Tg zebrafish expressing two independent indicators of SV recycling in motoneurons. A, Diagram represents the transgene construct introduced in the Tg (hb9:tTAad, TRE:TagRFP-P2A-VpHalo) zebrafish. The hb9 promoter drives the expression of the tTAad, which in turn induces the expression of TagRFP and VpHalo through its interaction with the tetracycline response element (TRE) composite promoter. B, Fluorescent stereoscopic images of TagRFP and pHluorin in an anesthetized Tg zebrafish at 4 dpf. Scale bar, 200 μm. C, Diagram represents VpHalo that resides on the SV membrane. D, Confocal microscopic images of TagRFP, pHluorin (pH 7.4), and postsynaptic AChR clusters visualized by α-BTX-CF633 in PFA-fixed Tg zebrafish. Bottom panels, z-stack images with higher magnification. Scale bars: 20 μm, 10 μm. E, Swimming was elicited by puff stimulus onto the tail of a 4 dpf zebrafish, and the images were captured at 1000 frames/s. Superimposed images at 4 ms intervals are shown for representative larvae from WT or Tg. Inset, An image with two frames superimposed at 1 ms interval, from which dθ corresponding to the head-turn speed (degree/ms) was measured. Scale bar, 1 mm. F, G, Maximum head-turn speed (F) and swimming speed calculated from the distance traveled during initial 30 ms (G) in WT (n = 8 fish) or Tg (n = 8 fish) fish. There was no significant difference between the two groups in head-turn speed (p = 0.39, unpaired t test) or swimming speed (p = 0.58, unpaired t test). H, Representative traces of mEPCs recorded from the fast muscle of WT or Tg zebrafish. I, J, Frequency (I) and amplitude (J) of mEPCs recorded from WT (n = 15 cells from 8 fish) or Tg (n = 14 cells from 6 fish) fish. No significant difference was found between the two groups in terms of frequency (p = 0.60, unpaired t test) or amplitude (p = 0.20, unpaired t test).
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