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) Area under the curve (AUC) calculated and averaged over two 5 min 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 hr after pentylenetetrazole (PTZ) exposure (PTZ rebound) (magenta, bottom). (H) AUC calculated over 1 hr 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<0.001, **p<0.01, *p<0.05, ns = not significant (p>0.05), 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) Area under the curve (AUC) calculated over 1 hr 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 hr 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 crispants (galr1aKO) larva (green, bottom). (M) AUC calculated over 1 hr recording, normalized to control (n control = 28; n galr1a crispants = 23). (N) Galanin transcript levels in pools of control vs. galr1aKO brains (n = 3). (O) Number of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right). (P) Average amplitude of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. (Q) Average duration of Ca2+ events above 5% ΔF/F0 (left) and 10% ΔF/F0 (right) per larva. Significance levels: ***p<0.001, **p<0.01, *p<0.05, ns = not significant (p>0.05), Wilcoxon-Mann-Whitney test (B, E, F, H, J, K, M, O, P), negative binomial regression (D).

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<0.001, **p<0.01, *p<0.05, ns = not significant (p>0.05), Wilcoxon-Mann-Whitney test (C, D), negative binomial regression (B).

Immunostaining confirms protein deficiency in gal -/- mutants whole-mount immunostaining of galanin localized in the diencephalon of 5 dpf zebrafish larvae. Antibodies used for staining included anti-galanin (green) and anti-ac. tubulin (acetylated tubulin, magenta). (A1) representative galanin staining in control larvae. (A2, A3) Single immunohistochemical channels. (B1) representative galanin staining in gal -/- larvae. (B2, B3) Single immunohistochemical channels. Scale bar = 25 μm

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) Area under the curve (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<0.001, **p<0.01, *p< 005, ns = not significant (p>0.05), Wilcoxon-Mann-Whitney test (B, C, E, F, H, I, K, L), negative binomial regression (D, J).

gal promotes seizures in pentylenetetrazole (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) Area under the curve (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, seven 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 crispants = 14). (P) Number of seizures per larva. (Q) Amplitude of seizures per larva. (R) Duration of seizures per larva. Significance levels: ***p<0.001, **p<0.01, *p<0.05, ns = not significant (p>0.05), Wilcoxon-MannWhitnMann Whitney (B, C, E, F, H, I, K, L, N, O, Q, R), negative binomial regression (D, J, P).