FIGURE SUMMARY
Title

Optical interrogation of neuronal circuitry in zebrafish using genetically encoded voltage indicators

Authors
Miyazawa, H., Okumura, K., Hiyoshi, K., Maruyama, K., Kakinuma, H., Amo, R., Okamoto, H., Yamasu, K., Tsuda, S.
Source
Full text @ Sci. Rep.

Transient expression of ASAP1. (a) Schematic diagram of plasmid construct for ASPA1. (b) Schematic diagram of the co-injection experiment. (c) ASAP1 was localized to the cellular membranes at 50% epiboly (arrowheads). (d) Lateral views of ASAP1-expressing embryos at 1 dpf. ASAP1 was expressed widely in the embryos.

Transgenic zebrafish showed membrane-localized ASAP1 in the neural tube. (a) Genotyping results of Tg(UAS:ASAP1) fish. Left: Tg(UAS:ASAP1) (Tg), right: wild-type (wt). The cropped gel image is shown. The full-length gel is presented in Supplementary Fig. S6a. (b) ASAP1 was distributed widely in the neural tube of Tg(elavl3:GAL4-VP16;UAS:ASAP1) fish at 1 dpf. (c) Dorsal view of the neural tube of Tg(elavl3:GAL4-VP16;UAS:ASAP1;UAS:lyn-RFP) embryos at 1 dpf. ASAP1 (green) was co-localized with Lyn-RFP (red: cell membranes) in the neural tube. Higher magnification images are shown in the lower panel. (d) Some of the ASAP1 positive cells had irregular shapes (arrowheads). Higher magnification images are shown in the upper right corner.

ASAP1 was expressed in neurons, including those of the cerebellum. (a) Horizontal sections of the neural tube of Tg(elavl3:GAL4-VP16;UAS:ASAP1) embryos stained with a HuC/D antibody and DAPI at 1 dpf. Hu-positive neurons (red) show a membrane-localized ASAP1 signal (arrowheads). Higher magnification images are shown in the lower panel. (b) ASAP1 was properly expressed in inferior olivary neurons. Dorsal view of the inferior olive (IO) of Tg(hspGFFDMC28C;UAS:ASAP1;UAS:RFP) fish (upper panel). Higher magnification images are shown in the lower panel. ASAP1 (green) is localized to the cell membranes of inferior olivary nuclei (red, arrowheads). Abbreviations indicate optic tectum (OT), and ear (E).

Spontaneous activity of spinal cord neurons was detected via ASAP1 imaging. (a) Changes in ASAP1 fluorescence were observed in the ventral region of the spinal cord. (left) Dorsal view of the ventral spinal cord of Tg (elavl3:GAL4-VP16;UAS:ASAP1) fish. The rostral side is to the left, and the area between 3–8 somites is shown. Regions of interest (ROIs) located between 5–7 somites are indicated by red (right side) and blue (left side) rectangles. (middle, right) Fluorescence changes of ASAP1 in the ROIs are shown in the middle panel. The images at two time points indicated by black arrowheads are shown in the right panel. White arrowheads indicate the activated neurons. Changes in fluorescence (−ΔF/F0) are indicated by the pseudocolor scale shown at right. (b) Fluorescence changes of ASAP1 were not observed in the dorsal region of the spinal cord. (left) Dorsal view of the same embryo shown in (a) with the image focus at the ventral region. An image at the time point indicated by the black arrowhead in the middle panel is shown to the right. (c) The spontaneous activity in the ventral region of the spinal cord was reduced by tetrodotoxin (TTX) treatment. The imaged plane and ROIs are in the same positions as in (a). (d) The frequency of the fluorescence changes was significantly higher in the ventral region. Similar results were obtained for both the left and right sides of the spinal cord (6 fish, *p < 0.05). (e) The spontaneous activity was almost eliminated by TTX treatment (6 fish, *p < 0.05). (f) Monitoring of spontaneous activity of individual cells (cells 1–8) by ASAP1 imaging. (top) A fluorescence image of the ventral spinal cord of Tg(elavl3:GAL4-VP16;UAS:ASAP1) fish. ROIs were located at the 8 cells (red: right side, blue: left side). Examples of activity patterns of the 8 cells for two conditions (before and after TTX treatment) are shown in the lower panel.

Neuronal activity in the cerebellum and optic tectum was detected by ASAP1 imaging. (a,b) Membrane-localized ASAP1 was observed in the cerebellum and optic tectum. Dorsal view of Tg(elavl3:GAL4-VP16;UAS:ASAP1) fish brain at 6 dpf (a) and 9 dpf (b). Higher magnification images are shown to the right. CE: cerebellum, OT: optic tectum. (ce) The evoked depolarizations in the cerebellum and optic tectum were detected by ASAP1 imaging. (c) Schematic diagram of the electrical stimulation system. (d) (Top left) A fluorescence image of the cerebellum of Tg(elavl3:GAL4-VP16;UAS:ASAP1) fish. (Bottom left) Images of changes in ASAP1 fluorescence before and 0.14 sec after the stimulation. Changes in fluorescence (−ΔF/F0) are indicated by the pseudocolor scale shown at right. (Right) ASAP1 signals produced by electrical stimulation of the cerebellum. Traces indicate signals detected at the 5 numbered locations indicated in the top-left image. ROI5 is located in the optic tectum. (e) Response map of the cerebellum. The peak amplitudes of the fluorescence changes in the cerebellar neurons upon stimulation are encoded by the pseudocolor scale shown at left. (f) The depolarizing signal measured in the cerebellum was reduced by tetrodotoxin (TTX) treatment (left bottom). Results from the same fish are shown in (d) and (f). Traces indicate signals detected at the location indicated in the top left image. (g) The mean peak values of the responses were significantly reduced after TTX treatment (*p < 0.01). This indicates that the depolarizing response represents neuronal activity in the cerebellum and optic tectum.

Transient expression of QuasAr2 and generation of QuasAr2 transgenic line. (a) Schematic diagrams of plasmid constructs for QuasAr2. (b) Lateral views of mOrange-expressing embryos at 1 dpf. (c) mOrange2 was localized to cell membranes in the neural tube, but QuasAr2 was not detected. Dorsal view of the neural tube of the injected embryos. (d) High magnification images of (c). Arrowheads indicate the localization of the fluorescence signal in cell membrane. (e) Representative images of the filopodia which are mOrange positive (arrowheads). (f) Genotyping results of UAS:QuasAr2 transgenic zebrafish. Tg: Tg(UAS:QuasAr2), wt: wild-type. gDNA isolated from the embryos of Tg(UAS:QuasAr2) F0 fish crossed with wild-type fish was used as the template for Tg. The cropped gel image is shown. The full-length gel is presented in Supplementary Fig. S6.

ASAP1 (green) is distributed in cerebellar granule cells (red). Dorsal view of the cerebellum of Tg(hspGFFDMC152B;UAS:RFP;UAS:ASAP1) fish observed at 5 dpf. Higher magnification views are shown in the lower panel.

3D images of Tg(hspGFFDMC28C;UAS:ASAP1) fish observed at 6 dpf. ASAP1 (green) expression was observed in climbing fibers (arrowheads). a: dorsal view, b: left view.

A confocal image (z-stack) of the cerebellar region of Tg(elavl3:GAL4-VP16; UAS:ASAP1) fish at 5 dpf. Dorsal view is shown. Axon bundles (arrowheads) were observed at the caudal region of the cerebellum.

The entire gel images of the cropped data shown in Fig. 2a (a) and Supplementary Fig. S1f (b). Genotyping results of (a) Tg(UAS:ASAP1) and (b) Tg(UAS:QuasAr2) transgenic zebrafish. A: Tg(UAS:ASAP1), Q: Tg(UAS:QuasAr2), w: wild-type.

Acknowledgments
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