Fig 2: 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. Vhold = -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.

Fig 3: tPA-Cer is localized to large MFBs and astrocytes in the CA3 stratum lucidum lamina of the hippocampus. Subcellular colocalization of tPA in stratum lucidum of the hippocampus was investigated using high-resolution confocal microscopy. Images (63×) are representative regions of interest from the stratum lucidum lamina of tPABAC-Cer transgenic mice and are presented as 5-µm z-stacks (?z = 0.5 µm) visualized in orthogonal YZ and XZ slices (A–C) and magnified 3D maximum intensity projections (A’–C’), for each of the respective colocalization markers. For quantification of colocalization using the Manders coefficient (D, E) entire images were concatenated from 5-µm z-stacks (512 × 512 pixels; ?z = 0.5 µm) that were independently acquired two to four times (ZnT3: mice n = 10, lines 863 and 876; EAAT2: mice n = 6–8, lines 863 and 876, respectively; MAP2: mice n = 4, lines 863 and 876). tPA-Cer puncta was found to colocalize with the zinc transporter-3 (ZnT3; red), which has previously been observed exclusively in MFBs, as indicated visually by the white overlay in the orthogonal sections and 3D max projection (Manders M1: 0.704 and 0.649 for lines 863 and 876, respectively; Costes probability p value = 95%; D, E). Partial colocalization was weakly observed in astrocytes visualized with the astrocytic glutamate transporter EAAT2 (orange; B, B’), which is in agreement with the lower quantified colocalization coefficient (Manders M1: 0.183 and 0.198 for lines 863 and 876, respectively; Costes probability p value = 95%; D, E). To confirm the presynaptic localization of tPA to MFBs, sections were also stained for the dendritic marker MAP2, which detects the dendritic thorny excrescences of CA3 pyramidal neurons that MFBs encase. Orthogonal YZ and XZ slices and 3D max projections of tPA-Cer and MAP2 (yellow; C, C’) showed no colocalization (Manders M1: 0.032 and 0.060 for lines 863 and 876, respectively; Costes probability p value = 0.0%; D, E). ZnT3, zinc-transporter 3; EAAT2, excitatory amino acid transporter 2; MAP2, microtubule associated protein 2. Scale bars: 5 µm (A–C) and the units on the scale boxes are given in µm (A’–C’).

Fig 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. Vhold = -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.

Fig 5: Complete blockade of GLT-1 transporters reverses glutamate uptake facilitation at 100 Hz in the ACC. a Representative responses of iGluSnFr signal in the barrel cortex following synaptic stimulation (single stimulation, 10 × 50 Hz and 10 × 100 Hz) during baseline (black traces) and after application of DHK (300 µM, blue traces). b Decay kinetics of extrasynaptic glutamate transients are similarly and significantly slower in the presence of DHK (300 µM) following single pulse stimulation (n = 10, *P < 0.05), 50 Hz stimulation (n = 10, **P < 0.01) and following 100 Hz stimulation (n = 10, N = 3, **P < 0.001). c Same as (a) for the ACC. d Decay kinetics of extrasynaptic glutamate transients following single pulse and 50 Hz stimulation are not affected by the presence of DHK (n = 7, P > 0.05). However, following 100 Hz stimulation the decay kinetics become significantly slower in the presence of DHK (n = 7, N = 3, ***P < 0.0001). Traces normalized to the peak. n = number of slices, N = number of mice. Data are mean ± SEM. Two-way RM ANOVA test