gal Expression Correlates with Whole-Brain Activity

A) Representative calcium signals (elavl3:GCaMP5G) recorded across the brain of 5 dpf control (eaat2a+/+) larva (blue, top) and eaat2a-/- mutant without seizure activity (red, bottom). B) AUC calculated and averaged over two five-minutes time windows per animal and normalized to control (n control = 25; n eaat2a-/- = 12). C) Galanin transcript levels in pools of control vs. eaat2a-/- brains (n = 3). D) Number of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right). E) Average amplitude of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. F) Average duration of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. G) Representative calcium signals (elavl3:GCaMP6f) recorded across the brain of 5 dpf control (blue, top) and larvae 2 hours after PTZ exposure (PTZ rebound) (magenta, bottom). H) AUC calculated over 1 h recording, normalized to control (n control = 21; n PTZ rebound = 21). I) Galanin transcript levels in pools of control vs. PTZ rebound brains (n = 3). G) Number of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right). J) Average amplitude of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. K) Average duration of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. Significance levels: ***p < .001, **p < .01, *p < .05, ns = not significant (p > .05), Wilcoxon-Mann-Whitney test (B, E, H, J, K), negative binomial regression (D, I), Student’s t-test (C, F).

gal Controls Whole-Brain Activity

A) Representative calcium signals (elavl3:GCaMP5G) recorded across the brain of 5 dpf control (blue, top) and hsp70l:gal sibling (orange, bottom). B) AUC calculated over 1 h recording, normalized to control (n control = 25; n hsp70l:gal = 29). C) Galanin transcript levels in pools of control vs. hsp70l:gal brains (n = 3). D) Number of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right). E) Average amplitude of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. F) Average duration of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. G) Representative calcium signals (elavl3:GCaMP5G) recorded across the brain of 5 dpf control (blue, top) and gal-/- larva (yellow, bottom). H) AUC calculated over 1 h recording, normalized to control (n control = 24; n gal-/- = 24). I) Number of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right). J) Average amplitude of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. K) Average duration of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. L) Representative calcium signals (elavl3:GCaMP6f) recorded across the brain of 5 dpf control injected (blue, top) and galr1a crispant (galr1aKO) larva (green, bottom). M) AUC calculated over 1 h recording, normalized to control (n control = 28; n galr1a crispant = 23). N) Number of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right). O) Average amplitude of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. P) Average duration of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. Significance levels: ***p < .001, **p < .01, *p < .05, ns = not significant (p > .05), Wilcoxon-Mann-Whitney test (B, E, F, H, J, K, M, O, P), negative binomial regression (D, I, N), Student’s t-test (C).

gal Exerts a Modest Effect on Seizure Activity in eaat2a-/- Mutants

A) Averaged calcium signals (elavl3:GCaMP5G) for spontaneous seizures recorded across the brain of 5 dpf control (eaat2a-/-) larva (blue, 35 events) and eaat2a-/-;hsp70l:gal (orange, 39 events) aligned by 50% of maximum amplitude. Shaded area represents SEM. B) Time to peak calculated from beginning of aligned seizure until maximum ΔF/F0 signal. C) AUC calculated over spontaneous seizures normalized to control (n control = 35; n eaat2a-/-;hsp70l:gal = 39). D) Number of spontaneous seizures per larva. E) Amplitude of spontaneous seizures per larva. F) Duration of spontaneous seizures per larva. G) Averaged calcium signals (elavl3:GCaMP5G) for spontaneous seizures recorded across the brain of 5 dpf control (eaat2a-/-) larva (blue, 77 events) and eaat2a-/-;gal-/- (yellow, 89 events) aligned by 50% of maximum amplitude. Shaded area represents SEM. H) Time to peak calculated from beginning of aligned seizure until maximum ΔF/F0 signal. I) AUC calculated over spontaneous seizures normalized to control (n control = 77; n eaat2a-/ - ;gal-/- = 89). J) Number of spontaneous seizures per larva. K) Amplitude of spontaneous seizures per larva. L) Duration of spontaneous seizures per larva. Significance levels: ***p < .001, **p < .01, *p < .05, ns = not significant (p > .05), Wilcoxon-Mann-Whitney test (B, C, E, F, H, I, K, L), negative binomial regression (D, J

gal Promotes Seizures in PTZ Exposed Larvae

A) Averaged calcium signals (elavl3:GCaMP6f) for seizures elicited by 20 mM PTZ recorded across the brain of 5 dpf control (blue, 76 events) and hsp70l:gal (orange, 113 events) aligned by 50% of maximum amplitude. Shaded area represents SEM. B) Time to peak calculated from beginning of aligned seizure until maximum ΔF/F0 signal. C) AUC calculated over seizures normalized to control (n control = 76; n hsp70l:gal = 113). D) Number of seizures per larva. E) Amplitude of seizures per larva. F) Duration of seizures per larva. G) Averaged calcium signals (elavl3:GCaMP6f) for seizures elicited by 20 mM PTZ recorded across the brain of 5 dpf control (blue, 29 events) and gal-/- (yellow, 10 events) aligned by 50% of maximum amplitude. Shaded area represents SEM. H) Time to peak calculated from beginning of aligned seizure until maximum ΔF/F0 signal. I) AUC calculated over seizures normalized to control (n control = 29; n gal-/- = 10). J) Number of seizures per larva. K) Amplitude of seizures per larva. L) Duration of seizures per larva. M) Averaged calcium signals (elavl3:GCaMP6f) for seizures elicited by 20 mM PTZ recorded across the brain of 5 dpf control injected (blue, 7 events) and galr1a crispants (galr1aKO, green, 14 events) aligned by 50% of maximum amplitude. Shaded area represents SEM. N) Time to peak calculated from beginning of aligned seizure until maximum ΔF/F0 signal. O) AUC calculated over seizures normalized to control (n control = 7; n galr1a crispant = 14). P) Number of seizures per larva. Q) Amplitude of seizures per larva. R) Duration of seizures per larva. Significance levels: ***p < .001, **p < .01, *p < .05, ns = not significant (p > .05), Wilcoxon-MannWhitney test (B, C, E, F, H, I, K, L, N, O, Q, R), negative binomial regression (D, J, P).

Heat Shock Decreases Brain Activity of 5 dpf Wild Type Larvae

A) Representative calcium signals (elavl3:GCaMP5G) recorded across the brain of 5 dpf unexposed wild type larva (blue, top) and 5 dpf wild type larvae with a 37°C heat shock for either 10 min (steelblue blue, middle) or 30 min (light blue, bottom) with different recovery times at room temperature. Significance levels: ***p < .001, **p < .01, *p < .05, ns = not significant (p > .05), Wilcoxon-Mann-Whitney test (C, D), negative binomial regression (B).