Characterization of epileptic phenotype in stxbp1b mutant zebrafish

(A) Representative spontaneous high-velocity convulsive behavior captured during high-speed imaging (250 fps) of a single stxbp1b mutant larva freely swimming in embryo media at room temperature. Note these were rare events that occur with a velocity near, or greater than, the acquisition speed of the camera (QImaging Optimos cMOS).

(B) Representative 15 min local field potential recordings from randomly selected larvae from a cross of stxbp1b^+/- adult breeders. LFP recordings were obtained from a glass microelectrode positioned under a microscope in the midbrain of agarose-embedded larvae at 5 dpf. Larvae were freed from the agarose and genotyped post hoc. Note the presence of small amplitude events building to a large amplitude multi-spike ictal event with postictal depression in the stxbp1b mutant larvae (see inset) but not the WT sibling. These recordings are representative of the spontaneous and unprovoked seizure activity associated with this zebrafish line.

(C) Confocal images taken with a 2-photon microscope of a representative neurod1:GCaMP6f expressing larval zebrafish at three different levels through the central nervous system. Scale bar, 100 μm

Measuring cascades in zebrafish optic tectum

(A) Calcium imaging in neurod1:GCaMP6f-expressed larval zebrafish. Neuronal dynamics within optic tectum (highlighted by white dash line) were extracted for cascade measurement.

(B) Calcium traces (ΔF/F) from pixels within the optic tectum in a stxbp1b mutant fish on 6 days post fertilization (dpf). A pixel is considered to be active when ΔF/F crosses the threshold of 5 times signal standard deviation (+5 SD). One pixel is 2.67 × 2.67 μm area. Simultaneously recorded local field potential (LFP) recording from midbrain is shown below. Scale bars as indicated in figure.

(C and D) Binarized activity from episodes indicated in (B) (underlined in red). One normal cascade was revealed in (C), and multiple cascades were revealed in (D), including an abnormally large and long cascade located in the left optic tectum. The last frame represents the summation of the binary time series stack of the corresponding episode showing the spatial mapping of the cascades. The cumulative intensity of activation is color coded as shown in the color bar. Scale bars, 100 μm.

Cascade quantification

(A) Cascade size versus duration on different days post fertilization (dpf). Blue dots represent WT siblings, and orange dots represent stxbp1b mutants. Dashed lines are the ellipse contour of the average maximum value of cascade size and duration (blue, WT siblings; orange, stxbp1b mutants). For WT siblings, n = 2843, 4188, and 2690 cascades on 5, 6, and 7 dpf, respectively. For stxbp1b mutants, n = 4212, 4214, and 2668 cascades on 5, 6, and 7 dpf, respectively. Data from 12 fish per day for each condition is plotted.

(B) Probability distribution of cascade sizes on different dpf. The plots show the cumulative distributions of the corresponding data (blue, WT siblings; orange, stxbp1b mutants) on different dpf. Measurement of the number of cascade, maximum cascade size, and maximum cascade duration (per 5 min recording) on different dpf is shown. n = 12 fish per day for each condition. Data are represented as mean ± SD. Statistical significance is indicated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; Student's t test.

Probability distribution of cascade size and duration

(A) Probability distribution of cascade sizes on different days post fertilization (dpf). The insert plots show the cumulative distributions of the corresponding data (blue, WT siblings; orange, stxbp1b mutants).

(B) Probability distribution of cascade durations on different dpf. p values of KS tests are indicated in figure. Data from 12 fish per day for each condition is plotted.

Cumulative distribution of cascade size and duration

(A) Representative stack summation of binary time series from WT siblings on different days post fertilization (dpf). Each tile represents one fish. The cumulative intensity of activation is color coded as shown in the color bar.

(B) Cumulative distribution of cascade sizes and durations from WT siblings on different dpf (light blue, 5 dpf; blue, 6 dpf; dark blue, 7 dpf).

(C) Representative time series stack summation from stxbp1b mutants on different dpf.

(D) Cumulative distribution of cascade sizes and durations from stxbp1b mutants on different dpf (yellow, 5 dpf; orange, 6 dpf; dark red, 7 dpf). Scale bars, 100 μm. Data from 12 fish per day for each condition is plotted.

Gap junction blockers reduce neuronal cascade intensity

(A) Representative stack summation of binary time series from stxbp1b mutants before (top row) and after (bottom row) heptanol treatment. The cumulative intensity of activation is color coded as shown in the color bar.

(B) Comparison of normalized (norm.) number (num) of cascade, maximum (max) cascade size, and maximum cascade duration (dur) before and after heptanol treatment. Data were normalized to the average value from recordings before drug treatment. (C and D) Results from propofol experiments on stxbp1b mutants. Scale bars, 100 μm. n = 5 fish for each drug test. Statistical significance is indicated as ∗p < 0.05, ∗∗p < 0.01; Student's t test.

Neuronal ensembles revealed in stxbp1b mutants.

(A) Representative neuron activation raster plot (top; red dots, ensemble neurons) and number of coactive neurons (bottom; ensembles were indicated by red arrow) from a WT sibling. (B) Left, spatial mapping of neuronal 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. Right, stack summation of calcium activity time series. The fluorescence intensity is color coded as shown in the color bar.

(C and D) Representative data from a stxbp1b mutant.

(E) Comparison of number (num) of ensembles.

(F) Comparison of average (avg.) ensemble size. Scale bars, 100 μm. n = 5 fish each condition: for WT, 3, 1, and 1 fish from 5, 6, and 7 dpf, respectively; for mutants, 2, 2, and 1 fish from 5, 6, and 7 dpf, respectively. Data are represented as mean ± SD. Statistical significance is indicated as ∗p < 0.05, ∗∗p < 0.01; Student's t test.