Transgenic zebrafish showing membrane-localized ArcLight and spontaneous activity in the neural tube.

(a) Schematic diagram of plasmid construct for ArcLight. (b) Lateral views of ArcLight-expressing embryos at 1 dpf. ArcLight is distributed widely in the neural tube of Tg(elavl3:GAL4-VP16;UAS:ArcLight) fish. (c) Dorsal view of the neural tube of Tg(elavl3:GAL4-VP16;UAS:ArcLight;UAS:lyn-RFP) embryos at 1 dpf. ArcLight (green) is co-localized with Lyn-RFP (red: cell membranes) in the neural tube (arrowheads). Higher magnification images are shown in the lower panel. (d-f) Spontaneous activity of spinal cord neurons is detected by ArcLight imaging. (d) Dorsal view of the ventral spinal cord of Tg (elavl3:GAL4-VP16;UAS:ArcLight-A242) fish at 20 hpf. The rostral side is to the left, and the area between 3–8 somites is shown. A schematic diagram of the imaging system is shown on the right. Changes in the fluorescence of ArcLight (−ΔF/F0) are indicated by the pseudocolor scale shown at right. White arrowheads indicate the activated neurons. Regions of interest (ROIs) located between 5–7 somites are indicated by red (right side) and blue (left side) rectangular in (d). Fluorescence changes of ArcLight in the ROIs are shown on the right. The time point of the image is indicated by a black arrowhead. (f-g) The spontaneous activity in the spinal cord mostly disappeared by tetrodotoxin (TTX) treatment (control 0.14±0.10 Hz, TTX 0.01±0.02 Hz, p<0.05, right side: control 0.14±0.10 Hz, TTX 0.01±0.02 Hz, *: p<0.05, Wilcoxon signed-rank test, 6 fish). The imaged plane and ROIs are in the same positions as in (d).

Monitoring spontaneous activity of individual cells in the spinal cord by ArcLight imaging.

(a) A fluorescence image of the ventral spinal cord of Tg(elavl3:GAL4-VP16;UAS:ArcLight) fish at 21 hpf. ROIs are located at the 18 cells (red: right side, blue: left side). Higher magnification views are shown on the right. (b) Changes in ArcLight signal at the two time points indicated in (c). (c) Activity patterns of the 18 cells are shown. Higher magnification views of the ArcLight signal of cells 4 and 13 are shown at the bottom. (d). (e, f) Hierarchal clustering analysis of the voltage dynamics in the 18 cells shows the synchronous activity in the ipsilateral neuron pairs (e), and also several groups with higher synchrony within them (f).

Voltage imaging of spinal cord neurons at a high spatiotemporal resolution at the cellular compartment level by ArcLight.

(a) Lateral views of the spinal cord of Tg(elavl3:GAL4-VP16;UAS:ArcLight) fish at 20 hpf. A schematic diagram of the imaging system is shown on the right. (b) Changes in ArcLight signal are observed at soma and axons in three different ROIs shown in (a). Higher resolution observations are shown in Supplementary Fig. S2 and S3.

Single cell labeling of neurons by ArcLight enables simultaneous recording of voltage and morphology.

(a) Schematic diagram of the co-injection experiment. (b) A stacked confocal image of the spinal cord region of the injected embryo at 20 hpf. The left side view is shown. Arrowheads and arrows indicate the soma and axons of ArcLight positive cells, respectively. (c) Voltage dynamics of a primary motor neuron are indicated by a yellow arrowhead in (b). ROIs at soma and axons are shown in red and blue, respectively. (d, e) Voltage imaging of the spinal cord interneurons. A higher magnification view is shown in (e). ArcLight signals from three cells are shown on the right. In addition to periodic depolarization, hyperpolarization (white arrowheads) and subthreshold-like signals (black arrowheads) are observed.

Long-term voltage imaging of the spinal cord PMNs shows the emergence of population activity.

(a) Left views of the spinal cord of ArcLight positive fish from 18 hpf to 19.5 hpf. (b) Enlarged views of (a). (c) Voltage dynamics obtained from the axons of the two cells shown in (a). Black and white arrowheads indicate small depolarization and immature firing, respectively.

Characteristic patterns of depolarization observed during the development of PMNs population activity.

Quantitative analysis indicates that coupled activity among PMNs emerged via small depolarization and immature firing. (a,b) (a) The relationship between axon length and firing frequency. (b) The relationship between axon length and coordination with neighboring neurons. Developmental process of each PMN is indicated by lines. Developmental stages are indicated by dots colors. (c) The relationship between the axonal length and existence of small depolarization (no depolarization: 13.76±3.75 μm, small depolarization: 11.97±1.67 μm, Firing: 27.12±1.38 μm, 5 fish, 30 cells, **p<0.01, Mann–Whitney U test). (d) The relationship between axonal length and immature firings (no depolarization: 10.48±2.38 μm, small depolarization: 22.42±2.69 μm, Firing: 26.06±1.44 μm, 5 fish, 30 cells, **p<0.01, Mann–Whitney U test). (e) Comparison of coupling rates between PMNs with small depolarizations and those with firings (immature and mature firings) (small depolarization: 0.24±0.07, firing: 0.93±0.03, 4 fish, 18 cells,, **p<0.01, Mann– Whitney U test). (f) Comparison of coupling rates between PMNs exhibiting immature and mature firings (immature firing: 0.58±0.10, mature firing: 0.94±0.03, 4 fish, 18 cells, **p<0.01, Mann–Whitney U test).

Changes in firing properties in spinal cord neuron populations.

(a) Dorsal view of ArcLight positive spinal cord at 20 hpf. (b) Voltage dynamics of each neuron are shown at 20 and 23 hpf. The position of ROIs is indicated in (a). (c) Cross-correlation analysis of ipsilateral and contralateral pairs of spinal cord neurons at 20 and 23 hpf. (d) Probability of altering activity at 23-24 hpf was significantly reduced compared to that at 23-24 hpf. 20–21 hpf: 98.9±1.10%; 23–24 hpf: 98.9±1.10%, 5 fish; 23–24 hpf: 79.8%±5.71%, 4 fish; p<0.05, Mann–Whitney U test.