Article Figures & Data
Figures
- Figure 1.
High-speed confocal calcium imaging at different scales. A, Experimental setup. Simultaneous LFP recording and fast confocal imaging (20–30 fps) in agar-embedded larval zebrafish exposed to pancuronium (300 µM) with PTZ (10 mM) or 4-AP (4 mM). B, Representative high-resolution imaging of neurod1:GCaMP6f expressed larval zebrafish on 5–6 dpf. C, Experimental workflow. Recordings were obtained ∼40 min after drug application, and 5× and 20× objectives were used for whole-brain and neuron-level imaging, respectively. D, Schematic illustration depicting sub-regions of the larval zebrafish brain. E, ROIs and representative calcium traces (ΔF/F) of PTZ-induced ictal-like events; 1, 2: pallium; 3, 4: habenula; 5, 6: neuropil; 7, 8: SPV; 9, 10: cerebellum; 11, 12: hindbrain. F, Neuronal microcircuits within optic tectum and representative calcium traces of individual SPV neurons with PTZ-induced ictal-like event break-ins (underlined in red). Cerebellum was included as an indicator of ictal-like events that involve all brain regions. Scale bars as indicated in figure.
- Figure 2.
Correlation between LFP and calcium transients. A, Representative simultaneous calcium traces (green) from the cerebellum and LFP (black) recorded from optic tectum/cerebellum with recurrent PTZ-induced ictal-like seizures. Scale bars as indicated in figure. B, Fluorescence images of calcium activity during baseline and peak ictal-like event. The intensity of fluorescence is color coded as shown in the color bar. Events 1 and 2 as noted in A. Asterisk indicates the LFP recording site. C, D, Correlation between LFP and calcium transients in ictal-like event amplitude (C) and duration (D). Corresponding correlation coefficient R 2 is indicated in figure; n = 54 events from 10 PTZ-treated larvae.
- Figure 3.
Brain-wide network synchronization. A, Representative whole-brain average fluorescence changes (ΔF/F; green) and LFP (black) recorded from control, 4-AP, and PTZ-treated larvae. B, Comparison of normalized AUC of fluorescence changes (norm. ΔF/F AUC). C, Representative fluorescence changes of each brain region under different conditions. ROIs refer to Figure 1E. D, Correlation matrices of whole-brain network activity for representative fish. E, Comparison of global synchronization (sync.) index. Scale bars as indicated in figure; n = 4, 7, and 7 fish for control, 4-AP, and PTZ condition, respectively. Error bars indicate SD. Statistical significance is indicated as ***p < 0.001.
- Figure 4.
Seizure propagation across the whole-brain. A, Representative calcium trace for each brain region within an PTZ-induced ictal-like event. Event onset time points were determined by the local maxima of the time derivative of moving average (of 50 data points) of ΔF/F. Scale bars as indicated in figure. B, Plot of normalized (norm.) relative lag from the earliest onset. C, Color-coded onset time mapping in a representative fish brain (average of four ictal events). D, Normalized relative lag for three quantile groups (first third early onset, second third middle onset, and third third late onset). E, Probability of brain regions falling into each quantile group. χ2 test X2(10) = 375.1, p < 0.001. Error bars indicate SD; n = 30 events from 7 PTZ-treated larvae.
- Figure 5.
Neuronal ensembles in optic tectum microcircuits. A, Representative calcium traces (gray, individual SPV neuron; green, cerebellum), neuron activation raster plot (red, ensemble neurons) and number of coactive neurons (ensembles were indicated by red arrow) of control, 4-AP, and PTZ-treated fish. Cerebellum activity was shown as an indicator of ictal-like events that involve all brain regions. B, C, Histogram (B) and distributions (C) of the neural firing rate (event count per 5 min); n = 75, 76, and 68 neurons for control, 4-AP, and PTZ-treated fish, respectively. D, Spatial mapping of ensembles depicted in A. Dots in the same color represent coactive neurons, and lines are distances from ensemble neurons to the corresponding ensemble centroid. Each ensemble is indicated by a color. Scale bars as indicated in figure. E, Distribution of coactive neuron numbers as percentage of frames (6000 frames for each recording). F, G, Histogram (F) and distributions (G) of distances from ensemble neurons to the corresponding ensemble centroid; n = 17, 36, and 42 ensemble neurons for control, 4-AP, and PTZ-treated fish, respectively. Error bars indicate SD. Statistical significance is indicated as ***p < 0.001.
Movies
- Movie 1.
Representative whole-brain imaging with 5× objective from control fish. Movie played at 5× speed.
- Movie 2.
Representative whole-brain imaging with 5× objective from 4-AP fish. Movie played at 5× speed.
- Movie 3.
Representative whole-brain imaging with 5× objective from PTZ fish. Movie played at 5× speed.
- Movie 4.
Representative imaging in optic tectum microcircuits with 20× objective from control fish. Movie played at 5× speed.
- Movie 5.
Representative imaging in optic tectum microcircuits with 20× objective from 4-AP fish. Movie played at 5× speed.
- Movie 6.
Representative imaging in optic tectum microcircuits with 20× objective from PTZ fish. Movie played at 5× speed.
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