Two‐day‐old zebrafish NMJs from double stmn2−/− and stmn2b−/− zebrafish display increased pre‐ and postsynaptic puncta and decreased quantal size of mEPCs. (A) Representative immunofluorescent images of zebrafish NMJs from wild type and double stmn2a−/− and stmn2b−/− embryos. Scale bars represent 25 µm. Quantification of orphaned presynaptic Syt2 puncta (red arrowheads) (B) and postsynaptic AchR puncta (green arrows) (C). Mean number of orphaned presynaptic terminals ± standard error and sample sizes were as follows: wild type (7.7 ± 0.9 terminals, n = 13); stmn2a−/− (10.7 ± 0.7 terminals, n = 18); stmn2b−/− (8.4 ± 1.1 terminals, n = 14); double stmn2a−/− and stmn2b−/− (11.7 ± 0.6 terminals, n = 16). Mean number of orphaned postsynaptic terminals ± standard error and sample sizes were as follows: wild type (4.2 ± 0.9 terminals, n = 13); stmn2a−/− (5.1 ± 0.5 terminals, n = 18); stmn2b−/− (4.9 ± 0.7 terminals, n = 14); double stmn2a−/− and stmn2b−/− (6.4 ± 0.5 terminals, n = 16). Statistical differences in (B) were as follows: wild type versus stmn2a−/−, p = 0.047; wild type versus double stmn2a−/− and stmn2b−/−, p = 0.004. Statistical difference in (C) was as follows: wild type versus double stmn2a−/− and stmn2b−/−, p = 0.011. (D) Example mEPC events in wild type and double stmn2a−/− and stmn2b−/− fish. (E) Quantification of mean mEPC amplitude. Mean mEPC amplitude was found to be reduced (p = 0.003) in double stmn2a−/− and stmn2b−/− zebrafish in comparison to recordings obtained from wild type fish. (F) Quantification of mean mEPC frequency. No significant difference was observed between wild type and double stmn2a−/− and stmn2b−/− groups (p = 0.148). Samples sizes for mEPC recordings were as follows: wild type = 13; double stmn2a−/− and stmn2b−/− = 10. Data points represent individual means from zebrafish recordings with black bar representing the overall group mean ± standard error. Kruskal–Wallis multiple comparisons tests were used to determine significance (B and C) or a t‐test (E and F). mEPCs, miniature endplate currents. *p < 0.05, **p < 0.01.
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