Effect of NMDA receptor signaling suppression and zinc treatment on slc13a5 mutants. (A-C) Quantification of the amplitude and frequency of calcium events (ΔF/F > 2) and representative single neuron calcium traces in the MB at 3 dpf. Memantine treatment and grin1a;grin1b MO injections significantly reduced the amplitude and frequency of calcium events in 5a−/−;5b−/− larvae compared to vehicle-treated or control MO-injected 5a−/−;5b−/− larvae. Vehicle-treated/control MO-injected WT larvae, n = 5; vehicle-treated/control MO-injected 5a−/−;5b−/− larvae, n = 5; memantine-treated 5a−/−;5b−/− larvae, n = 6; grin1a;grin1b MO-injected 5a−/−;5b−/− larvae, n = 6. (D-F) Quantification of the amplitude and frequency of calcium events (ΔF/F > 2) and representative single neuron calcium traces in the MB at 3 dpf. ZnCl2 treatment significantly reduced the amplitude and frequency of calcium events in 5a−/−;5b−/− larvae compared to vehicle-treated 5a−/−;5b−/− larvae. Vehicle-treated WT larvae, n = 4; vehicle-treated 5a−/−;5b−/− larvae, n = 9; ZnCl2-treated 5a−/−;5b−/− larvae, n = 6. (G) Quantification of basal respiration before and after treatment with memantine and MK801 at 6 dpf. Memantine and MK801 treatments rescue compromised basal respiration in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 6; vehicle-treated 5a−/−;5b−/−, n = 6; memantine-treated 5a−/−;5b−/−, n = 12; MK801-treated 5a−/−;5b−/−, n = 6 (individual values plotted from five cycles). (H) Quantification of ATP-linked respiration before and after treatment with memantine and MK801 at 6 dpf. Memantine and MK801 treatments rescue compromised ATP-linked respiration in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 5; vehicle-treated 5a−/−;5b−/−, n = 9; memantine-treated 5a−/−;5b−/−, n = 10; MK801-treated 5a−/−;5b−/−, n = 5 (individual values plotted from three cycles). (I) Quantification of total mitochondrial respiration before and after treatment with memantine and MK801 at 6 dpf. Memantine and MK801 treatments rescue compromised total mitochondrial respiration in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 10; vehicle-treated 5a−/−;5b−/−, n = 10; memantine-treated 5a−/−;5b−/−, n = 6; MK801-treated 5a−/−;5b−/−, n = 6 (individual values plotted from three cycles). (J) Quantification of non-mitochondrial respiration before and after treatment with memantine and MK801 at 6 dpf. Memantine and MK801 treatments rescue compromised non-mitochondrial respiration in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 8; vehicle-treated 5a−/−;5b−/−, n = 5; memantine-treated 5a−/−;5b−/−, n = 6; MK801-treated 5a−/−;5b−/−, n = 6 (individual values plotted from three cycles). (K) Quantification of basal respiration before and after treatment with ZnCl2 at 6 dpf. ZnCl2 treatment rescues compromised basal respiration in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 5; vehicle-treated 5a−/−;5b−/−, n = 4; ZnCl2-treated 5a−/−;5b−/−, n = 6 (individual values plotted from five cycles). (L) Quantification of ATP-linked respiration before and after treatment with ZnCl2 at 6 dpf. ZnCl2 treatment rescues compromised ATP-linked respiration in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 5; vehicle-treated 5a−/−;5b−/−, n = 4; ZnCl2-treated 5a−/−;5b−/−, n = 6 (individual values plotted from three cycles). (M) Quantification of total mitochondrial respiration before and after treatment with ZnCl2 at 6 dpf. ZnCl2 treatment rescues compromised total mitochondrial respiration in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 5; vehicle-treated 5a−/−;5b−/−, n = 4; ZnCl2 -treated 5a−/−;5b−/−, n = 6 (individual values plotted from three cycles). (N) Quantification of non-mitochondrial respiration before and after treatment with ZnCl2 at 6 dpf. ZnCl2 treatment rescues compromised non-mitochondrial respiration in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 5; vehicle-treated 5a−/−;5b−/−, n = 4; ZnCl2 -treated 5a−/−;5b−/−, n = 6 (individual values plotted from three cycles). (O) Quantification of total distance traveled in 10 min in acoustic startle before and after treatment with memantine at 5 dpf. Memantine treatment rescues impaired startle response in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 16; vehicle-treated 5a−/−;5b−/−, n = 58; memantine-treated 5a−/−;5b−/−, n = 43. (P) Quantification of total distance traveled in 10 min in acoustic startle before and after treatment with ZnCl2 at 5 dpf. ZnCl2 treatment rescues impaired startle response in 5a−/−;5b−/− larvae. Vehicle-treated WT, n = 9; vehicle-treated 5a−/−;5b−/−, n = 17; memantine-treated 5a−/−;5b−/−, n = 10. Data are Mean ± S.E.M. and Mean ± S.D., * P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001- Unpaired t test. MO, morpholino. The data underlying this figure can be found in S1 Data.
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