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. 2021 Oct 14;11:529. doi: 10.1038/s41398-021-01651-w

Fig. 4. Effects of altered GABAergic signalling on locomotor activity in 5 dpf old zebrafish larvae.

Fig. 4

A gad1b exon–intron structure with coding (grey) and non-coding exons (white). gad1b splice-inhibiting morpholino (MO, green) and corresponding primer binding sites are indicated. Binding of the gad1b-MO causes retention of intron 8. B, top and centre The reduction of the gad1b wildtype transcript at 1 dpf (black asterisks, 208 bp) and the presence of the misspliced transcript at 1 and 5 dpf (green asterisk, 500 bp) was verified by RT-PCR. A primer off-target (black triangle) was detected below the wildtype gad1b transcript. Control PCRs on beta-actin (above, 239 bp) and lbx1a (below, 353 bp) confirmed comparable cDNA levels and absent genomic DNA contamination for all samples. B, bottom The predicted amino acid sequence of the misspliced gad1b transcript suggests a premature termination of translation in gad1b intron 8 (asterisk). C Overviews of a 5 dpf old gad1b morphant (MO) and an uninjected control larva (WT). Scale bar, 1 mm. D Locomotor activity of 5 dpf old gad1b morphant larvae (MO, green) compared to uninjected controls (WT, light blue). Locomotor activity was assessed by mean velocity in low or high activity, or combined (total), total distance swum, duration or events of inactivity, low and high activity. E Maximum mean velocity (of 5 min) reached within 8 h of tracking (peak velocity in cm/s) after incubation (Post) in 100 mM Gad-inhibitor L-allylglycine (dark red) or Danieau’s solution (control, dark blue). Maximum mean velocity (of 5 min) of the same individuals before incubation in L-allylglycine (Pre, light red) or Danieau’s solution (Pre, light blue). Line plot displays activity changes of individual larvae. F Locomotor activity of 5 dpf old wildtype larvae injected with 10 mM GABA-A-receptor antagonist SR-95531 (red) or water (light blue) at one-cell stage. G Effect of 0.1 mM GABA-B-receptor antagonist CGP-55845 on locomotor activity of 5 dpf old wildtype larvae (red) in comparison to Danieau’s solution incubated controls (light blue). D, F, G Raw data was standardised using z-score transformation. *P < 0.05, **P < 0.01, ***P < 0.001.