Figure 5. Sesn3 modulates PTZ-induced c-fos expression, locomotor convulsions and Module-1 genes in zebrafish.

(a) Left, Sesn3 promotes convulsive locomotor response of zebrafish larvae exposed to PTZ. Three days post fertilization (d.p.f.) zebrafish larvae were incubated with and without 20 mM PTZ for 1 h and locomotor activity was monitored continuously. Larvae microinjected with Sesn3 morpholinos exhibited a sustained reduction in locomotor activity throughout the period of PTZ incubation, in comparison with control morphant larvae. Both Sesn3 morphant and control morphant larvae (n = 12) exhibited similarly low levels of locomotor activity in the absence of PTZ. Right, Sesn3 morpholinos reduced the cumulative locomotor activity of zebrafish exposed to 20 mM PTZ (black columns) without appreciably affecting basal locomotor activity of larvae incubated in the absence of PTZ (white columns). (b) Co-injecting Sesn3 morpholinos with synthetic Sesn3 mRNA showed that Sesn3 mRNA rescued the locomotor activity phenotype (total distance swam, y axis). For each group, 16–18 larvae were analysed. Black bars, 1 h PTZ treatment (20 mM). (c) Sesn3 morpholinos attenuate seizure-induced expression of the synaptic activity-regulated gene c-fos. Left and central images, dorsal views of the brains of 3 d.p.f. control morphant (top) and Sesn3 morphant larvae (bottom) maintained for 1 h in the absence and presence of 20 mM PTZ, after which larvae were fixed and analysed for c-fos expression by whole-mount in situ hybridization. Red arrowheads: position of the transverse sections of the brains; scale bar, 200 μm. Following PTZ treatment (20 mM, 1 h), quantitative PCR (qPCR) analysis revealed that Sesn3 morphant larvae exhibited significantly lower mRNA expression of c-fos in the brain than control morphant larvae (c, right panel). (d) PTZ-induced transcriptional response of Module-1 genes was significantly lower in Sesn3 morphants as compared with uninjected larvae; six samples were used in the qPCR experiments (one sample = 15–20 pooled larvae). (e) Upregulation of Module-1 genes upon injection of synthetic mRNA (1 ng) in zebrafish embryos (n = 30) as compared with uninjected control embryos (n = 30). Total RNA was extracted 28 h post fertilization and qPCR experiments were performed for the two pools of embryos using six technical replicates. Data reported as means±s.e.m. were determined by the 2^−ΔΔCt method and normalized to the housekeeping gene β-actin. P-values calculated by t-test (two-tailed) adjusting for unequal variances across different groups. NS, not significant (P>0.05).