Fig. 6

Treatment with the mGluR agonist L-AP4 increased myelin-related genes and rescues CNS myelination in iGluSnFR⁽⁺⁾ zfl. (a) CNRQ values representing mRNA quantification of mbpa, plp1b, ß-actin, and ef1a isolated from 1mM L-AP4, sham vehicle-injected and untreated zfl (wild type) at 5 dpf (injected at 3 dpf). mRNA levels were normalized against ß-actin and ef1a housekeeping gene expression. Both myelin genes, mbpa and plp1b, show a significant increase in expression in L-AP4 injected zfl compared to sham-injected or uninjected control larvae. Both housekeeping genes though do not show any significant difference in L-AP4 versus sham-injected or uninjected controls. Data were evaluated by one-way ANOVA followed by Bonferroni's multiple comparisons. * indicates a p-value < 0.05; ** indicates a p-value < 0.01. Values show mean ± SEM. mbpa: L-AP4 versus Ctrl **p < 0.01; L-AP4 versus uninj. **p < 0.01; F = 18.60; df = 11 and plp1b: L-AP4 versus Ctrl **p < 0.01; L-AP4 versus uninj. **p < 0.01; F = 10.74; df = 11; ß-actin: L-AP4 versus Ctrl p = 0.6673; L-AP4 versus uninj.: p = 0.6673; F = 0.4233; df = 11 and ef1a: L-AP4 versus Ctrl p = 0.8338; L-AP4 versus uninj. p = 0.8338; F = 0.1855; df = 11. N = 4 independent experiments indicated by different shapes. (b) Two-photon in vivo microscopic images of Tg(Olig2:dsRed) proximal zfl spinal cords of 5 dpf larvae after L-AP4 ventricle injections at 3 dpf where analyzed in lateral view. For representative example images, please refer to Figure S6a. A significant increase in total dorsal OPCs after L-AP4 injection could be seen in iGluSnFR⁽⁻⁾ zfl compared to sham-injected iGluSnFR⁽⁻⁾ larvae. *p = 0.0377; unpaired two-tailed t test; t = 2.158. iGluSnFR⁽⁺⁾ zfl at 5 dpf like before at 3 dpf (see Figure 2) showed a significant ***p = 0.001; unpaired two-tailed t test; t = 3.643 decrease in dorsal OPCs compared to iGluSnFR⁽⁻⁾ zfl. Injection of 1mM L-AP4 at 3 dpf into the ventricle of iGluSnFR⁽⁺⁾ zfl though could partly but significantly *p = 0.0309; unpaired t test; t = 1.959 rescue the OPC reducing effect of iGluSnFR at 5 dpf. Dot plot graphs show averages of dorsal OPC numbers (mean ± StDev). N = 3 independent experiments indicated by different shaped dots, with a total (n) of 13–19 larvae. (c) Low-resolution epi-fluorescence microscopic images of Tg(CaudinK:mem-TdTomato) proximal zfl spinal cords of 5 dpf larvae after L-AP4 or sham ventricle injections at 3 dpf where analyzed in lateral view. For representative example images, please refer to Figure S6b. A significant increase in tdTomato red myelin reporter fluorescence after L-AP4 injection could be seen in iGluSnFR⁽⁻⁾ zfl compared to sham-injected iGluSnFR⁽⁻⁾ larvae ***p < 0.0001; one-way ANOVA followed by Bonferroni's multiple comparisons; F = 82.24; df = 69. The same is true for a comparison with completely uninjected iGluSnFR⁽⁻⁾ siblings. Control iGluSnFR⁽⁺⁾ zfl at 5 dpf like before (see Figure 3c) showed a significant ***p < 0.0001; unpaired two-tailed t test; t = 9.378; decrease in tdTomato reporter fluorescence compared to control iGluSnFR⁽⁻⁾ zfl. Injection of 1mM L-AP4 at 3 dpf into the ventricle of iGluSnFR⁽⁺⁾ zfl though could completely and significantly ***p < 0.0001; rescue this myelin-reducing effect of iGluSnFR at 5 dpf; one-way ANOVA followed by Bonferroni's multiple comparisons; F = 172.2; df = 54. Dot plot graphs show averages of red tdTomato myelin reporter fluorescence (mean ± StDev). N = 3 independent experiments indicated by different shaped dots, with a total (n) of 15–17 larvae