Niklaus S , Cadetti L , Vom Berg-Maurer CM , Lehnherr A , Hotz AL , Forster IC , Gesemann M , Neuhauss SCF .

	Figure 1. Transcript expression of excitatory amino acid transporter 2 (eaat2) paralogs. A, B, eaat2a mRNA is strongly expressed in the inner nuclear layer (INL) in the retina, in both 5-d postfertilization (5-dpf) larvae (A) and adult retina (B). Additionally, extremely low transcript levels can be found in photoreceptors (B). C, D, mRNA of eaat2b is expressed in photoreceptors and weakly in the INL throughout different developmental stages (C, in 5-dpf larvae; D, in adult retinal sections). Small inset in C shows eaat2b in situ staining in an eye of a whole-mount larva that has been only shortly stained, to better visualize expression in the INL. Scale bar in A is 100 µm; also applies to C. Scale bar in B corresponds to 50 µm; also applies to D.
	Figure 2. Protein expression of EAAT2 paralogs. A–F, Double immunostaining of EAAT2a (green) and glutamine synthetase (magenta) in adult (A) and larval (5 dpf; D) retinal sections confirm expression of EAAT2a in Müller glia cells. Separated channels are shown in B (adult), E (5 dpf; EAAT2a, green channel only), C (adult), and F (5 dpf; glutamine synthetase, magenta channel only). Scale bar in A is 30 µm; also applies to B and C. Scale bar in D is 50 µm; also applies to E and F. G–N, EAAT2b protein is expressed in a dotted manner in the outer plexiform layer (OPL) in all cone pedicles, but it is not expressed in rods. EAAT2b antibody staining (magenta) on adult retinal sections stained with Zpr-1 (red-green double cones, G) and on retinal sections of zebrafish expressing GFP in blue cones (H), UV cones (I), and rods (J) confirms that EAAT2b is cone specific and is spared from rod spherules. K–M show zoom-ins of the cone pedicles expressing EAAT2b (magenta) in red-green double cones (K), blue cones (L), and UV cones (M). N shows larval (5 dpf) expression of EAAT2b in magenta together with a nuclear counterstain (DAPI, blue). Scale bars in G–J are 7 µm. Scale bars in K–M are 2 µm. Scale bar in N is 30 µm.
	Figure 3. Confirmation of knockdown. A–C, Immunostaining of EAAT2a on WT (A) and EAAT2a morphant [B, 1.3 ng EAAT2a morpholino (MO) 1; C, 1.8 ng EAAT2a MO 2] retinal sections (5 dpf). D, Box-and-whisker plot of analysis of fluorescence of WT, EAAT2a MO 1, and EAAT2a MO 2 injected animals stained with anti-EAAT2a antibody. Statistical analysis reveals a highly significant (p < 0.001) reduction of fluorescence for both MOs. E–G, Retinal sections of WT (E) and EAAT2b morphant (F, 1.8 ng EAAT2b MO 1; G, 9 ng EAAT2b MO 2) larvae stained with anti-EAAT2b antibody. H, Fluorescence was measured in the OPL, and background fluorescence (taken from area in INL) was subtracted. Fluorescence of WT and morphant immunostaining is plotted in a box-and-whisker plot and shows a significant (p < 0.01) and slightly significant (p < 0.05) decrease in fluorescence in animals injected with 1.8 ng EAAT2b MO 1 and 9 ng EAAT2b MO 2, respectively. EAAT2a WT, n = 6; EAAT2a MO 1, n = 8; EAAT2a MO 2, n = 8; EAAT2b WT, n = 10; EAAT2b MO 1, n = 10; EAAT2b MO 2, n = 10. Scale bar in A is 30 μm; also applies to B and C. Scale bar in E is 10 μm; also applies to F and G.
	Figure 4. Retinal histology of EAAT2 morphant zebrafish larvae. Histologic analysis of retinal sections of WT, EAAT2a, and EAAT2b morphant zebrafish larvae (5 dpf) stained with Richardson–Romeis (A–C and G–I). Immunostaining of glutamine synthetase (green) labeling Müller glia cells counterstained with Bodipy (magenta; D–F) of WT and EAAT2a morphant (E, EAAT2a MO 1; F, EAAT2a MO 2) 5-dpf retinal sections. Anti–Zpr-1 immunostaining (labeling red and green cones, shown in green) on WT (J) and EAAT2b morphant (K, EAAT2b MO 1; L, EAAT2b MO 2) retinal sections counterstained with Bodipy (magenta). Knockdown of neither EAAT2a (B, EAAT2a MO 1; C, EAAT2a MO 2) nor EAAT2b (H, EAAT2b MO 1; I, EAAT2b MO 2) causes any defect in retinal lamination. Thickness of the retina was assessed on WT and morphant larvae and did not reveal any significant difference in the thickness of the retina in either EAAT2a or EAAT2b morphants (M, O), yielding p values of 0.997 and 0.935 for EAAT2a MO 1 and EAAT2a MO 2, respectively, and 0.658 and 0.922 for EAAT2b MO 1 and EAAT2b MO 2 (all in comparison to WT). Moreover, knockdown of EAAT2a does not significantly influence Müller glia cell length (N), nor does the loss of EAAT2b result in cone length alteration (P). Statistical analysis of the cell length yielded p values of 0.969 and 0.989 for EAAT2a MO 1 and EAAT2a MO 2, respectively, and 0.911 and 0.631 for EAAT2b MO 1 and EAAT2b MO 2 (in comparison to WT). All scale bars are 50 µm. Scale bar in A also applies to B and C; scale bar in D also applies to E and F; scale bar in G also applies to H and I; and scale bar in J also applies to K and L.
	Figure 5. Box-and-whisker plots of ERG b-wave amplitudes of EAAT2a and EAAT2b morphant zebrafish larvae and representative ERG traces. A, Knockdown of EAAT2a results in a highly significant (p < 0.001) reduction of the ERG b-wave amplitude in comparison to both WT and control injected animals throughout all light intensities (log-4 to log0). For EAAT2a MO 2, we could demonstrate a dose dependence, resulting in a highly significant difference (p < 0.001) between the low dose (1.8 ng) and the high dose (3.6 ng) for the bright light intensities (log-1 and log0) and a significant difference (p < 0.01) for the medium light intensities (log-3 and log-2). WT, n = 33; control MO, n = 25; EAAT2a MO 1, n = 23; 1.8 ng EAAT2a MO 2, n = 27; 3.6 ng EAAT2a MO 2, n = 24. B, Knockdown of EAAT2b only mildly interferes with the ERG b-wave. There is an overall tendency in EAAT2b-depleted animals to have a slightly reduced ERG b-wave amplitude. This results in a slight statistical significance (p < 0.05) between WT and EAAT2b morphant (MO 1) at log-4 and a highly significant (p < 0.001) reduction at log-3 and log-2. Further, there is a slightly significant (p < 0.05) reduction between EAAT2b morphants (MO 1) and control morphants for log-2. When using EAAT2b MO 2, we obtained a significant (p < 0.01) reduction of the ERG b-wave amplitude in comparison to control morphants at log0 and in comparison to WT at log-2. WT, n = 39; control MO, n = 11; EAAT2b MO 1, n = 39; EAAT2b MO 2, n = 16. C, The function of EAAT2b could be demonstrated by double knockdown of both EAAT2 paralogs. Under such conditions, when glutamate uptake by Müller glia cells was impaired, we could show an even further reduction of the ERG b-wave amplitude in the double morphants in comparison to EAAT2a morphant larvae [slightly significant (p < 0.05) at log0, significant (p < 0.01) at log-1, and highly significant (p < 0.001) at log-3 and log-2]. EAAT2a MO 1, n = 23; EAAT2b MO 2, n = 16; double MO, n = 32. D–F, Representative ERG traces of control MO injected larvae (D), EAAT2a morphant (E, high dose of MO 2), and EAAT2b morphant (MO 2) larvae (F). Yellow bar represents light stimulus (starting at time 0, ending at 100 ms).
	Figure 6. Time-to-peak analysis of ERG recordings. Box-and-whisker plots of time (ms) from onset of light stimulus (0 ms) to the peak of the b-wave. EAAT2a morphant larvae display changed ERG kinetics. At low light levels (A), low and high doses of EAAT2a MO 2 result in significant (p < 0.01) and highly significant (p < 0.001) increases in the time to peak, respectively. At bright light levels (B), EAAT2a morphant (MO 1) larvae show a significant (p < 0.01) increase in the time to peak, whereas both levels of MO 2 result in a highly significant (p < 0.001) increase in the time to peak. ERG response of EAAT2b morphant fish was not decelerated in dim light conditions (C) or in bright light (D).
	Figure 7. Two-electrode voltage clamp recordings from EAAT2a- and EAAT2b-expressing oocytes. A, B, Glutamate-evoked currents normalized to the saturating current induced by 500 µM glutamate in EAAT2a-expressing (A) and EAAT2b-expressing (B) oocytes (n = 8) and fitted with the Michaelis–Menten equation. The oocytes were voltage clamped at –50 mV. For clarity, fitted curves were plotted only up to 100 µM. In the insets, inward currents were induced by increasing concentrations of l-glutamate (1, 5, 25, 100, and 500 µM) in representative oocytes. The arrows indicate when glutamate was applied. Scale bar is 20 nA. C, D, Voltage dependence of EAAT2a-mediated (C) and EAAT2b-mediated (D) currents (n = 5) induced by 100 µM l-glutamate in control solution (black) and a chloride-free solution (red). The data from each cell were normalized to the response elicited by 100 µM l-glutamate in control solution at –100 mV. Insets show I-V recordings from representative oocytes in normal buffer. Data under both conditions are recorded from the same cells; oocytes are from three different batches. E–H, TBOA reveals a leak current. TBOA (100 mM) was applied alone (black arrow) in control medium to EAAT2a (E) and EAAT2b (F) injected oocytes. In EAAT2b injected oocytes, it evoked an outward current. When oocytes were dialyzed with a chloride-free solution for 24 h and the control perfusing medium was exchanged with a solution containing SCN- as the main negative ion, TBOA induced an inward current in EAAT2a and EAAT2b injected oocytes (G, H). Blue arrows indicate wash from TBOA. Scale bar is 10 nA.
	Figure 8. Schematics of photoreceptor synapse in light and dark. Illustration of changes in the photoreceptor synapse between light and dark in WT (A) and EAAT2a (B) and EAAT2b (C) morphants. EAAT2a on Müller glia cells is responsible for the uptake of the main load of glutamate. The uptaken glutamate in Müller cells is recycled via the glutamate–glutamine cycle. In EAAT2a morphants (B), the main load of glutamate is not being taken up and the cleft glutamate concentrations remain high, even during bright light stimuli. This may lead to binding of glutamate to postsynaptic receptors and therefore to a decreased ON-response in comparison to WT animals. The presynaptic transporter EAAT2b has a large Cl- conductance with a large leak current in absence of glutamate. During a light stimulus, when photoreceptors hyperpolarize and very few glutamate remains in the synaptic cleft, Cl- leaves the photoreceptor (leak current) and brings back the membrane potential closer to the dark resting potential. Because of the lack of such a leak current in EAAT2b morphants, cones remain in a slightly more hyperpolarized state.

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