon terminals at sites near ribbons (arrows). Scale bar: 2.5 μm.

EAAT2 regulates signal transmission at RB→AII ribbon synapses. A, A schematic diagram showing the optogenetic study of neurotransmission between RBs and AII amacrine cells. ChR2 was expressed predominantly in RBs by cre-dependent recombination in adult Pcp2-cre::Ai32 mouse retinas. With all the synaptic transmission between photoreceptors and BCs is blocked pharmacologically, brief flashes of 470-nm LED light can directly activate ChR2+ RBs and induce postsynaptic responses in AIIs, which mainly reflect neurotransmitter release from RBs. The electrical coupling between ChR2+ ON cone bipolar cells and AIIs is negligible under this experimental condition (Liang et al., 2021). R, rod. B, The EPSCs recorded in AIIs, which were evoked by 470-nm LED light stimulation, were enhanced by 200 μm DHK, a selective EAAT2 blocker. V_hold = −80 mV. C, The ChR2-evoked EPSCs were reduced by 10 μm GT949, a positive allosteric modulator of EAAT2. D, E, Summary data showing the effects of GT949 (n = 10) and DHK (n = 7) on the peak amplitude of AII EPSCs. F, DHK reduced the time to peak of EPSCs slightly, but not significantly (n = 7, p = 0.0531). G, DHK reduced the rise time of EPSCs (n = 7). H, DHK did not change the decay time (tau) of EPSCs (n = 7). I–L, DHK did not affect the frequency, amplitude, rise time, or tau of mEPSCs recorded in AIIs (n = 7). mEPSCs, miniature EPSCs. M, The voltage changes in ChR2+ RBs, which were evoked by brief flashes of 470-nm LED light, were increased by 200 μm DHK. N, DHK increased the voltage changes in RBs evoked by light flashes (n = 6). O, DHK did not influence the resting membrane potentials of RBs (n = 6). The data were represented as mean ± SEM. Wilcoxon signed-rank test or Student’s t test was used where appropriate. *p < 0.05, **p < 0.01; ns, not significantly different. See also Table 4.

Pharmacological blockade of all EAATs has a significant effect on signal transmission at RB→AII synapses. A, The EPSCs recorded in AII amacrine cells, which were evoked by activating ChR2+ RBs with 470-nm LED light stimulation, were enhanced by 50 μm TBOA, a nonselective blocker of all EAATs. V_hold = −80 mV. R, rod; RB, rod bipolar cell; ChR2, channelrhodopsin-2. B, TBOA increased the peak amplitude of ChR2-evoked EPSCs (n = 7). C, The relative effects of TBOA on the peak amplitude and current integral of AII EPSCs (n = 7). The peak amplitudes/integrals were normalized to the peak amplitude/integral under control condition in each cell before averaging across cells. D, Comparison of the relative effects of DHK (n = 7), a selective EAAT2 blocker, and TBOA (n = 7) on the peak amplitude of AII EPSCs. E–G, TBOA changed the time to peak, rise time and tau of EPSCs (n = 7). H–K, TBOA did not influence the frequency, amplitude, or rise time of mEPSCs recorded in AIIs while increasing the tau slightly (n = 7). mEPSCs, miniature EPSCs. L, The voltage changes in ChR2+ RBs, which were evoked by brief flashes of 470-nm LED light, were increased by 50 μm TBOA. Note that, in the presence of TBOA, a large, long-lasting AHP (arrow) could be recorded in each RB following the light-evoked depolarization. M, TBOA increased the initial voltage changes in RBs evoked by light flashes (n = 5). N, Comparison of the relative effects of DHK (n = 6) and TBOA (n = 5) on light-evoked voltage changes in RBs. The data were represented as mean ± SEM. Wilcoxon signed-rank test or Student’s t test was used where appropriate. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significantly different. See also Table 5.

EAAT1 in Müller cells does not influence neurotransmission at RB→AII synapses. A, The EPSCs recorded in AII amacrine cells, which were evoked by activating ChR2+ RBs with 470-nm LED light stimulation, were not affected by 50 μm UCPH101, a selective blocker of EAAT1 expressed exclusively in Müller cells. V_hold = −80 mV. R, rod; RB, rod bipolar cell; ChR2, channelrhodopsin-2. B, C, UCPH101 did not change the peak amplitude or time to peak of ChR2-evoked EPSCs (n = 8). D–G, UCPH101 did not influence the frequency, amplitude, rise time, or tau of mEPSCs recorded in AIIs (n = 8). mEPSCs, miniature EPSCs. The data were represented as mean ± SEM. Wilcoxon signed-rank test or Student’s t test was used where appropriate. ns, not significantly different. See also Table 6.

EAAT5 plays a predominant role in regulating neurotransmission at RB→AII synapses. A, The EPSCs recorded in AII amacrine cells, which were evoked by 470-nm LED light stimulation of ChR2-expressing RBs, were increased slightly by co-application of DHK (200 μm) and UCPH101 (50 μm; n = 8), selective blockers of EAAT2 and EAAT1, respectively, and then enhanced more strongly by application of TBOA (50 μm; n = 3), a nonselective blocker of all EAATs. V_hold = −80 mV. R, rod; RB, rod bipolar cell; ChR2, channelrhodopsin-2. B, Magnification of the traces shown in A.