Fig 6

Assessment of calcium events, pERK levels, NMDA receptor expression and extracellular zinc levels in 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. 5a^−/−;5b^−/− larvae show a significant increase in calcium events compared to WTs. WT, n = 5; 5a^−/−;5b^−/−, n = 6. (D) Whole-brain activity MAP-map depicting significant changes in pERK signal, calculated using Mann-Whitney U statistic Z score. The significance threshold was set based on an FDR whereby 0.05% of control pixels is set as significant. Voxels exhibiting significantly higher intensity values of pERK are denoted in green and those exhibiting significantly lower pERK intensity values are depicted in magenta, in the slc13a5 mutants compared to WTs at 5 dpf. slc13a5 mutants show an enhanced neural activity via increased pERK levels in different regions of the brain (green) compared to WT. The lower pERK intensity shown in the eyes (magenta) could be a background autofluorescence captured while imaging. WT, n = 9; 5a^−/−;5b^−/−, n = 8. (E) Section of 3 dpf MB region of WTs; α-Slc13a5, α-Grin1. (E’, E”, E’’’) Higher magnification of dashed boxes in E. White arrowheads point to Slc13a5⁺/Grin1⁺ cells in MB. (F, F’, F”, F’’’) Slc13a5 expression shown in the brain section from E. (G, G’, G”, G’’’) Grin1 expression shown in the brain section for E. (H, I) 5 dpf WTs and 5a^−/−;5b^−/− larvae; Grin1 (green), NeuN (red). (J, K) Quantification of the fluorescence intensity (CTCF) of Grin1 (α-Grin1) in the neurons (NeuN⁺) of optic tectum. 5a^−/−;5b^−/− larvae show a significant increase in Grin1 expression in the neurons of optic tectum compared to WTs at 3 dpf and 5 dpf. WT, n = 4; 5a^−/−;5b^−/−, n = 4 at 3 dpf. WT, n = 4; 5a^−/−;5b^−/−, n = 6 at 5 dpf. (L) 50 hpf larvae stained with CellMask Orange and Palm-ZP1. (M, M’, N, N’, Q) Quantification of the fluorescence intensity (CTCF) ratios of Palm-ZP1:CellMask Orange in the plasma membrane. 5a^−/−;5b^−/− larvae show a significant decrease in Palm-ZP1:CellMask Orange CTCF ratio in the plasma membrane compared to WTs. Untreated WT, n = 4; Untreated 5a^−/−;5b^−/−, n = 4. White arrows indicate the plasma membrane regions used for CTCF measurements. (O, O’, P, P’, Q) Quantification of the fluorescence intensity (CTCF) ratios of Palm-ZP1:CellMask Orange in the plasma membrane. ZnCl₂-treated WTs show a significant increase in Palm-ZP1:CellMask Orange CTCF ratio in the plasma membrane compared to untreated WTs. Untreated WT, n = 4; ZnCl₂-treated WT, n = 5. Similarly, ZnCl₂-treated 5a^−/−;5b^−/− larvae show a significant increase in Palm-ZP1:CellMask Orange CTCF ratio in the plasma membrane compared to untreated 5a^−/−;5b^−/− larvae. Untreated 5a^−/−;5b^−/−, n = 4; ZnCl₂-treated 5a^−/−;5b^−/−, n = 4. White arrows indicate the plasma membrane regions used for CTCF measurements. (R) Untreated WTs (i.e., in E3 water) and DMSO-treated WTs (vehicle for Palm-ZP1) showed no significant changes in Palm-ZP1:CellMask Orange CTCF ratios in the plasma membrane. Untreated WTs, n = 4; DMSO-treated WTs, n = 3. Data are Mean ±  S.E.M., ns: no significant changes observed, * P ≤  0.05, **P ≤  0.01, ***P ≤  0.001- Unpaired t test. FDR, false discovery rate. CTCF, corrected total cell fluorescence. The data underlying this figure can be found in S1 Data.