Zebrafish behavioural profiling identifies GABA and serotonin receptor ligands related to sedation and paradoxical excitation

McCarroll, M.N., Gendelev, L., Kinser, R., Taylor, J., Bruni, G., Myers-Turnbull, D., Helsell, C., Carbajal, A., Rinaldi, C., Kang, H.J., Gong, J.H., Sello, J.K., Tomita, S., Peterson, R.T., Keiser, M.J., Kokel, D.
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GABAAR PAMs enhance acoustic startle in zebrafish. Zebrafish were treated with the indicated compounds and analyzed for changes in behavioral responses. a The scatter plot quantifies acoustic startle response as a z-score (y-axis) in zebrafish treated with the indicated CNS-depressants (x-axis) at the indicated concentrations (colorbar). Each point represents the average of n = 12 wells and 6 experimental replicates (also listed in Supplementary Table 1). b These plots show how the indicated compounds impact zebrafish motor activity (y-axis) over time (x-axis) (n = 12 wells, shaded boundary = 95% confidence interval; nMI, normalized motion index). Colored bars above the x-axis represent the timing and duration of low-volume acoustic stimuli (gray bars) and violet light stimuli (purple bars). The vertical dotted line indicates where the first assay ends and the second begins. c Representative images of animals treated with the indicated compounds. Time stamps indicate the time elapsed from the initial presentation of a low-volume acoustic stimulus. d These plots compare the motor activity (y-axis) over time (x-axis) of animals treated with DMSO (gray) or etomidate (red) (n = 50 larvae). Consecutive stimuli (n = 60) are indicated by vertical gray bars. e Dose-response curve showing phenoscores at the indicated concentrations (each point represents n = 12 wells/dose, error bars: ± SD). f Bar plot showing normalized response to the indicated stimulus (tap or violet light) of animals treated with DMSO, 6 μΜ propofol, or 6 μΜ etomidate (n = 12 wells, error bars: ± SD) for the indicated durations. g Average phenoscores (y-axis) of zebrafish treated with the indicated compounds. Dashed lines intersecting the y-axis at 0.51 and 0.71 correspond respectively to 1% and 5% significance cutoffs, as determined from statistical simulations. Compounds are grouped by ligand class: (1) GABABR agonist, (2) GABAAR orthosteric agonist, (3) PAM of δ-subunit containing GABAARs, (4) GABAAR BZ-site PAM, (5) GABAAR non-BZ-site PAM, (6) GABAAR neurosteroid PAM, (7) GABAAR anesthetic PAM

A high-throughput behavioral screen identifies GABAergic compounds. Zebrafish were treated with various compounds and analyzed for anesthetic-related behaviors. a This scatter plot compares phenoscores of individual wells treated with DMSO or etomidate (6.25 μΜ) (Z-factor = 0.7, n = 944 wells). b This contour plot scores each well from the large-scale behavior-based chemical screen (11,679 compounds, 2336 DMSO controls) by its phenoscore (y-axis) and immobilization index (x-axis). Labels indicate regions with 125 hit compounds (green), 44 toxic compounds (red), and the remaining screening compounds and DMSO controls (blue and gray, respectively). c Structural clustering of the top 125 hit compounds (y-axis) forms 14 clusters using a Tanimoto similarity metric (x-axis). d Example structures of selected compounds in the indicated clusters. e This scatter plot shows a 80.7% reproducibility rate for 57 primary hit compounds. Each point represents the average phenoscore of n = 12 wells at the indicated concentrations (colorbar). The first column represents DMSO controls; the order of other compounds are listed in Supplementary Table 4. f Human GABAAR activation (y-axis) was measured by FLIPR analysis. Of 47 hit compounds, 23 potentiated GABAARs. Compounds 7013338 and 5942595 potentiated GABAARs significantly greater than positive controls (red asterisk = 7013338, two red asterisks = 5942595, P< 0.0001, two-tailed t-test, n = 2–4 replicates as indicated). The hit threshold was defined as 2× the average DMSO control group. Picrotoxin, BGC 20-761, progesterone, and DMSO were used as negative controls (x-axis) while etomidate, tracazolate, propofol, diazepam, and thiopental were used as positive controls. Arrows indicate compounds that were predicted by SEA to bind GABAARs (red arrows) and compounds that bound to TSPO in vitro (green arrowheads)

Hit compounds activate hindbrain neurons. Animals were exposed to the indicated drugs and stimuli and analyzed for pERK levels as a readout of neuronal activity. a Plots showing motor activity (y-axis) over time (x-axis) for animals treated with the indicated compounds (n = 25–50 larvae) in response to the indicated acoustic (blue) or violet light (purple) stimuli. b, d, f, h, j Confocal projections showing the average fluorescent intensity of image registered larval brains stained with α-pERK (n = 10 larvae/condition). Larvae were treated with the indicated compounds and exposed to the low-amplitude acoustic stimulus once every 10 s for 10 min, except for (b, no stimulus) and (f, violet light exposure). c, e, g, i, k Brain activity maps showing significant ΔpERK signals using the Z-brain online reference tool (n = 5–10 animals/condition). The heatmap indicates positive (green), negative (purple), and nonsignificant (black) changes in pERK labeling (P< 0.0005, Mann–Whitney U test). All activity maps are comparisons between the indicated treatment conditions. Abbreviations: tel, telencephalon; ot, optic tectum; hb, hindbrain; ob, olfactory bulb; nm, neuromast; ap, area postrema; pg, pineal gland

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