Brenet et al., 2019 - Defective Excitatory/Inhibitory Synaptic Balance and Increased Neuron Apoptosis in a Zebrafish Model of Dravet Syndrome. Cells   8(10) Full text @ Cells

Figure 1

Correlation between LFP and calcium activity in scn1Lab model. (A) Schematic of the experimental setup to simultaneously record local field potential and calcium activity of a larval zebrafish brain. (B,C) Calcium images showing respectively the baseline activity (B) and the seizure activity (C). The fluorescence intensity is color-coded as shown in the color bar below the images. (D,E) Representative 20 min calcium recording of immobilized and paralyzed 4 dpf control (D) (N = 9) and scn1Lab (E) (N = 9) larvae. Each calcium event is shown with an orange arrowhead. (F) Number of events, calcium increases greater that 0.04 ΔF/F0 amplitude, during 1 h recording. (G) Representative LFP, recorded in the left neuropil of the optic tectum, and calcium traces, from the optic tectum, of a paralyzed and immobilized 4 dpf scn1Lab larva. (H) Correlation between LFP and calcium events duration (n = 46). N = number of larvae and n = number of events. Error bars on all graphs represent standard error of the mean (SEM). ***, p < 0.001. p-value was determined using Student’s unpaired t-test.

Figure 2

Defects of excitatory/inhibitory balance in the Scn1Lab-depleted larvae. (A,B) 20 µm coronal sections of 5 dpf control (A) (N = 5, n = 15) and scn1Lab (B) (N = 5, n = 15) larvae stained with PSD-95, an excitatory post-synaptic scaffolding protein. (D,E) 20 µm coronal sections of 5 dpf control (D) (N = 5, n = 20) and scn1Lab (E) (N = 5, n = 19) larvae stained with gephyrin, an inhibitory post-synaptic scaffolding protein. Images were acquired with a SP8 Leica laser scanning confocal microscope equipped with a 40x/oil 1.3 objective. Scale bar 5 µm. (C,F) Quantification of PSD-95 (C) and gephyrin (F) puncta density. (G) PSD-95/gephyrin puncta density ratio. N = number of larvae and n = number of sections. Error bars on all graphs represent standard error mean (SEM). **, p < 0.01. p-values were determined using the Mann-Whitney test.

EXPRESSION / LABELING:
Antibodies:
Fish:
Knockdown Reagent:
Anatomical Term:
Stage: Day 5
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Day 5

Figure 3

Evolution of the excitatory-inhibitory neuronal population in the scn1Lab model. (A1-3,B1-3) Dorsal view of 3 dpf Tg[Gad1b:GFP; Vglut2a:DsRed] control (A1-3) (N = 8) and scn1Lab (B1-3) (N = 8) living larvae. (A1,B1) Inhibitory neurons (Gad1b:GFP) in 3 dpf control (A1) and scn1Lab (B1) larvae. (A2,B2) Excitatory neurons (Vglut2a:DsRed) in 3 dpf control (A2) and scn1Lab (B2) larvae. (A3,B3) Merged image of 3 dpf control (A3) and scn1Lab (B3) larvae. (C) Dorsal view schematic of a 4 dpf larva. The red rectangle shows the region of interest. (E1-3,F1-3) Dorsal view of 4 dpf Tg[Gad1b:GFP; Vglut2a:DsRed] control (E1-3) (N = 8) and scn1Lab (F1-3) (N = 8) living larvae. (E1,F1) Inhibitory neurons (Gad1b:GFP) in 4 dpf control (E1) and scn1Lab (F1) larvae. (E2,F2) Excitatory neurons (Vglut2a:DsRed) in 4 dpf control (E2) and scn1Lab (F2) larvae. (E3,F3) Merged image of 4 dpf control (E3) and scn1Lab (F3) larvae. (H1-3,I1-3) Dorsal view of 5 dpf Tg[Gad1b:GFP; Vglut2a:DsRed] control (H1-3) (N = 8) and scn1Lab (I1-3) (N = 8) living larvae. (H1,I1) Inhibitory neurons (Gad1b:GFP) in 5 dpf control (H1) and scn1Lab (I1) larvae. (H2,I2) Excitatory neurons (Vglut2a:DsRed) in 5 dpf control (H2) and scn1Lab (I2) larvae. (H3,I3) Merged image of 5 dpf control (H3) and scn1Lab (I3) larvae. (D,G,J) Number of neurons in control and scn1Lab larvae at 3 dpf (D), 4 dpf (G) and 5 dpf (J). (K,L,M) Number of inhibitory and excitatory neurons in control and scn1Lab larvae at 3 dpf (K), 4 dpf (L) and 5 dpf (M). All images are representative 20 µm stacks acquired using LSM 880 Zeiss laser scanning confocal microscope equipped with a WPApo 20x/1.0 objective. Scale bar 40 µm. N = number of embryos. Error bars on all graphs represent standard error mean (SEM). * p < 0.05; ** p < 0.01; ***,p < 0.001 indicate a statistically significant difference between control and scn1Lab larvae. Statistically significant difference between inhibitory and excitatory neuronal population is indicated as $$, p < 0.01; $$$, p < 0.001. p-values were determined using Student’s unpaired t-test.

Figure 4

Increased neuronal death in Scn1Lab-depleted larvae. (A,B) Dorsal view of 4 dpf living control (A) (N = 15) and scn1Lab (B) (N = 15) larvae stained with acridine orange; 100 µm stack. Scale bar 40 µm. (C) Dorsal view schematic of 4 dpf larva. The red rectangle shows the region of interest for global cell death quantification. (D) Number of acridine orange-positive cells in 4 dpf brain. (E,F) Dorsal view of 4 dpf control (E) (N = 24) and scn1Lab (F) (N = 22) brain stained with activated caspase-3 antibody; 100 µm stack. Scale bar 40 µm. (G) Number of activated caspase-3-positive cells in 4 dpf brain. (H-I) Dorsal view of 4 dpf Z-VAD-treated control (H) (N = 18) and scn1Lab (I) (N = 16) larvae stained with acridine orange; 100 µm stack. Scale bar 40 µm. (J) Number of acridine orange-positive cells in 4 dpf brain. (K) Dorsal view schematic of 4 dpf larva. The red rectangle shows the region of interest for activated caspase-3 signal colocalization with inhibitory and excitatory neurons quantification. (L) Number of inhibitory (Gad1b:GFP) and excitatory (Vglut2a:DsRed) neurons also showing activated caspase-3 labelling in control and scn1Lab larvae. (M1-4, N1-4) Dorsal view of 4 dpf Tg[Gad1b:GFP; Vglut2a:DsRed] control (M1-4) (N = 14) and scn1Lab (N1-4) (N = 14) brain stained with activated caspase-3 antibody; representative 15 µm stack. Scale bar 10 µm. (M1,N1) Inhibitory neurons (Gad1b:GFP) in control (M1) and scn1Lab (N1) larvae. (M2,N2) Excitatory neurons (Vglut2a:DsRed) in control (M2) and scn1Lab (N2) larvae. (M3,N3) Activated caspase-3 staining in control (M3) and scn1Lab (N3) larvae. (M4,N4) Merged images of control (M4) and scn1Lab (N4) larvae. Excitatory neurons with activated caspase-3 expression are shown by arrowheads, while inhibitory neurons with activated caspase-3 expression are shown by arrows. Images were all acquired with an SP8 Leica laser scanning confocal microscope equipped with 40x/water 1.1 objective. N = number of embryos. Error bars on all graphs represent standard error mean (SEM). *, p < 0.05; ***, p < 0.001 indicate a statistically significant difference between control and scn1Lab larvae. A statistically significant difference between inhibitory and excitatory neuronal population is indicated as $$$, p < 0.001. p-values were determined using Student’s unpaired t-test.

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