Rapid behavior-based identification of neuroactive small molecules in the zebrafish

Abstract

Neuroactive small molecules are indispensable tools for treating mental illnesses and dissecting nervous system function. However, it has been difficult to discover novel neuroactive drugs. Here, we describe a high-throughput, behavior-based approach to neuroactive small molecule discovery in the zebrafish. We used automated screening assays to evaluate thousands of chemical compounds and found that diverse classes of neuroactive molecules caused distinct patterns of behavior. These 'behavioral barcodes' can be used to rapidly identify new psychotropic chemicals and to predict their molecular targets. For example, we identified new acetylcholinesterase and monoamine oxidase inhibitors using phenotypic comparisons and computational techniques. By combining high-throughput screening technologies with behavioral phenotyping in vivo, behavior-based chemical screens can accelerate the pace of neuroactive drug discovery and provide small-molecule tools for understanding vertebrate behavior.

Figure 1

The PMR and high—throughput behavioral barcoding. (a) Representative plot of the aggregate motor activity (in arbitrary units) over time (in seconds) from all animals in a single control well during the PMR assay. Prior to the first light stimulus (red bar at 10s), brief asynchronous movements in individual animals are recorded as narrow spikes with short (< 1 s) duration. A latency phase, lasting for approximately 1 s (marked with an arrow), exists between the first light pulse and the onset of the excitation phase. The PMR excitation phase is clearly distinguished from background as a large sustained increase in motor activity. Following the excitation phase, animals enter a refractory phase in which a second light pulse (red bar at 20 s) fails to elicit a response. (b) Nine zebrafish embryos in a single well of a 96—well plate. (c) Composite photograph of a 96—well microtiter plate prior to behavioral analysis. Each well contains 8—10 unhatched zebrafish embryos. (d) Fourteen features extracted from each PMR plot are converted into a `behavioral barcode.' Horizontal parallel lines in each of 7 time periods show the values of the motion index at the 75^th percentile (Q3), and 25^th percentile (Q1). Colors in the heat map represent their deviation from the average control phenotype. Purple represents increased activity and orange represents decreased activity. (e) PMR profiles and the respective behavioral barcodes of 96 untreated control wells.

Figure 2

Neuroactive chemicals cause specific patterns of behavior. (a—c) The photomotor response (PMR) in untreated control animals. Colors indicate motor activity levels as compared to the control population, with purple representing increased activity and orange representing decreased activity. (d—f) Isoproterenol, a psychostimulant, increases zebrafish motor activity (p = 6.53e-08). (g—i) Diazepam, an anxiolytic, reduces motor activity (p = 2.33e-07). (j—l) Apomorphine, a dopamine agonist, increases PMR latency period duration (p = 1.88e-06). (m—o) Digitoxigenin, a cardiotonic steroid, prolongs duration of the PMR excitation phase (p = 6.03e-07). (p—r) 6—Nitroquipazine, an SSRI, modulates the PMR refractory period (p = 6.73e-07). (c, f, i, l, o, r) Nine replicate behavioral barcodes from independent wells treated with the indicated compound.

Figure 3

Hierarchical clustering reveals that compounds cluster with functionally similar molecules. (a) A dendrogram of behavioral barcodes for all 1,627 hits clustered by phenotypic similarity. Candidate hits (y—axis) are clustered based on behavioral features (x—axis) from the PMR assay. Grey bars denote the presence of the indicated phenotypes in the ETR assay. The four clusters circled on the dendrogram are shown at higher resolution in panels b, c, d and e. Chemical names are colored based on annotated activity codes provided in parentheses. Compounds without functional annotation are labeled unknown (unk). (b) A phenocluster exhibiting excitation period prolongation is enriched for compounds that alter intracellular sodium levels (p = 4.71e-07). Inhibitors of Na⁺ channel inactivation (CHA) and the Na⁺/K⁺ pump (ATPPNAK) are colored pink and light green respectively. (c) A cluster exhibiting generalized stimulation is enriched for β-adrenergic receptor agonists (p = 9.94e-14)(ADR, dark green). (d) A cluster exhibiting prolonged latency is enriched for dopamine receptor agonists (p < 2.2e-16) (DOPA, red). (e) A cluster exhibiting attenuated excitation is enriched for adenosine receptor antagonists (p = < 2.2e-16) (ADS, orange). Indicated compounds (•) are structural analogs of the scaffold shown to the right of each cluster (f, g, h, i). PERM⁺, membrane permeability inhibitor; HIST, histaminergic; GABA, GABAergic; 5HT, serotonergic; EAA, glutamatergic; ES, esterase inhibitor.

Figure 4

Behavior—based discovery of novel neuroactive small—molecules. (a) Behavioral barcodes of chemicals causing the STR phenotype (p = 0.00034). Known AChE inhibitors are colored yellow. (b) Structures of eserine, a known AChE inhibitor, and two previously uncharacterized compounds that also cause the STR phenotype, STR—1 and STR—2. (c) AChE activity in vitro (black bars) or in vivo (gray bars). Data represent mean values ± s.d.. INSECT, insecticide; ACH, AChE inhibitor; P4501A1, cytochrome P450. (d) (e) Behavioral barcodes of structurally related MAG compounds. (e, f) Representative PMR plots of a control well compared to the effect of a MAG compound. Note the stimulus—dependent increase in excitation magnitude and duration. (g) Chemical structures of warfarin, a negative control, the known MAO inhibitor pargyline, and MAG—1. (h) MAO—B activity after treatment with the indicated compounds. Data points and error bars represent mean values ± s.d..

Figure 5

Chemical suppression of behavioral phenotypes. (a) Boxplots showing the mean excitation values for the 5 replicate wells shown in (b) and treated with DMSO alone, AzMet, an OP nerve poison, or AzMet co—treated with 2—PAM and atropine. (c) Boxplots showing the mean excitation values for the 5 replicate wells shown in (d) and treated with DMSO, clenbuterol (Clen), or clenbuterol and the beta—receptor antagonist bopindolol. For each treatment the box represents the middle half of the distribution of the data points stretching from the 25^th percentile to the 75^th percentile. The bold line across the box represents the median. The lengths of the lines above and below the box are defined by the maximum and minimum datapoint values.