www.sciencemag.org/cgi/content/full/327/5963/348/DC1

Supporting Online Material for

Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation

Jason Rihel,* David A. Prober, Anthony Arvanites, Kelvin Lam, Steven Zimmerman, Sumin Jang, Stephen J. Haggarty, David Kokel, Lee L. Rubin, Randall T. Peterson, Alexander F. Schier*

*To whom correspondence should be addressed. E-mail: [email protected] (A.F.S.); [email protected] (J.R.)

Published 15 January 2010, Science 327, 348 (2010) DOI: 10.1126/science.1183090

This PDF file includes:

Materials and Methods SOM Text Figs. S1 to S18 Table S1 References Supporting Online Material

Table of Contents

Materials and Methods, pages 2-4 Supplemental Text 1-7, pages 5-10 Text 1. Psychotropic Drug Discovery, page 5 Text 2. Dose, pages 5-6 Text 3. Therapeutic Classes of Drugs Induce Correlated Behaviors, page 6 Text 4. Polypharmacology, pages 6-7 Text 5. Pharmacological Conservation, pages 7-9 Text 6. Non-overlapping Regulation of Rest/Wake States, page 9 Text 7. High Throughput Behavioral Screening in Practice, page 10

Supplemental Figure Legends, pages 11-14 Figure S1. Expanded hierarchical clustering analysis, pages 15-18 Figure S2. Hierarchical and k-means clustering yield similar cluster architectures, page 19 Figure S3. Expanded k-means clustergram, pages 20-23 Figure S4. Behavioral fingerprints are stable across a range of doses, page 24 Figure S5. Compounds that share biological targets have highly correlated behavioral fingerprints, page 25 Figure S6. Examples of compounds that share biological targets and/or structural similarity that give similar behavioral profiles, page 26 Figure S7. Cluster analysis of multi-target compounds, page 27 Figure S8. Comparison of the effects of adrenergic drugs on rest/wake states in zebrafish to the effects in mammals, page 28 Figure S9. Comparison of the effects of serotonin drugs on rest/wake states in zebrafish to the effects in mammals, page 29 Figure S10. Comparison of the effects of dopamine drugs on rest/wake states in zebrafish to the effects in mammals, page 30 Figure S11. Comparison of the effects of GABAergic drugs on rest/wake states in zebrafish to the effects in mammals, page 31 Figure S12. Comparison of the effects of melatonin drugs on rest/wake states in zebrafish to the effects in mammals, page 32 Figure S13. Comparison of the effects of histamine drugs on rest/wake states in zebrafish to the effects in mammals, page 33 Figure S14. Comparison of the effects of adenosine drugs on rest/wake states in zebrafish to the effects in mammals, page 34 Figure S15. Comparison of the effects of glutamate drugs on rest/wake states in zebrafish to the effects in mammals, page 35 Figure S16. The L-type calcium channel inhibitor, YS-035, dose dependently increases rest with minimal effects on waking activity, page 36 Figure S17. Examples of immunomodulators that co-cluster as daytime-waking activity enhancers, page 37 Figure S18. ERG inhibitors, but not non-ERG blocking anti-histamine analogs, dose 1

dependently increase waking activity at night, page 38 Table S1. Summary of dose response experiments for selected compounds indicates fingerprints are stable across several concentrations, page 39

Supplemental References, pages 40-43.

Materials and Methods

Small molecule exposure. Larval zebrafish were raised on a 14 hour/10 hour light/dark cycle at 28.5oC. At four days post fertilization (dpf), a single larva was pipetted into each of 80 wells of a 96-well plate (7701-1651; Whatman, Clifton, NJ) containing 650 µL of standard embryo water (0.3 g/L Instant Ocean, 1 mg/L methylene blue, pH 7.0). Larvae were then exposed to small molecules beginning at ~96 hours post fertilization (hpf) by directly pipetting a 10 mM stock solution (in DMSO) into each well (10 animals per compound) for a final chemical concentration of ~10-30 μM and no more than 0.3% DMSO. This concentration of DMSO has no effect on behavior. Small molecules were obtained from the following libraries and screened at the following final concentrations: 1) Spectrum library from Microsource Discovery at ~30 µM (Gaylordsville, CT); 2) Prestwick library from Prestwick Chemical at ~15 µM (Illkirch, France); 3) neurotransmitter (cat#2810), ion channel (cat#2805), and orphan ligand libraries (cat#2825) from Biomol International at ~15 µM (now Enzo Life Sciences, International; Plymouth Meeting, PA); 4) Sigma LOPAC library at ~10 μM (Sigma-Aldrich, St. Louis, MO). Behavioral recording began at 11 PM, approximately 110 hpf.

Locomotor activity analysis. The behavioral assay is adapted from (S1). Locomotor activity was monitored for 60 hours using an automated video tracking system (Videotrack; Viewpoint Life Sciences, Montreal, Quebec, Canada) with a Dinion one- third inch Monochrome camera (model LTC0385; Bosch, Fairpoint, NY) fitted with a fixed-angle megapixel lens (M5018-MP; Computar) and infrared filter. The movement of each larva was recorded using the Videotrack quantization mode, and data from two cameras were collected in alternating minutes by one computer. The 96-well plate and camera were housed inside a custom-modified Zebrabox (Viewpoint, LifeSciences) that was continuously illuminated with infrared lights and illuminated with white lights from 9 AM to 11 PM. The 96-well plate was housed in a chamber filled with circulating water to maintain a constant temperature of 28.5oC. The Videotrack threshold parameters for detection used were: detection threshold, 40; burst, 25; freeze, 4; bin size, 60 seconds.

Data analysis. The data was processed using custom PERL, Visual Basic Macros for Microsoft (Seattle, WA) Excel, and Matlab (version R2007a; The Mathworks, Inc). Any one minute bin with less than 0.1 second of total movement was defined as one minute of rest; a rest bout was defined as a continuous string of rest minutes (S1). Rest latency was defined as the length of time from lights on or off to the start of the first rest bout. Total activity was defined as the average amount of detected activity in seconds, including all rest bouts. Waking activity was defined as the total amount of detected activity, excluding all one minute periods of rest. These parameters were calculated for each experimental day and night, standardized to camera-matched DMSO controls, and

2 expressed as +/- standard deviations from controls for hierarchical clustering (see below). We calculated the inter-assay variability as a coefficient of variation (standard deviation of all DMSO controls/mean of DMSO controls*100) for each measurement. For non-normal distributions, including rest length, rest latency, and wakingactivity, the coefficient of variation was calculated following a Box-Cox transformation to a normal distribution. The coefficient of variations (expressed as a %) are as follows: total rest, day 1, 42.3%, day 2, 42.1%, night 1, 24.4%, night 2, 19.9%; rest bouts, day 1, 27.3%, day 2, 21.9%, night 1, 16.7%, night 2, 11.3%; rest bout length, day 1, 14.6%, day 2, 12.9%, night 1, 15.6%, night 2, 11.3%; rest latency, day 1, 35.7%, day 2, 45.8%, night 1, 13.2%, night 2, 20.0%; total activity, day 1, 35.5%, day 2, 28.1%, night 1, 36.3%, night 2, 29.9%; waking activity, day 1, 15.5%, day 2, 17.9%, night 1, 18.9%, night 2, 15.6%. Much of the variability is due normal inter-individual variation.

To generate time series data for average rest per 10 minutes and average waking activity per 10 minutes (red traces, e.g. Figure 2B-F), these two parameters were averaged across all 10 replicate animals in 10 minute intervals and then normalized to camera matched controls by dividing each 10 minute rest and 10 minute waking activity measure by the mean daytime rest and activity for camera matched controls, respectively. For comparison, 10 representative sets of DMSO controls (10 larvae each set; e.g. blue traces in Figure 2B-F) are plotted in each time series. The same DMSO controls are shown in all graphs for clarity.

To identify compounds that significantly altered behavior, each parameter was compared to camera-matched DMSO controls by Student‟s t-test. Compounds were chosen for further analysis if either: 1) the compound altered the same parameter in the same direction at a p value < 10-5 for two consecutive days or nights, 2) the compound affected a behavioral parameter at p<10-10 within the first 24 experimental hours, or 3) the compound altered rest latency at p<0.001 for two consecutive days or nights but had little effect on other parameters. These parameters were chosen to select the largest manageable set of small molecules with the strongest and most consistent effects on behavior while minimizing false positives and compounds with spurious short- term behavioral alterations. Biological targets were assigned to compounds based on annotations in the Pharmaprojects, Drugbank, Drugs@FDA, and Leadscope Marketed Drugs databases, as well as information supplied by the library vendors and detailed manual literature searches for each compound.

Hierarchical and k-means clustering analysis. Clustering analysis was performed with Cluster 3.0 (adapted by Michiel de Hoon from Cluster, written by Michael Eisen, (S2)) and visualized using TreeView (written by Michael Eisen, (S2)). Hierarchical clustering was performed using Pearson uncentered correlation and average linkage. The parameters were weighted as follows: total rest, 1.0; rest bouts, 0.8; rest bout length 0.3, rest latency, 1.0; total activity and waking activity, 1.0. K-means clustering was performed using k=24 and Euclidean distance. To determine the relative position of the k-clusters, the average vector for each cluster was calculated, and these 24 vectors were hierarchically clustered using Euclidean distance.

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Correlation Analysis: All correlation analysis was performed in Matlab (version R2007a; The Mathworks, Inc). The weighted, uncentered Pearson‟s correlation coefficients were calculated for all compound pairs. Compounds were assigned to pharmacological classes as described above. Compounds were considered to share targets if both their target assignment and activity (i.e. agonist or antagonist) overlapped for at least one target.

Rank Correlation Analysis: To perform rank correlation analysis for adrenergic antagonists and ERG blocking drugs, the average weighted uncentered Pearson‟s correlation coefficient between each compound of the identified cluster and every other small molecule was calculated. Each small molecule was then assigned a rank based on their mean correlation, with rank 1 being the highest mean correlation. Compounds that showed ERG or adrenergic antagonist activity were identified by their pharmacological assignments, and the Kolmogorov-Smirnov statistic was used to assign the p value for whether ERG or adrenergic antagonist compounds were enriched among the higher ranking compounds. The rank correlations were graphically represented in Treeview (S2).

Dose Correlation Analysis. Compounds were ordered from Sigma-Aldrich (St. Louis, MO), dissolved in DMSO to a stock concentration of 30 or 45 mM and tested in the locomotor activity assay at final concentrations of 0.15, 0.45, 1.5, 4.5, 15, and 45 µM, or 0.1, 0.3, 1.0, 10, and 30 µM (0.3% DMSO final concentration). Each concentration was tested on 10-20 larvae. To generate the fingerprint for each concentration, the behavioral parameter measurements were standardized to camera-matched DMSO controls and expressed as +/- standard deviations from controls. Two-tailed t-test assessments (Bonferroni corrected for multiple comparisons) determined statistically significant differences of each measurement relative to camera-matched controls. The correlation matrices were generated by calculating the weighted, uncentered Pearson‟s correlation coefficient between the fingerprints for all concentration pairs. The fingerprint, significance, and correlation matrix heat maps were generated using Matlab (version R2007a; The Mathworks, Inc.).

MAO inhibitor assay. MAO-B activity was assayed as described (S3).

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Supplemental Texts 1-7

Supplemental Text 1. Psychotropic Drug Discovery The limitations of in vitro drug screening are particularly acute for the development of drugs that modulate the central nervous system (CNS). Surveys of drug discovery success show that CNS drugs take longer to develop (12.6 years versus 6.3 years for cardiovascular drugs) and fail to reach the market more often (7% success rate versus 15%) than other therapeutic indications (S4). The CNS poses a particular challenge to drug discovery because the brain cannot be modeled in vitro. Indeed, most modern psychotropic drugs were first developed for non-psychotropic uses and serendipitously discovered to have behavior-altering side effects (e.g. the MAO inhibitor, iproniazid, was developed to treat tuberculosis and found to alter mood, and the tricyclic antidepressant imipramine was first used as an anti-histamine, (S5)). Furthermore, CNS drugs tend to produce undesirable side effects, including dizziness, tremors, lethargy, and seizures, which are difficult to predict at early stages of the drug discovery pipeline. Also, the mechanism of action for many CNS drugs, including bipolar medications, antidepressants, and antipsychotics, is poorly understood and in many cases may depend on a compound‟s complex poly-pharmacological actions across multiple neurotransmitter systems (S6-7). Indeed, efforts to increase efficacy and reduce side effects through the development of newer generations of drugs with more selective target profiles has to date achieved only limited success (S8-9).

Alternatives or complements to in vitro target-based screens include phenotype-based, whole organism screens. Such screens have several advantages. First, whole- organism screens need not be limited to single, well-validated targets. In fact, whole organism screens can identify efficacious compounds with complex mechanisms of action. Second, unwanted side effects, an obstacle in any drug discovery pipeline, can be detected at earlier stages, even during the primary screening process. Third, compounds that produce a behavioral effect are bio-available, another major stumbling block to in vitro based drug discovery pipelines. In principle, whole organism phenotypic screening could replace serendipity, but the requirement for large numbers of phenotyped animals is usually cost and time prohibitive. Behavioral screening in zebrafish larvae offers a cost-effective model system for psychotropic drug characterization and discovery.

Supplemental Text 2. Dose Choosing the compound concentration is a challenge for any large-scale small molecule screen. Effective doses can vary among compounds, but it is impractical in most cases to screen at multiple concentrations. The decision to perform our screen in the 10-30 µM range was based on the following considerations. First, we were guided by several previous studies. Developmental screens in zebrafish used concentrations of 10-50 µM (S10-12), single compound behavioral studies in zebrafish were done at 1-100 µM (S13-19), and the connectivity map cell-based screen was performed at 10 µM (S20, 21). Second, we performed a pilot screen of 96 compounds and found that only 10% of

5 the compounds were overtly toxic at the 30 μM range, a percentage deemed acceptable for screening. Third, dose response curves on selected compounds indicated that a concentration of 10-30 µM was an effective range with consistent behavioral effects for most compounds (Table S1 and Fig. S4). Specifically, we found that fingerprints for the same compound were strongly correlated (25/29 cases) across the statistically effective concentration range (Fig. S4). These results show that the behavioral fingerprints are broadly stable across multiple doses.

Supplemental Text 3. Therapeutic Classes of Drugs Induce Correlated Behaviors To test the potential of the clustering analysis, we determined whether compounds that share targets are more likely to produce similar phenotypes (Fig. S5A-B). Expanding this correlation analysis to 50 categories of drugs, we found, with few exceptions, that the correlations among related classes of compounds were much larger than chance (Fig. S5C). In many cases, every intra-class pair-wise correlation was larger than the zero target correlation (see Figure Legend S5 for definitions of category abbreviations; + indicates agonist, - indicates antagonist), including the ADR-A2+ (median correlation = 0.853; range = 0.352-0.974), SNRI (median = 0.775; range = 0.453-0.956), 5HT-1A+ (median = 0.791; range = 0.463-0.958), NEURO- (median = 0.759; range = 0.587- 0.894), DOPA-D1+ (median = 0.885; range = 0.766-0.927), and CHA-NA+ (median = 0.833; range = 0.519-0.985) classes (Fig S5C). In other examples, intra-class pair-wise correlations were much larger except for a few outliers, including the SSRI (median = 0.767 ; range = 0.262-0.963), 5HT-1A- (median = 0.769; range = -0.02-0.930 ), 5HT-2- (median = 0.698; range = 0.187-0.977), DOPA-D2+ (median = 0.727; range = 0.072- 0.982), GABA- (median = 0.722; range = -0.05-0.972), HIST-H1-(ERG) (median = 0.722; range = 0.229-0.911), and NMDA- (median = 0.651; range = -0.419-0.971) classes (Fig. S5C). Not all classes had higher correlations than the no target class, including the adrenergic reuptake class (ADR-U; median = 0.257; range = -0.50-0.959) and the sodium channel antagonist class (CHA-NA-; median = -0.031; range = -0.513 – 0.913). This may reflect the heterogeneity of secondary targets for the compounds of these classes.

Supplemental Text 4. Polypharmacology. Many compounds have multiple, potentially non-overlapping targets. In such cases, one would expect that not all compounds that share targets produce similar phenotypes (Fig. S5A-C). For example, outliers of the correlation analysis might reflect the difficulty in assigning compounds to single classes when in fact they have multiple targets. This observation can be used to identify potential polypharmacological effects (Figs. S5C and S7). For example, NMDA antagonist (NMDA-) outliers included compounds that also bind to σ-opioid receptors (e.g. 3-methoxymorphinan), the GABA antagonist (GABA-) outlier tert-butyl-bicyclophosphorothionate (TBPS) also potently blocks chloride channels, the serotonin receptor 1A antagonist (5HT-1A-) outlier S(-)UH-301 is also a dopamine D2/3 receptor agonist, and the dopamine D2 receptor agonist (DOPA-D2+) outlier pergolide is also a serotonin receptor agonist (Fig S5C). Our correlation analysis can also uncover higher order associations among drugs with multiple targets, such as the high correlation between drugs that share dopamine reuptake inhibition and acetylcholine antagonism (Figure S7A-B). Furthermore, the clustering analysis can

6 reveal unexpected associations between annotated drug classes. For example, a cluster of known α1-adrenergic receptor antagonists (ifenprodil, terazosin, and 5-methyl- urapidil) also cluster with several serotonin receptor modulators, including NAN-190, RU-24969, WAY-100635, spirotraxine, and buspirone (Fig. S7C-D). Intriguingly, buspirone and spirotraxine do not strongly bind to α1-adrenergic receptors in vitro yet can show in vivo adrenergic activity in specific pharmacological contexts (22). These results show that behavioral profiling can uncover associations between multi-target drugs whose properties are not fully predicted by in vitro studies.

Supplemental Text 5. Pharmacological Conservation (Figs. S8-15). We systematically compared the behavioral effects of agonists and antagonists of the major neurotransmitter systems between zebrafish and mammals. In most cases, confidence in the conservation between zebrafish and mammals is enhanced when multiple compounds known to affect the same target elicit similar phenotypes and when agonist/antagonist pairs elicit opposing phenotypes. We discuss each class in more detail below.

Adrenaline (Fig. S8). In zebrafish, α2-adrenergic agonists decreased wakefulness with little effect on rest, while activation of β-adrenergic receptors selectively reduced total rest at night. Conversely, β-adrenergic receptor antagonists increased total rest. α1- adrenergic antagonists gave a “mixed” phenotype of increased waking activity but also increased rest. These effects are broadly consistent with those observed in mammals (S23-35).

Serotonin (Fig. S9). The analysis of serotonin-regulating drugs is complicated because most, if not all, serotonin-modulating drugs have considerable affinity for non-serotonin receptors, especially dopaminergic and adrenergic receptors. Thus, the drugs chosen for the table in Figure S9 combine serotonin-signaling selectivity, consistency of phenotype, and reproducibility (i.e. multiple hits within the screen of the same or similar compounds). As often as possible, the drugs shown have known mammalian phenotypes (S36-55). The complexity of the phenotypes we observed with serotonergic agonists and antagonists is similar to the controversial and contradictory nature of the mammalian literature pertaining to these drugs. This may reflect the difficulty in isolating the drugs‟ effects on serotonin signaling from other secondary effects, differences between short-term and long-term exposure, or complex signaling interactions among different populations of serotonergic neurons in the brain. For example, in mammals agonists and antagonists of serotonin receptor 1A both cause short-term reduction of sleep and increase in wakefulness followed by longer-term increases in sleep and reduced activity (S36-40, S45-47). In long-term experiments, both classes of drugs increased rest in zebrafish. Only buspirone had an observed short-term increase in waking during the first night of the experiment (black arrow in Fig. S9). Serotonin receptor 2/3 antagonists have paradoxical effects in mammals, increasing both exploratory behavior and sleep (S38, S44, S49-52). Similarly, both waking activity and total rest were increased in zebrafish. Finally, SSRIs consistently decreased waking activity and increased rest in zebrafish; in human patients, SSRIs

7 often improve insomnia in depressed patients but can have biphasic effects on sleep, with long-term decreases in sleep in some mammalian models (S53-55).

Dopamine (Fig. S10). With one notable exception, dopamine receptor agonists and antagonists produce similar phenotypes in mammals and zebrafish (S56-67). For example, the D2-receptor agonists consistently reduced waking activity and increased rest in zebrafish, as they do at low concentrations in mammals (S60-63). This effect was receptor subtype selective, as the D2/D3 receptor agonists quinelorane and quinpirole only reduced waking activity (S64, S65). As another example of conservation, D2-receptor antagonists, several of which are used as anti-psychotics, increase waking activity and rest in zebrafish and mammals (S57, S62, S67). Previous work indicated that antipsychotics produce locomotor defects in zebrafish larvae (S15- 16). The only clear examples of non-conserved pharmacology in our study were the dopamine D1 agonists (Fig. S10, marked in gray), all of which had little effect on waking activity but greatly increased total rest in zebrafish. In all mammalian species tested, dopamine D1 agonists increased waking (S56-59). This could indicate a distinct role for the zebrafish orthologs of D1 receptors in the control of behavior or that these dopamine D1 agonist drugs have altered target selectivity in zebrafish.

GABA (Fig. S11). While many of the GABA-regulating benzodiazepine drugs were toxic at the concentrations used in our screen, the GABA-A agonists GBLD-345 and avermectin B1 increased total rest and had only a small effect on waking activity (S68). Although only represented by a single hit in the screen, the GABA-B agonist CGP- 13501 (Fig. S11, marked in gray) slightly increased waking activity at night with very modest increases in rest during the day, even though this drug is hypnotic in mammals (S69). In contrast, many GABA-A antagonists dramatically increased waking activity, possibly reflecting pro-convulsant effects of these drugs on zebrafish larvae, which is consistent with the mammalian literature (S70-72) and with previous work in zebrafish (S13, S17, S19). Tracazolate, an anxiolytic acting as an allosteric modulator of GABA- A receptors, modestly increased waking activity in zebrafish; in mammals, tracazolate can both increase or decrease locomotor activity (S73, S74). Intriguingly, the neuroactive steroid GABA agonists allopregnanolone, pregnanolone, and alfadolone increased both total rest and waking activity specifically at night (S75-77). As neuroactive steroids have additional functions in mammalian brains, including modulation of NMDA receptors (S75), this mixed phenotype may reflect the polypharmacology of these compounds.

Melatonin (Fig. S12). Although only a few examples were present in our screen, melatonin-modulating drugs gave results that are consistent with those observed in mammals (S78-82) and previous work in zebrafish (S14). Melatonin slightly increased rest in our long-term experiments. In addition, the melatonin agonist 8-methoxy-1- propionamidotetralin decreased waking activity and increased rest. An exception was the agonist IIK7 (S78), which slightly increased waking activity at night and had small effects on rest.

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Histamine (Fig. S13). Histamine antagonists identified in our screen tended to increase rest, decrease waking activity, or both. This increased rest in zebrafish is consistent with the mammalian literature, although it should be noted that anti-histamine drugs that do not cross the blood-brain barrier in humans, such as loratadine, tend to be less sedating than the older generation of drugs that do cross the blood-brain barrier (S83- 87). Our work is also consistent with previous work on anti-histamines in zebrafish larvae (S13, S18). Notably, the anti-histamine diphenylpyraline increased waking activity, consistent with reports of psychostimulatory effects in rodents (S87). Also, as noted in the main text and Figures 4C-D and S18, anti-histamines that block the human ether-a-go-go related potassium channel (ERG) consistently increased waking activity at night, with modest effects on rest in zebrafish larvae.

Adenosine (Fig. S14). Consistent with mammals, adenosine receptor agonists increased rest in zebrafish larvae, while adenosine receptor A1 antagonists increased waking activity and reduced rest (S88-93). Although behavioral effects of the adenosine A3 antagonist MRS-1220 are unknown in mammals, it decreased waking activity and increased rest in zebrafish. This phenotype is consistent with its inhibitory effects on MAO (Figure 3D).

Glutamate (Fig. S15). All NMDA receptor antagonists identified in the screen dramatically increased waking activity during the day and night, consistent with the effects observed in mammals (S94, S95). Muscarinic glutamate receptor 5 (mGluR5) antagonists selectively increased rest, with little effect on waking activity. Antagonists of mGluR5 can block drug-induced hyperlocomotion, down-regulate bursting of prefrontal cortical neurons in awake rats, and have anxiolytic properties in rodents (S96, S97).

Supplemental Text 6. Non-overlapping Regulation of Rest/Wake States. Many pharmacological agents selectively altered waking activity, rest latency, or total rest. For example, verapamil related L-type calcium channel inhibitors increased rest with no effect on waking activity (Fig. S16), while β-adrenergic agonists, including clenbuterol and fenoterol, decreased rest only (Fig. S8). Non-competitive NMDA receptor antagonists, including the psychotomimetics dizocilpine (MK-801) and L- 701324, predominantly increased waking activity with minimal effects on total rest (Figs. 2C and S15), the D2/D3 agonist, ropinirole, predominantly decreased waking activity only (Fig. S10), and podocarpatrien-3-one analogs predominantly altered only rest latency (Fig. 4A). These single parameter effects can also be selective for day versus night. For example, many anti-inflammatory compounds only increased waking activity during the day (Figs. 2B, 4B and S17), while ERG-blocking drugs increased waking activity at night (Figs. 2E, 4C-D, S18). Finally, some compounds can affect waking activity and total rest in opposite directions (e.g. increase both waking activity and total rest). For example, the α1-adrenergic antagonists ifenprodil, the antimalarial quinacrine, and the steroidal GABA agonist alfadolone, increased both waking activity and total rest (Figs S7C, S11). These observations suggest that the behavioral parameters of waking activity, total rest, and rest latency during the day and the night can be under the control of distinct pharmacological mechanisms.

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Supplemental Text 7. High Throughput Behavioral Screening in Practice Behavioral profiling in zebrafish is highly efficient. Our current zebrafish behavior room (~200 sq. ft.) has 16 cameras that can each observe 80 wells of a 96-well plate, for a total of 1280 larvae. Using our original screening method of 10 larvae per compound and including 10 control wells for each camera, we can screen 112 compounds (16 cameras x 7 drugs per camera) every three days, or 224 compounds per week. Using the latest videotracking software, which can track all 96 wells of the plate, and cutting the number of larvae per drug to eight expands our capacity to 352 compounds per week (16 cameras X 11 drugs per camera X 2 runs per week). A single technician can set up the entire behavioral room in less than 4 hours, including fish husbandry, larval pipetting, drug dispensing, and software setup (an 8 hour commitment per week). Expanding the screen to 115 cameras and 5 technicians (a total commitment of 40 hours per week) would increase the throughput to more than 10,000 compounds per month and more than 100,000 per year.

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Supplemental Figure and Table Legends

Figure S1. Expanded hierarchical clustering analysis. This is an expanded version of Figure 2A in which the name of each chemical in the clustergram can be seen. Each row represents a different chemical, and each column represents a behavioral measurement. From left to right, these measurements are: the rest total, the number of rest bouts, the rest bout length, the rest latency, the total activity, and the waking activity. The black bars indicate night measurements; the white bars indicate day measurements. Yellow indicates that the value is increased relative to controls; blue indicates that the value is decreased, as in Figure 2A. The measurements are normalized as standard deviations from control values.

Figure S2. Hierarchical and k-means clustering yield similar cluster architectures. Each row represents a different chemical, and each column represents a behavioral measurement. From left to right, these measurements are: the rest total, the number of rest bouts, the rest bout length, the rest latency, the total activity, and the waking activity. The black bars indicate night measurements; the white bars indicate day measurements. Yellow indicates that the value is increased relative to controls; blue indicates that the value is decreased, as in Figure 2A. The measurements are normalized as standard deviations from control values.

Figure S3. Expanded k-means clustergram. This is an expanded version of Figure S2 in which the name of each chemical in the clustergram can be seen. Each row represents a different chemical, and each column represents a behavioral measurement. From left to right, these measurements are: the rest total, the number of rest bouts, the rest bout length, the rest latency, the total activity, and the waking activity. The black bars indicate night measurements; the white bars indicate day measurements. Yellow indicates that the value is increased relative to controls; blue indicates that the value is decreased, as in Figure 2A. The measurements are normalized as standard deviations from control values.

Figure S4. Behavioral fingerprints are stable across a range of doses. Select compounds were tested for behavioral effects at multiple concentrations (see Table S1 for additional examples and summary data). First, the fingerprints for each dose were calculated as standard deviations from DMSO controls („Fingerprint‟, left panels). In general, these fingerprints are stable across multiple concentrations until the dose is too low to elicit a phenotype. To assess the stability of single drug fingerprints over multiple concentrations, the Pearson uncentered correlation coefficient was calculated pairwise between each dose („correlation matrix‟, right panels). With the exception of pergolide and several other ergoline derivatives, which have distinct fingerprints at high versus low concentrations, these examples have relatively stable fingerprints from the highest dose tested (45 μM) to the lowest effective doses (150 nM for NAN-190 and

11 betamethasone, 450 nM for ropinirole, 1.5 µM for clonidine and buspirone), as indicated by the high correlations (red boxes). See Materials and Methods for additional information.

Figure S5. Compounds that share biological targets have highly correlated behavioral fingerprints. (A) The distribution of all pair-wise correlation coefficients (Pearson weighted, uncentered; see Materials and Methods) for compounds that share at least 1 target (blue) is shifted to higher values compared to compounds that share 0 targets (magenta). This skewing is strong despite the fact that these compounds can have many divergent targets in addition to the shared target. Each pair of compounds is represented once, even if they share multiple targets. (B) Box-whisker plot comparing correlations between compounds with 0 shared targets, compounds with at least 1 shared target, and repeats of the same compound from different chemical libraries. For each measure, the blue box represents the interquartile range between the 25th and 75th percentiles; the red line marks the sample median; the whiskers extend from the box ends to values within 1.5 times the interquartile distance; red crosses mark outlier values beyond the 1.5 interquartile distance from the box ends. (C) Box-whisker plots of correlation coefficients within biologically related classes of compounds. For comparison, the mean correlation of compounds that share 0 targets is plotted as a green line (correlation = 0.266). The number of compounds used for comparisons within each class is indicated (N). See supplemental text 3 for a detailed discussion of specific classes. For all classes, a (-) sign indicates an inhibitor/antagonist and a (+) sign indicates an agonist. The classes are: α-adrenergic-receptor 1, ADR-A1; α-adrenergic receptor-2, ADR-A2; β-adrenergic, ADR-B; noradrenaline reuptake inhibitor, ADR-U-; serotonin/noradrenaline reuptake inhibitor, SNRI; selective serotonin reuptake inhibitor, SSRI; serotonin receptor-1A, 5HT-1A; serotonin receptor-1D, 5HT-1D; serotonin receptor-2, 5HT-2; serotonin receptor-2/3, 5HT-2/3; serotonin receptor 4, 5HT-4; monoamine oxidase inhibitor, MAO-; neurotransmitter inhibitor (reserpine class), NEURO-; dopamine receptor, DOPA; dopamine D1 receptor, DOPA-D1; dopamine D2 receptor, DOPA-D2; dopamine D2/3 receptor, DOPA-D2/3; dopamine D3 receptor, DOPA-D3; dopamine D4 receptor, DOPA-D4; γ-butyric acid receptor, GABA; histamine H1 receptor, HIST-H1; ether-a-go-go-related potassium channel, ERG; adenosine signaling, ADS; metabotropic glutamate receptor 5, mGLUR5; N-methyl-D-aspartic acid receptor, NMDA; acetylcholinesterase inhibitor, ACH-ES- IRev, irreversible; Rev, reversible; muscarinic acetylcholine receptor; ACH-M; nicotinic acetylcholine receptor, ACH-N; acetylcholine synthesis, ACH-SY; L-type calcium channel; CHA-CA-L; sodium channel, CHA-NA; peroxisome proliferator-activated receptor, alpha, PPAR-A.

Figure S6. Examples of compounds that share biological targets and/or structural similarity that give similar behavioral profiles. (A) Structurally divergent selective serotonin reuptake inhibitors (SSRIs) similarly decrease waking activity and increase rest. (B) Structurally related insecticides dramatically increase waking activity independent of the time of day. The graphs represent the normalized waking activity and total rest for behavior-altering compounds (red trace; average of 10 larvae) and representative controls (10 blue traces; average of 10 larvae each).

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Figure S7. Cluster analysis of multi-target compounds. A dopamine reuptake inhibitor (DOPA-U) with muscarinic acetylcholine antagonist (ACH-M-) activity does not correlate well with (A) compounds with only DOPA-U activity or (B) compounds with only ACH-M- activity. It does, however, show high correlation with other compounds known to share both properties (B). (C) α1-adrenergic receptor antagonists (marked in blue) form a large cluster with serotonin modulators (marked in green) that are known to also have α1- adrenergic receptor antagonist activity in vivo. (D) Rank-correlations of compounds across the entire dataset compared to the α1-adrenergic receptor antagonist cluster of ifenprodil, terazosin, and 5-methyl-urapidil enriches for compounds with α1-adrenergic receptor antagonism. In the rank-graph, the black lines indicate compounds with known α1-adrenergic antagonism; red indicates high correlation, green indicates low correlation. Compounds with α1-adrenergic antagonism are highly enriched in the top ranks (p < 10-23; see Materials and Methods).

Figure S8-15. Comparison of the effects of neuroactive drugs on rest/wake states in zebrafish to the effects in mammals. The graphs represent the waking activity and rest for representative agonists and antagonists of each class (red trace; average of 10 larvae) and DMSO controls (10 blue traces; average of 10 larvae each). Each plotted example is typical for the entire class of compounds. The tables provide a more comprehensive list of agonists and antagonists of each receptor subtype. The tables summarize the overall direction and magnitude of the observed effects of the drugs on zebrafish wake and rest behavior (up arrow, increased; down arrow, decreased; a dashed line indicates no change; a small arrow indicates a smaller relative effect, while a large arrow indicates a larger relative effect). See supplemental text 5 for a more detailed discussion of each phenotype.

Figure S16. The L-type calcium channel inhibitor, YS-035, dose dependently increases rest with minimal effects on waking activity. (A) Waking activity and rest graphs are shown for three structurally related L-type calcium channel inhibitors (red trace = average of 10 larvae) and representative DMSO controls (10 blue traces = average of 10 larvae). (B) YS-035 increases the time spent at rest during both the day (red) and night (blue) in a dose dependent fashion. Each point indicates the average minutes of rest per hour for 10 larvae at each YS-035 concentration. The error bars represent +/- SEM. (C) YS-035 has little or no effect on waking activity at all concentrations tested, suggesting that muscle function is not dramatically perturbed. Each point indicates the average seconds of waking activity every 10 minutes during the day (red) or night (blue) for 10 larvae at each YS-035 concentration. Error bars represent +/- SEM (B, C).

Figure S17. Examples of immunomodulators that co-cluster as daytime-waking activity enhancers. (A) The table lists anti-inflammatory agents that selectively increased daytime waking activity (expanded examples from Figure 4B). (B) Waking activity and rest graphs are shown for compounds from each major class (red trace = average of 10 larvae) and representative controls (10 blue traces = average of 10 larvae).

Figure S18. ERG inhibitors, but not non-ERG blocking anti-histamine analogs, dose dependently increase waking activity at night. (A) Waking activity and rest graphs are

13 shown for two ERG-blocking compounds (red trace = average of 10 larvae) and representative controls (10 blue traces = average of 10 larvae). Both compounds increased waking activity at night without affecting rest. Psora-4, which blocks Kv1.3 shaker channels, increased nighttime waking activity and also strongly decreased rest, a phenotype distinct from ERG blockers. Thus, the ERG phenotype is not the result of general potassium channel misregulation. (B) Dose response curves for ERG blocking and ERG non-blocking analogs are shown. Each point indicates the waking activity at night (seconds per minute) averaged for 10 larvae. Cisapride and dofetilide, which have ERG blocking activity, dose-dependently increased nighttime wakefulness, while the non-ERG blocking histamine H1 receptor antagonists fexofenadine and cetirizine had no effect on wakefulness. Another non-ERG blocking anti-histamine, loratadine, reduced waking activity. Error bars represent +/- SEM. The 0 M concentration is a DMSO control.

Table S1. Summary of dose response experiments for selected compounds indicates fingerprints are stable across several concentrations. This table is an expansion of the dose response fingerprints shown in Fig. S4. Note that only ergoline-derived compounds (bottom of table) have complex dose dependent effects (i.e. different nontoxic fingerprints at high versus low concentrations). This complex dose dependence is likely due to the high affinity that many ergoline drugs have for multiple receptor families and sub-types, including dopamine, serotonin, and adrenergic receptors (for example, see (S98)).

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Figure S1

Rest Rest

Bout

Total

Bouts

Rest

Total

# Rest #

Length

Activity Waking Activity Activity Latency NAME Activity 1 Activity 2 Activity3 Activity 4 Senecrassidol 6-Acetate Glycine NMDA+ MG-624 ACH-N- Physcion BACT- Budesonide ARACH- LIPCOR+ Diclofenac Sodium ARACH- 14-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-one 5-beta-12-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-one Enoxolone 11-alpha-Acetoxykhivorin Dinitolmide Chlorthalidone DIUR+ 3-beta-Acetoxydeoxodihydrogedunin Kynurenine Estrone Acetate ESTR+ Betamethasone ARACH- LIPCOR+ IM- GluR+ Clenbuterol Hydrochloride ADR-B-2+ Telmisartan ANG-2-1- PPAR-G+ SB 206553 Hydrochloride 5HT-2-B/C- Psora-4 CHA-K- Fenoterol Hydrobromide ADR-B+ Ornithine alpha-Ketoglutarate Chlorophyllide Cu Complex Sodium Salt Oxiconazole Nitrate FUNG- Sulfaguanidine BACT- Benzyl Penicillin Potassium BACT- Retinoic Acid p-Hydroxyanilide APOP+ Pimozide DOPA-D-2- 5HT-2-A- ADR-A-1- DOPA-U- Chlorpromazine Hydrochloride DOPA-D-2- DOPA-D-1- 5HT1-2- HIST-H-1- Mitotane ESTR- CHA-NA? SB 228357 5HT-2-B/C- Maprotiline Hydrochloride ADR-U- 5HT-U-(weak) Amitryptiline Hydrochloride ADR-U- 5HT-U- CHA-K- R(-)-Isoproterenol (+)-Bitartrate ADR-B-2+ ADR-B-1+ Maprotiline Hydrochloride ADR-U- 5HT-U-(weak) Carbenoxolone Sodium I-OMe-Tyrphostin AG 538 KI-IGF-1- (-)-Eseroline Fumarate OPI-M+ ACH-(weak) Bacitracin BACT- Leflunomide DH-HYO- KI-GFPL- ALOX5- Meclizine Hydrochloride HIST- ACH- Pancuronium Bromide ACH-N- Fenoldopam DOPA-D-1-A+ ADR-A-2+ Chloroethylclonidine Dihydrochloide ADR-A-1- ADR-A-2+ Mecamylamine Hydrochloride ACH-N- 1-Benzyl-1-Methyl-4-Cyclopentylmethoxycarbonylpiperidinium Bromide ACH-T- Tegaserod Maleate 5HT-4+ 5HT-2-B- Austricine LP-P-A-2- Azobenzene Diflunisal SY-PG- COX- Piroxicam SY-PG- COX- Flecanide CHA-NA- Flunisolide ARACH- LIPCOR+ IM- GluR+ Atrazine HERB- Capsazepine VAN- CHA-Na+ Cyclosporin A IM- 2-Cyclooctyl-2-Hydroxyethylamine Hydrochloride NEURT- PNMT- Oxethazaine Spectinomycin Hydrochloide BACT- Tetrahydrotrimethylhispidin Theaflavin Digallate Nf-kB-? Equilin ESTR+ SKF 94836 ES-P-C-3- Aminophenazone Diplosalsalate ES-P-C-? Fosfosal ES-P-C- Clobetasol Propionate ARACH- LIPCOR+ IM- GluR+ Fenoprofen SY-PG- COX- Nicotine Ditartrate ACH-N+ (-)-Nicotine ACH-N+ Fenoprofen Calcium Salt Dihydrate SY-PG- COX- 5-Aminopentanoic Acid Hydrochloride Catechin Tetramethylether Pentamidine Isethionate DNA- Valproate Sodium HDAC- GABA+ Betamethasone ARACH- LIPCOR+ IM- GluR+ Mefloquine Hydrochloride CALM- Clobetasol Propionate ARACH- LIPCOR+ IM- GluR+ Flumethasone ARACH- LIPCOR+ IM- GluR+ Desoxycorticosterone Acetate COR-M+ MR+ Ethylene Glycol-Bis(2-Aminoethylether)-N,N,N,N-Tetraacetic Acid ACE-2- CHEL+ Dexamethasone ARACH- LIPCOR+ IM- GluR+ Propentofylline ES-P-C- ADS-/+ Bopindolol Malonate ADR-B-1- Betamethasone Valerate ARACH- LIPCOR+ IM- GluR+ Bretylium Tosylate ADR-B- NA/K-ATPase- Pancuronium Bromide ACH-N- Cepharanthine APOP- NF-KB- IM- Ursolic Acid ARACH- LIPCOR+ IM- GluR+ Benzamil Hydrochloride CHA-NA-Ca- Cefoxitin Sodium Salt SY-CW- BACT- Papaverine Hydrochloride ES-P-C-4- Medrysone ARACH- LIPCOR+ IM- GluR+ 6-alpha-Methylprednisolone ARACH- LIPCOR+ IM- GluR+ 6,7-Dichloroquinoxaline-2,3-dione EAA-G- NMDA- L-689560 EAA-G- NMDA- 7-Chlorokynurenic Acid EAA-G- NMDA- SDZ 220-581 EAA-G- NMDA- IEM-1460 EAA-G- AMPA-R- NMDA- Finasteride RED-5A- Bufexamac SY-PG- COX- OX-C- Ro 20-1724 ES-P-C-4- Cisapride 5HT-4+ ACH+(indirect) CHA-K- (+)-Tubocurarine Chloide ACH-N- ACH+ 5HT-3- Hydrocortisone ARACH- LIPCOR+ IM- GluR+ L-701324 EAA-G- NMDA- L-701324 EAA-G- NMDA- L-701324 EAA-G- NMDA- Sinapic Acid Methyl Ether Dizocilpine Maleate EAA-G- NMDA- (-)-MK-801 Hydrogen Maleate EAA-G- NMDA- (+)-MK-801 EAA-G- NMDA- Allopregnanolone GABA-A+ CHA-CA- Tetrandrine CHA-CA- CHA-K-(Ca+) Diphenylpyraline Hydrochloride HIST-H-1- Spiperone DOPA-D-2- 5HT-1-A- 5HT-2-A- TG003 KI-CLK- Hexylresorcinol Monobenzone SY-PG- 2,4-Dihydroxychalcone 4-Glucoside Astemizole HIST-1- CHA-K- 3-(1H-Imidazol-4-yl)Propyl Di(p-Fluorophenyl)Methyl Ether Hydrochloride HIST-H3-/+ Dichlorodiphenyltrichloroethane CHA-NA+ INSECT- Tracazolate Hydrochloride GABA-A- GABA-A+ 1,4-PBIT Dihydrobromide NO-S- Menadione VITA+ (-)-Mk-801 Hydrogen Maleate EAA-G- NMDA- S,S,S,-Tributylphosphorotrithioate ACH- Endosulfan GABA-A- Coumaphos ACH- Amoxapine ADR-U- 5HT-U- Megestrol Acetate ESTR+ PROG+ L-701252 EAA-G- NMDA- Finasteride RED-5A- ANDR- Rutilantinone BACT- Terfenadine HIST-1- CHA-K- N-Desmethylclozapine 5HT-2-C- ACH-M1+ Mianserin Hydrochloride ADR-U- ADR-A-2- HIST- 5HT-2-A/2-C- ODQ CYC-GU- Clorgyline Hydrochloride MAO-A- 15 Amoxapine ADR-U- 5HT-U- Figure S1(Cont) NAME Activity 1 Activity 2 Activity3 Activity 4 Loxapine Succinate DOPA-D-1- DOPA-D-2- 5HT-2- (indirect?) Propazine HERB- Bepridil Hydrochloride CHA-CA- CHA-NA- CHA-K- 1S,9R-Hydrastine GABA-A- Dacthal HERB- Chlordane GABA-A- INSECT- ATPP-Ca/MgATPP- -Na/K- 7-Oxocallitrisic Acid, Methyl Ester Ibudilast ES-P-C-4- Roxithromycin PRT-50S- NF-kB- LY-367265 5HT-2-A- 5HT-U- Naftopidil Dihydrochloride ADR-A-1- ADR-U-? 5HT-2-? Clozapine 5HT-2- DOPA-D-2- ADR- HIST-H-1- Halofantrine Hydrochloride CHA-K- Amperozide Hydrochloride 5HT-2- DOPA-D-2- Haloperidol DOPA-D-1-2- NMDA- CHA-K- Terfenadine HIST-1- CHA-K- Fiduxosin Hydrochloride ADR-A-1- Strobane CHA-NA+ INSECT- Oxatomide HIST-1- 3-alpha-Bis-(4-Fluorophenyl)Methoxytropane DOPA-U- ACH-M- Dihydroergotamine Mesylate 5HT-1-D+ 5HT-1-A+ ADR-A-2-ADOPA-D-2/D-3 Methiothepin Maleate 5HT-1-2- DOPA-D-2- Mianserin ADR-U- ADR-A-2- HIST- 5HT-2-A/2-C- Methoxychlor CHA-NA+ INSECT- Toxaphene CHA-NA+ INSECT- Heptachlor GABA-A- INSECT- ATPP-Ca/MgATPP- -Na/K- Aldrin GABA-A- INSECT- ATPP-Ca/MgATPP- -Na/K- Bromo-3-Hydroxy-4-(Succin-2-yl)-Caryolane gamma-Lactone Cyproterone Acetate ANDR- Promethazine Hydrochloride HIST-1- ACH-M- 5HT-2- calmodulin- Tyrphostin AG 494 KI-GFEP- 8-Cyclopentyl-1,3-Dipropylxanthine ADS-A-1- Flutamide ANDR- Derrusnin Pregnenolone GABA-A- Deptropine Citrate HIST- ACH-M- Clemastine HIST-1- ACH-M-? Spermine Tetrahydrochloride EAA-G- EAA-G+ NMDA- Homopterocarpin Aconitine CHA-NA+ Cinnarazine CHA-CA- HIST-H-1- DOPA-D-2-ACH-M- Verapamil Hydrochloride CHA-CA- Benztropine ACH-M- DOPA-U- HIST- (1S,9R)-Beta-Hydrastine GABA-A- Disulfoton ACH- INSECT- Naltriben Methanesulfonate OPI-D- Benzanthrone Vinpocetine CHA-NA- ES-P-C-1- Oxymetazoline Hydrochloride ADR-A-1+ ADR-A-2+ 3-Isobutyl-1-Methylxanthine ES-P-C- ADS- 2-[[B-(4-Hydroxyphenol)Ethyl]Aminoethyl]-1-Tetralone ADR-A-1- Niloticin Meclozine Dihydrochloride HIST- ACH- CGP-13501 GABA-B+ Mesulergine Hydrochloride DOPA-D-2+ 5HT-2-C- Thioridazine Hydrochloride DOPA-D-2- 5HT-2- ADR- CHA-K- Hydroxyprogesterone Caproate ESTR+ ESTR+ PR+ Nicolsamide KI-TYR- 2-Methoxyestradiol HIF-1- Tolnaftate FUNG- Danazol LHRH- ESTR- (1-[(4-Chlorophenyl)phenyl-methyl]-4-methylpiperazine) HIST- Beclomethasone Dipropionate ARACH- LIPCOR+ IM- GluR+ Astemizole HIST-1- CHA-K- Icilin TRP-M8+ Oxaprozin SY-PG- COX- Ro 25-6981 hydrochloride EAA-G- NMDA-NR2-B- Droperidol DOPA-D-2- CHA-K- Ethynodiol Diacetate PROG+ ESTR+ Bromocryptine Mesylate DOPA-D-2+ CREB+ SSTATIN Androsterone Acetate ANDR+ CHA-CA-? 3,4-Dimethoxyflavone AHR- Trihexyphenidyl Hydrochloride ACH-M-1- Vincamine ADR-A-1- CHA-NA- Cephaeline Dihydrochloride Heptahydrate 5HT-4+? LY-53857 5HT-2- Amodiaquine Dihydrochloride Risperidone DOPA-D-2- 5HT-2- ADR-B- HIST- Doxazosin Mesylate ADR-A-1- Alcuronium Chloride ACH-M- L-765314 ADR-A-1- Prazosin ADR-A-1- Benperidol DOPA-D-2- CHA-K- Saquinavir Mesylate PROT- TNF-alpha+ PROL- Butylparaben ESTR? Ketanserin Tartrate 5HT-2- DOPA? Papaverine Hydrochloride ES-P-C-4- Nomegestrol Acetate PROG+ ANDR- PD 168077 DOPA-D-4+ Quinacrine Hydrochloride ACH-M+ Lipase-A-2- OX-LP-5- Ifenprodil Tartrate ADR-A-1- EAA-G- NMDA- CHA-K(GIRK)- Phenamil CHA-NA- Epiandrosterone ESTR- CHA-CA- GABA-A+ Loperamide OPI+ L-701252 EAA-G- NMDA- BWB-70C OX-LP-5- Strophanthidinic Acid Lactone Acetate ATPP-Na/K- Domperidone DOPA-D-2- Spiperone DOPA-D-2- 5HT-1-A- 5HT-2-A- Spiperone Hydrochloride DOPA-D-2- 5HT-1-A- 5HT-2-A- Pancuronium Bromide ACH-N- Amsacrine Hydrochloride TO-2- Dihydroergotamine Methanesulfonate 5HT-1-D+ 5HT-1-A+ ADR-A-2-ADOPA-D-2/D-3 Medroxyprogesterone Acetate DEC-OR- ANDR- ESTR+ PR+ WB 4101 Hydrochloride ADR-A-1- 3-Methoxymorphinan Hydrochloride EAA-G- NMDA- OPI-S+ DOPA-U-? L-741626 DOPA-D-2- AMI-193 5HT-2- DOPA-D-2- Spiroxatrine 5HT-1-A- DOPA-D-2- ADR-A-2- AMI-193 5HT-2- DOPA-D-2- trans-Dehydroandrosterone ESTR+ ANDR+ GABA-A- DO 897/99 DOPA-D-2/D-3DOPA- -D-3+ 5-HA-1-A ADR-A-1 Triptophenolide Azaperone DOPA- HIST- ACH- WB4101 Hydrochloride ADR-A-1- R(+)-Terguride 5HT-2-B- DOPA+/partial Azaperone DOPA- HIST- ACH- SB 205384 GABA-A+ Alfadolone Acetate GABA-A+ 3-Methyl-6-(3-[Trifluoromethyl]Phenyl)-1,2,4-Triazolo[4,3-B]Pyridazine GABA-A+ S(-)-Lisuride DOPA-D-2+ 5HT-2-B- Dihydroergocristine DOPA-D-2+ ADR-A+/- 5HT-2- 4-Androstene-3,17-dione ESTR- ANDR+ Pirenperone 5HT-2- DOPA-D-2-? Ketanserin Tartrate 5HT-2- DOPA? LY-165163 5HT-1-A+ DOPA-D-2- Astemizole HIST-1- CHA-K- Chlormadinone Acetate ANDR- ESTR+ PR+ Totarol BACT- Metergoline Phenylmethyl Ester 5HT-1-D- 5HT-2- DOPA+ Acetopromazine Maleate Salt DOPA- Metergoline 5HT-1-D- 5HT-2- DOPA+ BP554 5HT-1-A+ R-(-)-Fluoxetine Hydrochloride 5HT-U- Methapyrilene Hydrochloride HIST-1- Paroxetine Hydrochloride Hemihydrate 5HT-U- GR 4661 5HT-1-D+ Doxazosin Mesylate ADR-A-1- TCPOBOP CAR+ 16 Figure S1(Cont) NAME Activity 1 Activity 2 Activity3 Activity 4 Prednicarbate ARACH- LIPCOR+ IM- GluR+ CNS-1102 EAA-G- NMDA- Yohimbine Hydrochloride ADR-A-2- Opipramol Dihydrochloride 5HT-2- OPI-S+ HIST-H-1- DOPA-D-2- DCEBIO CHA-K+ Cefamandole Sodium SY-CW- BACT- (+)-Hydrastine GABA-A- Fusaric Acid DOPA-BH- MRS-1220 ADS-A3- Phenelzine Sulfate MAO- R(+)-UH-301 Hydrochloride 5HT-1-A+ Phenelzine Sulfate MAO- Tranylcypromine Hydrochloride MAO- DOPA-D-2? 5HT-2-A? L-750667 DOPA-D-4- Dl-p-Chlorophenylalanine Methyl Ester Hydrochloride 5HT-SY- (+/-)-Vesamicol Hydrochloride ACH-SY- Loratadine HIST-1- CHA-K(A5)- 1-(3-Chlorophenyl)Piperazine Dihydrochloride 5HT-1-2+ 5HT-3- Gedunol 1,3-Diethyl-8-Phenylxanthine ADS-A-1- ES-P-C-IV- Nf-kB Zardaverine ES-P-C-3- Dioxybenzone UV- Ciclopirox Olamine PERM- ATPP-Na/K- L-703606 Oxalate NK-1- Oxibendazole NEMO- MICROTUBE- Papaverine Hydrochloride ES-P-C-10 Niflumic Acid SY-PG- COX- AE2- Cyclopiazonic Acid CHA-CA-i- Tetradecylthioacetic Acid PPAR-A+ Fenofibrate PPAR-A+ Clioquinol FUNG- Spermine EAA-G- EAA-G+ NMDA- Tert-Butyl-Bicyclo[2.2.2]Phosphorothionate GABA-A- Indomethacin Morpholinylamide SY-PG- COX-1-2- Dihydrogeduninic Acid, Methyl Ester Chol-11-Enic Acid Diosmetin P450- Syrosingopine NEURT- Apomorphine Hydrochloride DOPA+ COMT- TH- 6-(5H)-Phenanthridinone PARP- Lysergol R(-)Apomorphine Hydrochloride Hemihydrate DOPA+ COMT- TH- Indole-3-Carbinol Clofibrate PPAR-A+ LP-P-A-2+ Avermectin B1 GABA-A+ Thioxolone ANC-2- CGS-12066A Maleate 5HT-1-B+ 5HT-1-D+ Dihydrocapsaicin VAN+ Suprofen Methyl Ester SY-PG- COX-1- COX-2- Pramoxine Hydrochloride Quipazine Dimaleate 5HT-2-A/3+ Pergolide Methanesulfonate DOPA-D-2+ Pergolide Methanesulfonate DOPA-D-2+ Fluspirilen DOPA-D-2- CY 208-243 DOPA-D-1+ 7-Hydroxy-DPAT DOPA-D-3+ DOPA-D-1-2/4+ 1-[1-(2-Benzo[B]Thienyl)Cyclohexyl)]Piperidine DOPA-U- BRL 15572 5HT-1-D- Mexiletine Hydrochloride CHA-NA- ADR-B- Adenosine ADS+ Gitoxin ATPP-NA/K- 1-Benzo[B]Thien-2-Yl-N-Cyclopropylmethylcyclohexanamine DOPA-U- Tetrac THYR+ Haloperidol DOPA-D-1-2- NMDA- CHA-K- Triflupromazine Hydrochloride DOPA-D-1/D-15HT- -2-B- ACH-M- Metergoline 5HT-1-D- 5HT-2- DOPA+ Imipramine Hydrochloride 5HT-U- ADR-U- ADS-A-1- RX 821002 Hydrochloride ADR-A-2- CV-3988 PAF- Totarol-19-Carboxylic Acid Clozapine 5HT-2- DOPA-D-2- ADR- HIST-H-1- Amitriptyline Hydrochloride 5HT-U- ADR-U- Norcyclobenzaprine 5HT-2-A- Estradiol PROG+ ESTR+ SXR+ Chrysin DIUR+ GW 9662 PPAR-G- IL-4- Chrysene-1,4-Quinone BACT- S(-)-3PPP Hydrochloride DOPA-D-2- 1-(2-Methoxyphenyl)-4-(4-Succinimidobutyl)Piperazine 5HT-1-A- WAY-100635 Maleate 5HT-1-A- DOPA-D-4+ NAN-190 Hydrobromide 5HT-1-A- ADR-A-1- Quinazolin-5(6H)-One Analog ADR-A-1- 5-Methyl-Urapidil ADR-A-1- 5HT-1-A- Terazosin Hydrochloride ADR-A-1- Quinacrine Dihydrochloride ACH-M+ Lipase-A-2- OX-LP-5- Quinacrine Dihydrochloride ACH-M+ Lipase-A-2- OX-LP-5- Ajmalicine Hydrochloride SY-CW- RU 24969 5HT-1-A/2-C+ Ifenprodil Tartrate ADR-A-1- EAA-G- NMDA- CHA-K(GIRK)- Ifenprodil Tartrate ADR-A-1- EAA-G- NMDA- CHA-K(GIRK)- ODQ CYC-GU- Quinelorane Dihydrochloride DOPA-D-2/3+ WAY-100635 Maleate Salt 5HT-1-A- DOPA-D-4+ Buspirone 5HT-1-A+ DOPA-D-2- PD 168077 Maleate DOPA-D-4+ Dihydroergocristine Methanesulfonate DOPA-D-2+ ADR-A+/- 5HT-2- Hycanthone H-89 KI-PKA- Nicergoline ADR-A-1- MK-912 ADR-A-2- Omeprazole ATPP-H/K- (+/-)-Methoxyverapamil Hydrochloride CHA-CA- 2-Methyl-6-(Phenylethynyl)Pyridine EAA-G- mGluR5- Spiroxatrine 5HT-1-A- DOPA-D-2- ADR-A-2- GR 103691 DOPA-D-3- Droperidol DOPA-D-2- LY 235959 EAA-G- NMDA- Coralyne Chloride Hydrate DNA- Trifluoperazine Hydrochloride DOPA-1-2- ADR-1- ATPase-K- Trifluoperazine Dihydrochloride DOPA-1-2- ADR-1- ATPase-K- YS-035 Hydrochloride CHA-CA- B-HT 920 DOPA-D-2+ ADR-A-2+ 5HT-3- 2,Beta-Dihydroxychalcone Estrone ESTR+ Phenazopyridine Hydrochloride Mesoridazine DOPA-D-2- 5HT-2-A-(indirect?)ADR-(indirect?) Reserpine NEURT- PD 169316 KI-P38- BMY 7378 Dihydrochloride 5HT-1-A+/- ADR-A-1-D- ADR-A-2-C- P-MPPF Dihydrochloride 5HT-1-A- (+/-)-Verapamil Hydrochloride CHA-CA- Rescinnamin ACE- Verapamil CHA-CA- Prometryn HERB- Cortexolone Maleate GlucoR- Reserpine NEURT- Nicergoline ADR-A-1- Dihydroergocristine DOPA-D-2+ ADR-A+/- 5HT-2- Risperidone DOPA-D-2- 5HT-2- ADR-B- HIST- Testosterone Propionate ANDR+ ESTR+ Piribedil Maleate DOPA-D-3/D-2+5HT-2-A/C- Lisuride DOPA-D-2+ 5HT-2-B- Carvedilol Tartrate ADR-B- ADR-1- Risperidone DOPA-D-2- 5HT-2- ADR-B- HIST- Phloretin CHA-CA- CHA-K-? Imipramine Hydrochloride 5HT-U- ADR-U- ADS-A-1- Domperidone Maleate DOPA-D-2-

17 Figure S1(Cont)

NAME Activity 1 Activity 2 Activity3 Activity 4 MDL 72832 5HT-1-A+ ADR-A-1? 8-Methoxy-2-Propionamidotetralin ML+ Bromocriptine Mesylate DOPA-D-2+ CREB+ SSTATIN N-Methylquipazine 5HT-3+ 3beta-Chloroandrostanone (-)-Quinpirole DOPA-D-2/3+ GR 127935 Hydrochloride 5HT-1-B/D- 5HT-1-D+? Dioxybenzone UV- Clemizole Hydrochloride HIST-1- Perphenazine DOPA-D-1-2- ADR-A-1- Paroxetine Hydrochloride 5HT-U- MDL 73005EF 5HT-1-A+ ADR-A-1- p-Iodoclonidine Hydrochloride ADR-A-2+ Ethynylestradiol 3-Methylether PROG+ ESTR+ Indirubin-3-Oxime KI-CD- KI-GSK- ARC 239 Dihydrochloride ADR-A-2-B- 5HT-1-A- Prochlorperazine Dimaleate DOPA-D-2- ADR-A-1- HIST-H-1- 5HT-2- Trazodone Hydrochloride 5HT-1-2- ADR-A-1- 5HT-2+(high5HT doses)-U- Desipramine Hydrochloride ADR-U- 5HT-U- ACH-M- ADR-B- Chloropyramine Hydrochloride HIST-H-1- Prazosin Hydrochloride ADR-A-1- Cinanserin 5HT-2- Diphenhydramine Hydrochloride 5HT-U- HIST-H-1- Harmane Hydrochloride GABA-A 5HT-2-A+ CHA- Trimeprazine Tartrate HIST-H-1- N-Butyl-N-Ethyl-2-(1-Naphthyloxy)Ethanamine 5HT-1-A+ 17alpha-Hydroxyprogesterone ESTR- PR+ Norethynodrel ESTR- PR+ 3-Hydroxyflavone Piperacetazine DOPA-D-1/D-25HT-? -2-B-? Zimelidine Dihydrochloride Monohydrate 5HT-U- R(+)-6-Bromo-APB Hydrobromide DOPA-D-1+ Methoxyverapamil CHA-CA- (+/-)-Chloro-APB Hydrobromide DOPA-D-1+ Clemizole Hydrochloride HIST-1- SIB 1757 EAA-G- mGluR5- Benfluorex Hydrochloride RED-HMG-? S-(-)-Eticlopride Hydrochloride DOPA-D-2/D-3- 1-[1-(2-Benzo[B]Thienyl)Cyclohexyl]Pyrrolidine DOPA-U- Ethinyl Estradiol PROG+ ESTR+ SXR+ Chlorpheniramine Maleate 5HT-U- ADR-U- HIST-H-1- 2-[(4-Phenylpiperazin-1Yl)Methyl]2,3-Dihydroimidazo[1,2C]Quinazolin-5(6H)-One ADR-A-1- 6-Nitroquipazine 5HT-U- Scoulerine GABA-A+ ADR-A-1- ADR-A-1- 5HT- Dextromethorphan Hydrobromide Monohydrate EAA-G- NMDA- OPI-S+ DOPA-U-? Perphenazine DOPA-D-1-2- ADR-A-1- Verapamil Hydrochloride CHA-CA- Vincamine ADR-A-1- CHA-NA- Promazine Hydrochloride DOPA-1-2/4- 5HT-2-A/C- ACH-M- ADR-A-1- NAN-190 5HT-1-A- ADR-A-1- L(-)-Vesamicol Hydrochloride ACH-SY- (+)-Chlorpheniramine Maleate 5HT-U- ADR-U- HIST-H-1- Fluvoxamine Maleate 5HT-U- Zimelidine Dihydrochloride 5HT-U- (+/-)-SKF 82958 DOPA-D-1+ DOPA-D-2+ SKF 83565 Hydrobromide DOPA-D-1+ BMY 7378 Dihydrochloride 5HT-1-A+/- ADR-A-1-D- ADR-A-2-C- S(-)-UH-301 Hydrochloride 5HT-1-A- Prochlorperazine Dimaleate DOPA-D-2- ADR-A-1- HIST-H-1- 5HT-2- 6-Nitro-Quipazine Maleate 5HT-U- SKF 86466 ADR-A-2- Quipazine 5HT-2-A/3+ Clovanediol Diacetate GBLD-345 GABA-A+ BZD+ (-)-trans-(1S,2S)-U-50488 Hydrochloride OPI-K+ Naftifine Hydrochloride EPOX-SQ- FUNG- (+/-)-SKF 38393, N-Allyl-, Hydrobromide DOPA-D-1+ Dydrogesterone PROG+ Brinzolamide ANC-2- (+/-)-Brompheniramine Maleate 5HT-U- ADR-U- HIST-H-1- Dyclonine Hydrochloride CHA-NA- beta-Caryophyllene Alcohol Biochanin A, Dimethyl Ether ESTR+ Picrotoxinin GABA- GLY-R- Estradiol Cypionate ESTR+ ESTR+ Chlorzoxazone CHA-K-(Ca)- Ticlopidine Hydrochloride P2Y-R-12- PLAT-AD- BIO KI-GSK-3- KI-PDK1- Iofetamine Hydrochloride 6-Methoxy-1,2,3,4-Tetrahydro-9H-Pyrido[3,4B] Indole MAO- Dyclonine Hydrochloride CHA-NA- SIB 1757 EAA-G- mGluR5- Enilconazole SY-CW- FUNG- Pelletierine Hydrochloride Imipramine Hydrochloride 5HT-U- ADR-U- ADS-A-1- Nefopam Hydrochloride 5HT-U- DOPA-U- ADR-U- COX- S(-)-DS 121 Hydrochloride DOPA-D-3- Hymecromone 3-[2-[4-(2-Methoxyphenyl)Piperazin-1-Yl]Ethyl]-1,5-Dimethylpyrimido[5,4-B]Indole-2,4-Dione ADR-A-1+ 5HT Ropinirole Hydrochloride DOPA-D-3/2+ Dipropyl-6,7-ADTN DOPA-D-2+ Fluvoxamine Maleate 5HT-U- R(-)-SCH-12679 Maleate DOPA-D-1- SCH 23390 DOPA-D-1- Quipazine Maleate 5HT-2-A/3+ YS-035 CHA-CA- Prazosin Hydrochloride ADR-A-1- Clorgyline Hydrochloride MAO-A- Fluoxetine Hydrochloride 5HT-U- Tropanyl 3,5-Dimethylbenzoate 5HT-3- Metixene Hydrochloride ACH-M- Oxybutynin Chloride ACH-M- 2-Chloroadenosine ADS+ Strophanthidin ATPP-Na/K- Atracurium Besylate ACH-N/M- Guanabenz Acetate ADR-A-2+ Guanabenz Acetate ADR-A-2+ Guanabenz Acetate ADR-A-2+ Amitraz ADR-A-2+ Arecaidine Propargyl Ester Hydrobromide ACH-M-2+ Methomyl ACH- INSECT- Guanfacine Hydrochloride ADR-A-2+ Clonidine ADR-A-2+ Guanfacine Hydrochloride ADR-A-2+ Guanabenz Acetate ADR-A-2+ Uk 14304 ADR-A-2+ R-(-)-Desmethyldeprenyl Hydrochloride MAO-B- 2,6-Dimethoxyquinone BACT- Baicalein PO-RDNA- Clonidine Hydrochloride ADR-A-2+ Tranylcypromine Sulfate MAO- DOPA-D-2? 5HT-2-A? Cinnarizine CHA-CA- HIST-H-1- DOPA-D-2-ACH-M- Thyroxine THYR+ (-)-Huperzine A ES-ACH- Tizanidine Hydrochloride ADR-A-2+ Propachlor HERB- Naftopidil Dihydrochloride ADR-A-1- ADR-U-? 5HT-2-? Amoxapine ADR-U- 5HT-U- Physostigmine Sulfate ES-ACH- SU 6656 KI-SRC-

18 Figure S2 Hierarchical Clustering K Hierarchical Clustering

Rest Total # Rest Bouts Rest Bout Length Rest Latency Activity Total Waking Activity Cluster # - Means Means Clustering (k=24) S.D. 8 7 6 5 4 3 2 1 24 18 17 16 15 14 13 12 11 10 | - 9 Rest -3 Total # Rest -2 Bouts -1 Rest Bout Length 0 Rest Latency 1 Activity 2 Total 3 Waking Activity 19

Figure S3

Rest

Rest

Total

Bouts Total

# Rest #

Latency

Waking Activity

Length

Activity Activity Rest Bout Rest NAME Activity 1 Activity 2 Activity3 Activity 4 SB 206553 Hydrochloride 5HT-2-B/C- Fenoterol Hydrobromide ADR-B+ Clenbuterol Hydrochloride ADR-B-2+ Maprotiline Hydrochloride ADR-U- 5HT-U-(weak) Maprotiline Hydrochloride ADR-U- 5HT-U-(weak) Retinoic Acid p-Hydroxyanilide APOP+ Betamethasone ARACH- LIPCOR+ IM- GluR+ Prednicarbate ARACH- LIPCOR+ IM- GluR+ Budesonide ARACH- LIPCOR+ Diclofenac Sodium ARACH- Monobenzone SY-PG- Spectinomycin Hydrochloride BACT- Sulfaguanidine BACT- Benzyl Penicillin Potassium BACT- Physcion BACT- Leflunomide DH-HYO- KI-GFPL- ALOX5- 1 Estrone Acetate ESTR+ I-OMe-Tyrphostin AG 538 KI-IGF-1- (-)-Eseroline Fumarate OPI-M+ ACH-(weak) Kynurenine Chlorophyllide Cu Complex Na Salt Enoxolone Chlorthalidone DIUR+ Dinitolmide SU 6656 KI-SRC- Physostigmine sulfate ES-ACH- 5-beta-12-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-one 14-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-one 3-beta-Acetoxydeoxodihydrogedunin 11-alpha-Acetoxykhivorin Carbenoxolone Sodium Glycine NMDA+ Senecrassidol 6-Acetate

Cisapride 5HT-4+ ACH+(indirect) CHA-K- Tegaserod Maleate 5HT-4+ 5HT-2-B- Ethylene Glycol-Bis(2-Aminoethylether)-N,N,N,N-Tetraacetic Acid ACE-2- CHEL+ Benztropine ACH-M- DOPA-U- HIST- MG-624 ACH-N- (+)-Tubocurarine Chloride ACH-N- ACH+ 5HT-3- Pancuronium Bromide ACH-N- Mecamylamine Hydrochloride ACH-N- 2 (-)-Nicotine ACH-N+ Nicotine Ditartrate ACH-N+ 1-Benzyl-1-Methyl-4-Cyclopentylmethoxycarbonylpiperidinium Br ACH-T- Chloroethylclonidine Dihydrochloride ADR-A-1- ADR-A-2+ Bretylium Tosylate ADR-B- NA/K-ATPase- Bopindolol Malonate ADR-B-1- Desoxycorticosterone Acetate COR-M+ MR+ Flunisolide ARACH- LIPCOR+ IM- GluR+ Clobetasol Propionate ARACH- LIPCOR+ IM- GluR+ Hydrocortisone ARACH- LIPCOR+ IM- GluR+ 6alpha-Methylprednisolone ARACH- LIPCOR+ IM- GluR+ Betamethasone Valerate ARACH- LIPCOR+ IM- GluR+ Clobetasol Propionate ARACH- LIPCOR+ IM- GluR+ Ursolic Acid ARACH- LIPCOR+ IM- GluR+ Dexamethasone ARACH- LIPCOR+ IM- GluR+ Medrysone ARACH- LIPCOR+ IM- GluR+ Betamethasone ARACH- LIPCOR+ IM- GluR+ Flumethasone ARACH- LIPCOR+ IM- GluR+ Cefoxitin Sodium Salt SY-CW- BACT- Mefloquine Hydrochloride CALM- Tetrandrine CHA-CA- CHA-K-(Ca+) Flecanide CHA-NA- Benzamil Hydrochloride CHA-NA-Ca- Pentamidine Isethionate DNA- Fenoldopam DOPA-D-1-A+ ADR-A-2+ Propentofylline ES-P-C- ADS-/+ Diplosalsalate ES-P-C-? Fosfosal ES-P-C- SKF 94836 ES-P-C-3- Ro 20-1724 ES-P-C-4- Papaverine Hydrochloride ES-P-C-4- Equilin ESTR+ Allopregnanolone GABA-A+ CHA-CA- Valproate Sodium HDAC- GABA+ Atrazine HERB- Diphenylpyraline Hydrochloride HIST-H-1- Cyclosporin A IM- Austricine LP-P-A-2- 2-Cyclooctyl-2-Hydroxyethylamine Hydrochloride NEURT- PNMT- Theaflavin Digallate Nf-kB-? Finasteride RED-5A- Bufexamac SY-PG- COX- OX-C- Diflunisal SY-PG- COX- Fenoprofen SY-PG- COX- Fenoprofen Calcium Salt Dihydrate SY-PG- COX- Piroxicam SY-PG- COX- Capsazepine VAN- CHA-Na+ 5-Aminopentanoic Acid Hydrochloride Catechin Tetramethylether Tetrahydrotrimethylhispidin Aminophenazone Oxethazaine Sinapic Acid Methyl Ether IEM-1460 EAA-G- AMPA-R- NMDA- 6,7-Dichloroquinoxaline-2,3-Dione EAA-G- NMDA- 7-Chlorokynurenic Acid EAA-G- NMDA- SDZ 220-581 EAA-G- NMDA- L-689560 EAA-G- NMDA-

(-)-MK-801Hydrogen Maleate EAA-G- NMDA- L-701324 EAA-G- NMDA- L-701324 EAA-G- NMDA- L-701324 EAA-G- NMDA- 3 Dizocilpine Maleate EAA-G- NMDA- (+)-MK-801 EAA-G- NMDA- Spiperone DOPA-D-2- 5HT-1-A- 5HT-2-A- Azobenzene

SB 228357 5HT-2-B/C- R(-)-Isoproterenol (+)-Bitartrate ADR-B-2+ ADR-B-1+ Amitryptiline Hydrochloride ADR-U- 5HT-U- CHA-K- Amoxapine ADR-U- 5HT-U- Telmisartan ANG-2-1- PPAR-G+ 4 Cefamandole Sodium SY-CW- BACT- TCPOBOP CAR+ Cinnarazine CHA-CA- HIST-H-1- DOPA-D-2- ACH-M- Psora-4 CHA-K- Pimozide DOPA-D-2- 5HT-2-A- ADR-A-1- DOPA-U- Mitotane ESTR- CHA-NA? Oxiconazole Nitrate FUNG- Ornithine alpha-Ketoglutarate

N-Desmethylclozapine 5HT-2-C- ACH-M1+ Coumaphos ACH- S,S,S,-Tributylphosphorotrithioate ACH- Fiduxosin Hydrochloride ADR-A-1- Mianserine Hydrochloride ADR-U- ADR-A-2- HIST- 5HT-2-A/2-C- Bepridil Hydrochloride CHA-CA- CHA-NA- CHA-K- Aconitine CHA-NA+ 5 Loxapine Succinate DOPA-D-1- DOPA-D-2- 5HT-2- (indirect?) Ibudilast ES-P-C-4- Endosulfan GABA-A- Clorgyline Hydrochloride MAO-A-

Amoxapine ADR-U- 5HT-U- ODQ CYC-GU- L-701252 EAA-G- NMDA- Megestrol Acetate ESTR+ PROG+ 6 1S,9R-Hydrastine GABA-A- Chlordane GABA-A- INSECT- ATPP-Ca/MgATPP- -Na/K- Dacthal HERB- Propazine HERB- 7-Oxocallitrisic Acid, Methyl Ester

Methiothepin Maleate 5HT-1-2- DOPA-D-2- Mianserin ADR-U- ADR-A-2- HIST- 5HT-2-A/2-C- Cyproterone Acetate ANDR- 7 (1S,9R)-Beta-Hydrastine GABA-A- Pregnenolone GABA-A- Astemizole HIST-1- CHA-K- Bromo-3-Hydroxy-4-(Succin-2-Yl)-Caryolane Gamma-Lactone 20

Figure S3(Cont)

Rest

Rest

Total

Total

Bouts

# Rest #

Latency

Waking Activity

Length

Activity Activity Rest Bout Rest NAME Activity 1 Activity 2 Activity3 Activity 4 Dichlorodiphenyltrichloroethane CHA-NA+ INSECT- Methoxychlor CHA-NA+ INSECT- 8 Strobane CHA-NA+ INSECT- Toxaphene CHA-NA+ INSECT- Aldrin GABA-A- INSECT- ATPP-Ca/Mg- ATPP-Na/K- Heptachlor GABA-A- INSECT- ATPP-Ca/Mg- ATPP-Na/K-

Disulfoton ACH- INSECT- 9 (+)-Hydrastine GABA-A- Dihydroergotamine Mesylate 5HT-1-D+ 5HT-1-A+ ADR-A-2-A DOPA-D-2/D-3 Amperozide Hydrochloride 5HT-2- DOPA-D-2- Clozapine 5HT-2- DOPA-D-2- ADR- HIST-H-1- 10 Cephaeline Dihydrochloride Heptahydrate 5HT-4+? Chlormadinone Acetate ANDR- ESTR+ PR+ Flutamide ANDR- Halofantrine Hydrochloride CHA-K- Haloperidol DOPA-D-1-2- NMDA- CHA-K- Chlorpromazine Hydrochloride DOPA-D-2- DOPA-D-1- 5HT1-2- HIST-H-1- 3-alpha-Bis-(4-Fluorophenyl)Methoxytropane DOPA-U- ACH-M- (-)-MK-801 Hydrogen Maleate EAA-G- NMDA- Spermine Tetrahydrochloride EAA-G- EAA-G+ NMDA- Epiandrosterone ESTR- CHA-CA- GABA-A+ CGP-13501 GABA-B+ 2-Methoxyestradiol HIF-1- Deptropine Citrate HIST- ACH-M- Astemizole HIST-1- CHA-K- Astemizole HIST-1- CHA-K- Clemastine HIST-1- ACH-M-? Oxatomide HIST-1- Promethazine Hydrochloride HIST-1- ACH-M- 5HT-2- calmodulin- Terfenadine HIST-1- CHA-K- 3-(1H-Imidazol-4-Yl)Propyl Di(P-Fluorophenyl)Methyl Ether HIST-H3-/+ Tyrphostin AG 494 KI-GFEP- Danazol LHRH- ESTR- 1,4-PBIT Dihydrobromide NO-S- Roxithromycin PRT-50S- NF-kB- Finasteride RED-5A- ANDR- Menadione VITA+ 2,4-Dihydroxychalcone 4-Glucoside Benzanthrone Derrusnin Hexylresorcinol Homopterocarpin

MDL 72832 5HT-1-A+ ADR-A-1? Metergoline Phenylmethyl Ester 5HT-1-D- 5HT-2- DOPA+ Metergoline 5HT-1-D- 5HT-2- DOPA+ GR 4661 5HT-1-D+ LY-367265 5HT-2-A- 5HT-U- 11 5HT-2-B- DOPA+/partial R(+)-Terguride LY-53857 5HT-2- Imipramine Hydrochloride 5HT-U- ADR-U- ADS-A-1- R-(-)-Fluoxetine Hydrochloride 5HT-U- Naftopidil Dihydrochloride ADR-A-1- ADR-U-? 5HT-2-? Naftopidil Dihydrochloride ADR-A-1- ADR-U-? 5HT-2-? Vincamine ADR-A-1- CHA-NA- Oxymetazoline Hydrochloride ADR-A-1+ ADR-A-2+ Yohimbine Hydrochloride ADR-A-2- Beclomethasone Dipropionate ARACH- LIPCOR+ IM- GluR+ Rutilantinone BACT- Verapamil Hydrochloride CHA-CA- Acetopromazine Maleate Salt DOPA- Azaperone DOPA- HIST- ACH- Triflupromazine Hydrochloride DOPA-D-1/D-1- 5HT-2-B- ACH-M- Haloperidol DOPA-D-1-2- NMDA- CHA-K- L-741626 DOPA-D-2- Thioridazine Hydrochloride DOPA-D-2- 5HT-2- ADR- CHA-K- Mesulergine Hydrochloride DOPA-D-2+ 5HT-2-C- CNS-1102 EAA-G- NMDA- trans-Dehydroandrosterone ESTR+ ANDR+ GABA-A- Tolnaftate FUNG- SB 205384 GABA-A+ 3-Methyl-6-(3-[Trifluoromethyl]Phenyl)-1,2,4-Triazolo[4,3-B]Pyridazine GABA-A+ Prometryn HERB- Meclozine Dihydrochloride HIST- ACH- Meclizine Hydrochloride HIST- ACH- 1-[(4-Chlorophenyl)Phenyl-Methyl]-4-Methylpiperazine HIST- Methapyrilene Hydrochloride HIST-1- Terfenadine HIST-1- CHA-K- TG 003 KI-CLK- Nicolsamide KI-TYR- Naltriben Methanesulfonate OPI-D- Icilin TRP-M8+ Niloticin Triptophenolide

LY-165163 5HT-1-A+ DOPA-D-2- BP554 5HT-1-A+ Metergoline 5HT-1-D- 5HT-2- DOPA+ Dihydroergotamine Methanesulfonate 5HT-1-D+ 5HT-1-A+ ADR-A-2-A DOPA-D-2/D-3 Ketanserin Tartrate 5HT-2- DOPA? 12 Pirenperone 5HT-2- DOPA-D-2-? Ketanserin Tartrate 5HT-2- DOPA? Paroxetine Hydrochloride Hemihydrate 5HT-U- Trihexyphenidyl Hydrochloride ACH-M-1- Quinacrine Hydrochloride ACH-M+ Lipase-A-2- OX-LP-5- Pancuronium Bromide ACH-N- Doxazosin Mesylate ADR-A-1- WB-4101 Hydrochloride ADR-A-1- Ifenprodil Tartrate ADR-A-1- EAA-G- NMDA- CHA-K(GIRK)- Nicergoline ADR-A-1- 3,4-Dimethoxyflavone AHR- Androsterone Acetate ANDR+ CHA-CA-? Totarol BACT- Phenamil CHA-NA- Azaperone DOPA- HIST- ACH- Spiperone Hydrochloride DOPA-D-2- 5HT-1-A- 5HT-2-A- Benperidol DOPA-D-2- CHA-K- Spiperone DOPA-D-2- 5HT-1-A- 5HT-2-A- Droperidol DOPA-D-2- CHA-K- DO 897/99 DOPA-D-2/D-3- DOPA-D-3+ 5-HA-1-A ADR-A-1 Risperidone DOPA-D-2- 5HT-2- ADR-B- HIST- S(-)-Lisuride DOPA-D-2+ 5HT-2-B- Dihydroergocristine DOPA-D-2+ ADR-A+/- 5HT-2- Dihydroergocristine DOPA-D-2+ ADR-A+/- 5HT-2- Bromocriptine Mesylate DOPA-D-2+ CREB+ SSTATIN PD 168077 DOPA-D-4+ Ro 25-6981 Hydrochloride EAA-G- NMDA-NR2-B- Butylparaben ESTR? Hydroxyprogesterone Caproate ESTR+ ESTR+ PR+ Alfadolone Acetate GABA-A+ Reserpine NEURT- Saquinavir Mesylate PROT- TNF-alpha+ PROL- Nomegestrol Acetate PROG+ ANDR- Ethynodiol Diacetate PROG+ ESTR+ Oxaprozin SY-PG- COX-

Spiroxatrine 5HT-1-A- DOPA-D-2- ADR-A-2- AMI-193 5HT-2- DOPA-D-2- AMI-193 5HT-2- DOPA-D-2- WB-4101 Hydrochloride ADR-A-1- 13 Amoxapine ADR-U- 5HT-U- 8-Cyclopentyl-1,3-Dipropylxanthine ADS-A-1- Strophanthidinic Acid Lactone Acetate ATPP-Na/K- Vinpocetine CHA-NA- ES-P-C-1- Medroxyprogesterone Acetate DEC-OR- ANDR- ESTR+ PR+ Domperidone DOPA-D-2- 3-Methoxymorphinan Hydrochloride EAA-G- NMDA- OPI-S+ DOPA-U-? L-701252 EAA-G- NMDA- 4-Androstene-3,17-Dione ESTR- ANDR+ Tracazolate Hydrochloride GABA-A- GABA-A+ Loperamide OPI+ BWB70C OX-LP-5-

21

Figure S3(Cont)

Rest

Rest

Total

Total

Bouts

# Rest #

Latency

Waking Activity

Length

Activity Activity Rest Bout Rest NAME Activity 1 Activity 2 Activity3 Activity 4 1-(2-Methoxyphenyl)-4-(4-Succinimidobutyl)Piperazine 5HT-1-A- Spiroxatrine 5HT-1-A- DOPA-D-2- ADR-A-2- WAY-100635 Maleate Salt 5HT-1-A- DOPA-D-4+ RU-24969 5HT-1-A/2-C+ CINANSERIN 5HT-2- 14 Opipramol Dihydrochloride 5HT-2- OPI-S+ HIST-H-1- DOPA-D-2- Norcyclobenzaprine 5HT-2-A- Alcuronium Chloride ACH-M- Quinacrine Dihydrochloride ACH-M+ Lipase-A-2- OX-LP-5- Quinacrine Dihydrochloride Dihydrate ACH-M+ Lipase-A-2- OX-LP-5- Pancuronium Bromide ACH-N- 2-[4-(2-Methoxyphenol)Piperazin-1-Yl]Methyl-6-Methyl2,3Dihydroimidazo[1,2C]Quinazolin-5(6H)OneADR-A-1- 2-[[B-(4-Hydroxyphenol)Ethyl]Aminoethyl]-1-Tetralone ADR-A-1- Doxazosin Mesylate ADR-A-1- Ifenprodil Tartrate ADR-A-1- EAA-G- NMDA- CHA-K(GIRK)- Ifenprodil Tartrate ADR-A-1- EAA-G- NMDA- CHA-K(GIRK)- L-765314 ADR-A-1- Nicergoline ADR-A-1- Prazosin ADR-A-1- 5-Methyl-Urapidil ADR-A-1- 5HT-1-A- MK-912 ADR-A-2- RX 821002 Hydrochloride ADR-A-2- Testosterone Propionate ANDR+ ESTR+ Cepharanthine APOP- NF-KB- IM- Omeprazole ATPP-H/K- Gitoxin ATPP-NA/K- (+/-)-Methoxyverapamil Hydrochloride CHA-CA- Mexiletine Hydrochloride CHA-NA- ADR-B- ODQ CYC-GU- Coralyne Chloride Hydrate DNA- Fusaric Acid DOPA-BH- Domperidone Maleate DOPA-D-2- Droperidol DOPA-D-2- Risperidone DOPA-D-2- 5HT-2- ADR-B- HIST- Dihydroergocristine Methanesulfonate DOPA-D-2+ ADR-A+/- 5HT-2- GR 103691 DOPA-D-3- 1-Benzo[B]Thien-2-Yl-N-Cyclopropylmethylcyclohexanamine DOPA-U- 2-Methyl-6-(Phenylethynyl)Pyridine EAA-G- mGluR5- LY-235959 EAA-G- NMDA- 3-Isobutyl-1-Methylxanthine ES-P-C- ADS- Papaverine Hydrochloride ES-P-C-4- Ajmalicine Hydrochloride SY-CW- Amsacrine hydrochloride TO-2- Amodiaquine Dihydrochloride Hycanthone

Trazodone Hydrochloride 5HT-1-2- ADR-A-1- 5HT-2+(high doses)5HT-U- P-MPPF Dihydrochloride 5HT-1-A- WAY-100635 Maleate 5HT-1-A- DOPA-D-4+ MDL-73005EF 5HT-1-A+ ADR-A-1- BMY 7378 Dihydrochloride 5HT-1-A+/- ADR-A-1-D- ADR-A-2-C- 15 GR 127935 Hydrochloride 5HT-1-B/D- 5HT-1-D+? Clozapine 5HT-2- DOPA-D-2- ADR- HIST-H-1- N-Methylquipazine 5HT-3+ (+)-Chlorpheniramine Maleate 5HT-U- ADR-U- HIST-H-1- Diphenhydramine Hydrochloride 5HT-U- HIST-H-1- Fluvoxamine Maleate 5HT-U- Imipramine Hydrochloride 5HT-U- ADR-U- ADS-A-1- Paroxetine Hydrochloride 5HT-U- Zimelidine Dihydrochloride Monohydrate 5HT-U- Prazosin Hydrochloride ADR-A-1- Terazosin Hydrochloride ADR-A-1- Vincamine ADR-A-1- CHA-NA- 3-[2-[4-(2-Methoxyphenyl)Piperazin-1-Yl]Ethyl]-1,5Dimethylpyrimido[5,4-B]Indole-2,4-Dione ADR-A-1+ 5HT p-Iodoclonidine Hydrochloride ADR-A-2+ ARC 239 Dihydrochloride ADR-A-2-B- 5HT-1-A- Carvedilol Tartrate ADR-B- ADR-1- Desipramine Hydrochloride ADR-U- 5HT-U- ACH-M- ADR-B- (+/-)-Verapamil Hydrochloride CHA-CA- Methoxyverapamil CHA-CA- Phloretin CHA-CA- CHA-K-? Verapamil Hydrochloride CHA-CA- YS-035 CHA-CA- YS-035 Hydrochloride CHA-CA- Apomorphine Hydrochloride DOPA+ COMT- TH- Trifluoperazine Dihydrochloride DOPA-1-2- ADR-1- ATPase-K- Trifluoperazine Hydrochloride DOPA-1-2- ADR-1- ATPase-K- Piperacetazine DOPA-D-1/D-2-? 5HT-2-B-? (+/-)-SKF 38393, N-Allyl-, Hydrobromide DOPA-D-1+ Perphenazine DOPA-D-1-2- ADR-A-1- Perphenazine DOPA-D-1-2- ADR-A-1- Mesoridazine DOPA-D-2- 5HT-2-A-(indirect?)ADR-(indirect?) Prochlorperazine Dimaleate DOPA-D-2- ADR-A-1- HIST-H-1- 5HT-2- Risperidone DOPA-D-2- 5HT-2- ADR-B- HIST- Quinelorane Dihydrochloride DOPA-D-2/3+ S(-)-Lisuride DOPA-D-2+ 5HT-2-B- B-HT 920 DOPA-D-2+ ADR-A-2+ 5HT-3- Dipropyl-6,7-ADTN DOPA-D-2+ Piribedil Maleate DOPA-D-3/D-2+ 5HT-2-A/C- 17alpha-Hydroxyprogesterone ESTR- PR+ Norethynodrel ESTR- PR+ Estrone ESTR+ Harmane Hydrochloride GABA-A 5HT-2-A+ CHA- Scoulerine GABA-A+ ADR-A-1- ADR-A-1- 5HT- Cortexolone Maleate GlucoR- Clemizole Hydrochloride HIST-1- Chloropyramine Hydrochloride HIST-H-1- Indirubin-3-Oxime KI-CD- KI-GSK- PD 169316 KI-P38- H-89 KI-PKA- Reserpine NEURT- Diosmetin P450- GW 9662 PPAR-G- IL-4- Ethynylestradiol 3-Methylether PROG+ ESTR+ Dioxybenzone UV- 2beta-Dihydroxychalcone 3-Hydroxyflavone Phenazopyridine Hydrochloride Totarol-19-Carboxylic Acid

1-(3-Chlorophenyl)Piperazine Dihydrochloride 5HT-1-2+ 5HT-3- NAN-190 5HT-1-A- ADR-A-1- S(-)-UH-301 Hydrochloride 5HT-1-A- Buspirone 5HT-1-A+ DOPA-D-2- 16 R(+)-UH-301 Hydrochloride 5HT-1-A+ BMY 7378 Dihydrochloride 5HT-1-A+/- ADR-A-1-D- ADR-A-2-C- Quipazine 5HT-2-A/3+ Quipazine Dimaleate 5HT-2-A/3+ Dl-p-Chlorophenylalanine Methyl Ester Hydrochloride 5HT-SY- 6-Nitroquipazine 5HT-U- Chlorpheniramine Maleate 5HT-U- ADR-U- HIST-H-1- Fluvoxamine Maleate 5HT-U- 6-Nitroquipazine Maleate 5HT-U- Zimelidine Dihydrochloride 5HT-U- Rescinnamin ACE- L(-)-Vesamicol Hydrochloride ACH-SY- 2-[(4-Phenylpiperazin-1-Yl)Methyl]-2,3-Dihydroimidazo[1,2-C]Quinazolin-5(6H)-One ADR-A-1- SKF 86466 ADR-A-2- Verapamil CHA-CA- Promazine Hydrochloride DOPA-1-2/4- 5HT-2-A/C- ACH-M- ADR-A-1- (+/-)-Chloro-APB Hydrobromide DOPA-D-1+ (+/-)-SKF 82958 DOPA-D-1+ DOPA-D-2+ R(+)-6-Bromo-APB Hydrobromide DOPA-D-1+ SKF 83565 Hydrobromide DOPA-D-1+ Prochlorperazine Dimaleate DOPA-D-2- ADR-A-1- HIST-H-1- 5HT-2- S-(-)-Eticlopride Hydrochloride DOPA-D-2/D-3- L-750667 DOPA-D-4- PD 168077 Maleate DOPA-D-4+ 1-[1-(2-Benzo[B]Thienyl)Cyclohexyl]Pyrrolidine DOPA-U- Dextromethorphan Hydrobromide Monohydrate EAA-G- NMDA- OPI-S+ DOPA-U-? SIB 1757 EAA-G- mGluR5- Syrosingopine NEURT- 6(5H)-Phenanthridinone PARP- Ethinyl Estradiol PROG+ ESTR+ SXR+ Benfluorex Hydrochloride RED-HMG-? Suprofen Methyl Ester SY-PG- COX-1- COX-2- Dihydrogeduninic Acid, Methyl Ester 22

Figure S3(Cont)

Rest

Rest

Total

Total

Bouts

# Rest #

Latency

Waking Activity

Length

Activity Activity Rest Bout Rest NAME Activity 1 Activity 2 Activity3 Activity 4 NAN-190 Hydrobromide 5HT-1-A- ADR-A-1- N-Butyl-N-Ethyl-2-(1-Naphthyloxy)Ethanamine 5HT-1-A+ CGS-12066A Maleate 5HT-1-B+ 5HT-1-D+ Quipazine Maleate 5HT-2-A/3+ Tropanyl 3,5-Dimethylbenzoate 5HT-3- (+/-)-Brompheniramine Maleate 5HT-U- ADR-U- HIST-H-1- Amitriptyline Hydrochloride 5HT-U- ADR-U- Fluoxetine Hydrochloride 5HT-U- Imipramine Hydrochloride 5HT-U- ADR-U- ADS-A-1- Nefopam Hydrochloride 5HT-U- DOPA-U- ADR-U- COX- Methomyl ACH- INSECT- Metixene Hydrochloride ACH-M- 17 Oxybutynin Chloride ACH-M- Arecaidine Propargyl Ester Hydrobromide ACH-M-2+ Atracurium Besylate ACH-N/M- Prazosin Hydrochloride ADR-A-1- Amitraz ADR-A-2+ Clonidine ADR-A-2+ Clonidine ADR-A-2+ Guanabenz Acetate ADR-A-2+ Guanabenz Acetate ADR-A-2+ Guanabenz Acetate ADR-A-2+ Guanabenz Acetate ADR-A-2+ Guanfacine Hydrochloride ADR-A-2+ Guanfacine Hydrochloride ADR-A-2+ Tizanidine Hydrochloride ADR-A-2+ UK 14304 ADR-A-2+ 2-Chloroadenosine ADS+ Adenosine ADS+ MRS-1220 ADS-A3- Brinzolamide ANC-2- Strophanthidin ATPP-Na/K- 2,6-Dimethoxyquinone BACT- Chrysene-1,4-Quinone BACT- Cinnarizine CHA-CA- HIST-H-1- DOPA-D-2- ACH-M- Chlorzoxazone CHA-K-(Ca)- Dyclonine Hydrochloride CHA-NA- Dyclonine Hydrochloride CHA-NA- Chrysin DIUR+ R(-)-SCH-12679 Maleate DOPA-D-1- SCH 23390 DOPA-D-1- S(-)-3PPP hydrochloride DOPA-D-2- (-)-Quinpirole DOPA-D-2/3+ Bromocriptine Mesylate DOPA-D-2+ CREB+ SSTATIN S(-)-DS 121 Hydrochloride DOPA-D-3- Ropinirole Hydrochloride DOPA-D-3/2+ SIB 1757 EAA-G- mGluR5- Naftifine Hydrochloride EPOX-SQ- FUNG- (-)-Huperzine A ES-ACH- Biochanin A, Dimethyl Ether ESTR+ Estradiol Cypionate ESTR+ ESTR+ Picrotoxinin GABA- GLY-R- GBLD-345 GABA-A+ BZD+ Propachlor HERB- Clemizole Hydrochloride HIST-1- Trimeprazine Tartrate HIST-H-1- BIO KI-GSK-3- KI-PDK1- 6-Methoxy-1,2,3,4-Tetrahydro-9H-Pyrido[3,4B] Indole MAO- Phenelzine sulfate MAO- Phenelzine Sulfate MAO- Tranylcypromine Hydrochloride MAO- DOPA-D-2? 5HT-2-A? Tranylcypromine Sulfate MAO- DOPA-D-2? 5HT-2-A? Clorgyline Hydrochloride MAO-A- R-(-)-Desmethyldeprenyl Hydrochloride MAO-B- 8-Methoxy-2-Propionamidotetralin ML+ (-)-trans-(1S,2S)-U-50488 Hydrochloride OPI-K+ Ticlopidine Hydrochloride P2Y-R-12- PLAT-AD- CY-3988 PAF- Baicalein PO-RDNA- Dydrogesterone PROG+ Estradiol PROG+ ESTR+ SXR+ Enilconazole SY-CW- FUNG- Tetrac THYR+ Thyroxine THYR+ 3beta-Chloroandrostanone beta-Caryophyllene Alcohol Clovanediol Diacetate Hymecromone Iofetamine hydrochloride Pelletierine Hydrochloride Pramoxine Hydrochloride

Fluspirilen DOPA-D-2- 18 Pergolide Methanesulfonate DOPA-D-2+ BRL 15572 5HT-1-D- CY 208-243 DOPA-D-1+ Pergolide Methanesulfonate DOPA-D-2+ 19 7-Hydroxy-DPAT DOPA-D-3+ DOPA-D-1-2/4+ 1-[1-(2-Benzo[B]Thienyl)Cyclohexyl)]Piperidine DOPA-U- Dihydrocapsaicin VAN+

20 Clioquinol FUNG- Cyclopiazonic Acid CHA-CA-i- Papaverine Hydrochloride ES-P-C-10 Tert-Butyl-Bicyclo[2.2.2]Phosphorothionate GABA-A- 21 Oxibendazole NEMO- MICROTUBE- Indomethacin Morpholinylamide SY-PG- COX-1-2-

(+/-)-Vesamicol Hydrochloride ACH-SY- 1,3-Diethyl-8-Phenylxanthine ADS-A-1- ES-P-C-IV- Nf-kB 22 Ciclopirox Olamine PERM- ATPP-Na/K- DCEBIO CHA-K+ Spermine EAA-G- EAA-G+ NMDA- Zardaverine ES-P-C-3- Loratadine HIST-1- CHA-K(A5)- L-703606 Oxalate NK-1- Fenofibrate PPAR-A+ Tetradecylthioacetic acid PPAR-A+ Niflumic Acid SY-PG- COX- AE2- Dioxybenzone UV- Gedunol

Thioxolone ANC-2- R(-)Apomorphine Hydrochloride Hemihydrate DOPA+ COMT- TH- Avermectin B1 GABA-A+ 23 Clofibrate PPAR-A+ LP-P-A-2+ Indole-3-Carbinol Lysergol 24 Chol-11-Enic Acid

23 Figure S4 Correlation Fingerprint Matrix

NAN190 µM NAN190 3 11 1 3 S.D. 45 1 22 45 152 11 152 3 0.5 4.5 0 0.5 0 1.54 4.53 -1

5 -1 190 0.45 -2 4 - -2 1.5 0.156 0 0 -3-3 S.D.

5 10 15 20 0.455 correlation

0.156

NAN -0.5

1 2 3 4 5 6 -0.5

rest rest total

rest rest

# rest # bouts

total µM

45 15

length

activity waking activity

4.5 1.5

latency

rest bout rest

0.45 0.15

betamethasone µM betamethasone 1 3 1 451 3 S.D. 1 2 45 2 2 15 2 11 15 3 0.50.5 4.5 0 0 3 1.54 4.5 -1

5 -1 0.45 4 -2 1.5 -2 0.156 0 0 -3

5 10 15 20 -3 S.D. 0.455 correlation

0.156 -0.5-0.5

1 2 3 4 5 6

rest rest total

rest rest

# rest # bouts

total 15

µM 45

length

waking waking activity

activity

4.5 1.5

latency

rest bout rest

Betamethasone

0.45 0.15

ropinirole µM ropinirole 3 11 1 3 S.D. 45 1 2 45 2 152 1 152 3 1 4.5 0.50.5 00 1.54 4.53 -1

5 -1 0.45 4 -2-2 1.5 0.156 0 0 -3

5 10 15 20 -3 S.D. 0.455 correlation

0.156 -0.5-0.5

1 2 3 4 5 6

Ropinirole

rest rest total

rest rest

# rest # bouts

total µM

45 15

length

waking waking activity

activity

4.5 1.5

latency

rest bout rest

0.45 0.15

clonidine-dosestable clonidine correlation stablity µM 3 1 1 1 3 S.D. 1 45 2 45 2 152 11 15 2 3 0.5 4.5 0 0.5 0 3 1.54 4.5 -1

5 -1 0.45 4 -2 1.5 -2 0 0.156 0 -3

5 10 15 20 -3 S.D. 0.45 5 correlation 0.15 6 -0.5

Clonidine -0.5

1 2 3 4 5 6

rest rest total

rest rest

# rest # bouts

total µM

45 15

length

waking waking activity

activity

4.5 1.5

latency

rest bout rest

0.45 0.15

buspirone buspirone 3 1 1 45 3 S.D. 1 45 2 45 15 2 15 2 11 15 4.5 0.50.5 4.5 0 1.51.5 0 4.5 3 -1-1 0.450.45 4 -2 1.5 -2 0 0 0.150.15 -3-3 S.D. 0.45 5

5 10 15 20 correlation 0.15 6 -0.5-0.5

Buspirone 1 2 3 4 5 6

rest rest total

rest rest

# rest # bouts

total µM

45 15

length

waking waking activity

activity

4.5 1.5

latency

rest bout rest

0.45 0.15

pergolide pergolide µM 3 1 3 S.D. 1 4545 1 22 45 1515 11 152 4.54.5 0.50.5 0 1.51.5 0 4.53 -1-1 0.45 0.45 1.54 -2-2 0.150.15 0 0 -3-3 S.D. 0.455

5 10 15 20 correlation

0.156 -0.5-0.5

Pergolide 1 2 3 4 5 6

rest rest total

rest rest bouts

# rest # µM

total

45 15

length

1.5 4.5

waking waking activity

activity

latency

rest bout rest

0.45 0.15

24 Figure S5

A. 6 B. 0 Targets Shared >=1 Target Shared 1

5 0.8

0.6

0.4 4

0.2

0 3 -0.2 Correlation

Percent -0.4 2 -0.6

-0.8

1 -1 0 targets1 >=1 target2 repeats3 (2 sources) 0 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Correlation

C. 1

0.5

0 Correlation

-0.5 ------2 L B U U N 4+ M - - B+ A+ N+ - - - - M+ - - A1 A2 1A - 1D H1 - D1 D2 D3 - SY NA - A2+ 1A+ Rev 1D+ D1+ D2+ D3+ D4+ ------2/3+ NA+ - - IRev ------SSRI SNRI ADS 1A+/ - D2/3 2B/C MAO CA ADS+ D2/3+ - - (ERG) - - - 5HT GABA DOPA - NMDA 5HT GABA+ ADR DOPA+ ES ADR ACH ACH ADR ES ACH 5HT - 5HT ACH NEURO ADR ADR 5HT - 5HT ACH CHA 5HT DOPA HIST ADR PPAR CHA H1 mGLUR5 5HT 5HT DOPA DOPA DOPA - DOPA DOPA DOPA DOPA CHA DOPA DOPA ACH ACH HIST 24 4 12 3 3 10 8 10 9 7 2 4 3 13 3 2 3 8 4 3 2 8 7 24 2 2 12 2 3 2 3 9 8 8 9 2 2 3 18 3 3 7 3 7 2 2 8 6 5 3 N Class 25 Figure S6 A. Waking Activity Rest O Average Waking Activity ,3584,6-Nitroquipazine Sleep,3584,6-Nitroquipazine 33 44 N+ 3.5 - 2.5 O 33 2 SSRIs 2 2.5 1.5 22 N N 1.5

6-nitroquipazine 11 Normalized Waking ActivityWaking Normalized Normalized Sleep/10 minutes Sleep/10 Normalized 11 0.5 HN 0.5

0 00 00 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours)

Average Waking Activity ,1552,*(D)Zimelidine Dihydrochloride Sleep,1552,*(D)Zimelidine Dihydrochloride Br 33 44 3.5 2.5

33 2 N 2 2.5

1.5 22

1.5

zimelidine 11 Normalized Waking ActivityWaking Normalized Normalized Sleep/10 minutes Sleep/10 Normalized 11 0.5 0.5 0 00 0 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) N

Average Waking Activity ,491,(+)-Chlorpheniramine Maleate 3 Sleep,491,(+)-Chlorpheniramine Maleate 3 4 4 Cl 2.5 3.5 N 3 2 3 2 2.5

1.5

22 Normalized Rest Normalized

chlorpheniramine 11 1.5 Normalized Waking ActivityWaking Normalized

Normalized Sleep/10 minutes Sleep/10 Normalized 1 0.5 1 0.5

0 00 5 10 15 20 25 30 35 40 45 50 00 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours)

Normalized Waking Activity Waking Normalized Average Waking Activity ,666,Fluvoxamine Maleate Sleep,666,Fluvoxamine Maleate N 33 44 O 3.5 2.5 33 2 2 2.5 F F fluvoxamine 1.5 22 1.5 1

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized 11 0.5 F 0.5 N O 0 00 NH 00 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 2 Experimental Time (Hours) Experimental Time (Hours) Time Time B.

Average Waking Activity ,4915,*(H)Chlordane Average Waking Activity ,4797,Heptachlor 55 55 4.5 4.5 44 44 chlordane 3.5 heptachlor 3.5 Cl Cl 33 Cl 33 2.5 2.5 Cl 22 Cl 22

1.5 1.5 Normalized Waking ActivityWaking Normalized Normalized Waking ActivityWaking Normalized Cl Cl 11 11 0.5 0.5

0 Cl 0 00 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Cl Experimental Time (Hours) Experimental Time (Hours) Cl Cl Cl Cl Cl Cl Average Waking Activity ,4783,Strobane Average Waking Activity ,4804,Aldrin strobane 55 aldrin 55 4.5 4.5 Cl 44 Cl 44 3.5 Cl 3.5 Cl 33 Cl 33 Cl 2.5 2.5

2 22 Cl 2

1.5 1.5 Normalized Waking ActivityWaking Normalized Cl ActivityWaking Normalized Cl Cl 11 11 0.5 0.5

00 0 Cl Cl 0 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Cl Cl

Average Waking Activity ,4907,*(D)Toxaphene Average Waking Activity ,4782,Endosulfan 55 55

4.5 4.5 Normalized Activity WakingNormalized toxaphene Activity WakingNormalized endosulfan Cl 44 44 3.5 Cl 3.5 Cl Cl Cl 33 33 2.5 Cl 2.5 22 O 22

1.5 1.5 Normalized Waking ActivityWaking Normalized Normalized Waking ActivityWaking Normalized Cl 11 O S 11 Cl 0.5 Cl 0.5 0 00 Cl 00 5 10 15 20 25 30 35 40 45 50 O Cl 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Cl Time Cl Time Cl

26 C. D. DOPA-U/ A. B.

DOPA-U ACH-M- Figure S7

Rest 1 1 3α Total - - [1 [1 cyclohexyl N 1 cyclohexyl - - - - bis - (2 (2 benzo[B]thien # Rest cyclopropyl cyclohexanamine - - - benzo[B] benzo[B] Bouts methoxytropane (4 - Rest Bout fluorophenyl) ] ] pyrrolidine

Length piperidine thienyl thienyl methyl DOPA

Rest ACH - Latency 2 - yl 4.5 3.5 2.5 1.5 0.5 ) ) - 4 3 2 1 0.5

Activity - M -

Total U/ 3α-bis-(4-fluorophenyl)1 - methoxytropane 1.5 Waking 1-benzo[B]thien-2-yl- 2

Activity DOPA N-cyclopropyl methyl

cyclohexanamine2.5 Name WAY Quinelorane ODQ Ifenprodil Ifenprodil RU Ajmalicine Quinacrine Quinacrine Terazosin 5 Quinazolin NAN MK Nicergoline H Hycanthone Dihydroergocristine PD 168077 Buspirone - 3 - methyl

89 1-[1-(2-benzo[B]thienyl) - - 24969 912 - 3.5 - 190

- cyclohexyl]pyrrolidine 100635 7 6 5 4 3 2 1 U

- urapidil 1-[1-(2-benzo[B]thienyl)4 - 5(6h)

cyclohexyl]piperidine4.5

1 - -0.2 0 0.2 0.4 0.6 0.8 one Target/Indication 5 agonist dopamine cyclase guanylyl alpha alpha 5 not annotated antimalarial antimalarial alpha alpha alpha 5 alpha1 alpha1 PKA antagonist not annotated alpha agonist dopamine 5 - - - - HT1A HT1A antagonist HT1A agonist HT1A antagonist HT1A HT1A agonist ACH-M- DOPA-U/ ACH-M------2 1 1 antagonist 1 antagonist 1 antagonist 1 antagonist 1 antagonist 1 antagonist 3α - - antagonist antagonist - bis - methoxytropane (4 trihexyphenidyl - fluorophenyl) benztropine 3 alcuronium oxybutynin deptropine metixene Rank Name Name Rank 55 54 49 47 45 43 42 38 37 36 33 32 30 27 26 23 21 20 18 17 16 15 13 10 8 7 6 4 3 2 1 7 6 5 4 3 2 1 4

DOPA

Risperidone Spiroxatrine Terazosin Domperidone Amodiaquine Quinacrine BMY Nicergoline Perphenazine Quinazolin M77 Prazosin NAN RU NAN Dihydroergocristine P ARC239 WAY H Vincamine Ifenprodil 5 Buspirone MK Ajmalicine L Droperidol WAY Ifenprodil Quinacrine 3α-bis-(4-fluorophenyl) 1 - - - -

methyl 765314 methoxytropane MPPF 89 - - 24969 912 - - - - - 190 190 7378 2 100635 100635 deptropine - 5 U/ACH - urapidil - 5(6h) benztropine3 Mean - 4 one -

M alcuronium 6 - 0.750 0.755 0.769 0.770 0.778 0.781 0.781 0.785 0.789 0.789 0.793 0.793 0.797 0.802 0.802 0.809 0.810 0.811 0.813 0.813 0.817 0.817 0.818 0.822 0.826 0.828 0.831 0.835 0.848 0.848 0.851 Correlation trihexyphenidyl5 ACH

metixene6 7 548 1. - M oxybutynin7 -

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1 27 0.4 0.6 0.8 1 -0.4 -0.2 0 0.2 - - 0 0.2 0.4 0.6 0.8 1 0.4 0.2 Correlation Figure S8 Adrenergic Signaling Waking Activity Rest Average Waking Activity ,3739,Clonidine Sleep,3739,Clonidine 33 α2-adenergic agonists 33 2.5 (Clonidine) 2.5 22 22 Effects in Selected

1.5 1.5 Agonists Wake Rest mammals References

11 11

Normalized Waking ActivityWaking Normalized Normalized Sleep/10 minutes Sleep/10 Normalized 0.5 0.5 Alpha2 0 0 00 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Clonidine

β-AverageAdrenergic Waking Activity ,5356,Clenbuterol agonists Hydrochloride Sleep,5356,Clenbuterol Hydrochloride 33 33 Guanabenz sedating, (S23-S27) 2.5 (Clenbuterol) 2.5 Guanfacine analgesia

2 2 - 2 2 Tizanidine 1.5 1.5 UK 14304

11 11

Normalized Waking ActivityWaking Normalized Normalized Sleep/10 minutes Sleep/10 Normalized 0.5 0.5 Beta

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 R(-)-Isoproterenol stimulant, (S28-S30) Experimental Time (Hours) Experimental Time (Hours) - Sleep,915,Ifenprodil Tartrate Clenbuterol insomnia α1 Averageantagonists Waking Activity ,915,Ifenprodil Tartrate 33 33 Fenoterol 2.5 (Ifenprodil) 2.5 22 22 Antagonists 1.5 1.5

11 11

Normalized Waking ActivityWaking Normalized Normalized Sleep/10 minutes Sleep/10 Normalized

Normalized Rest Normalized Alpha1 0.5 0.5 Nicergoline 0 0 00 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Prazosin increased wake, β-adrenergic antagonists Ifenprodil mixed effects (S30-33) Average Waking Activity ,4571,Carvedilol Tartrate Sleep,4571,Carvedilol Tartrate 3 3 Normalized Waking Activity Waking Normalized 3 (Carvedilol) 3 5-methyl-urapidil on REM sleep

2.5 2.5 Doxazosin 22 22

1.5 L-765314 1.5

11 1

Normalized Waking ActivityWaking Normalized 1

Normalized Sleep/10 minutes Sleep/10 Normalized Beta 0.5 0.5 Bopindolol - reduced activity, (S34, S35) 00 0 0 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Carvedilol sedation Time Time

28 Figure S9

Serotonin Signaling Selected Waking Activity Rest Agonists Wake Rest Effects in mammals References 5HT-1A-receptor agonists 5HT-1A Average Waking Activity ,3696,Buspirone Sleep,3696,Buspirone 33 (Buspirone) 3.53.5 BMY-7378 - 3 2.5 3 BP-554 short term increases in 2.5 22 2.5 waking; long term increases (S36-40) 22 Buspirone in SWS (humans, rats, cats) 1.5 1.51.5 PAPP 11

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized 1 RU-24969 - 0.5 0.50.5 short term increases in 00 0 0 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) 5HT-1B/D - waking followed by general (S41,42) Average Waking Activity ,610,Cgs-12066A Maleate Sleep,610,Cgs-12066A Maleate 3.5 deactivation 33 5HT1B/D agonists 3.5 CGS-12066A

3 2.5 (CGS-12066A) 3 5HT-1D 2.52.5 22 Dihydroergotamine migraine treatment 22 1.5 potentiates 5HT-1A induced S43 1.51.5 GR 46611 1 locomotion in guinea pig 1 1

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized

0.5 0.50.5 5HT-2/3 short-term sleep 00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 suppression; long term (S38,S44) Experimental Time (Hours) Experimental Time (Hours) N-methylquipazine -/ Quipazine increase in SWS Average5HT1D Waking Activity ,692,Dihydroergotamineagonists Methanesulfonate Sleep,692,Dihydroergotamine Methanesulfonate 33 3.53.5

3 2.5 (Dihydroergotamine) 3 Antagonists 2.52.5 22 5HT-1A 22 1.5 1.51.5 MM-77 11

Normalized Waking ActivityWaking Normalized 1 reduced locomotion; Normalized Sleep/10 minutes Sleep/10 Normalized 1 NAN-190 0.5 0.50.5 initial sleep suppression -/ (S36,45-47) 0 0 P-MPPF followed by long term SWS 00 5 10 15 20 25 30 35 40 45 50 0 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) S(-)-UH-301 increase 5HT-2/3 agonists Spiroxatrine Average Waking Activity ,3616,Quipazine Sleep,3616,Quipazine 33 (Quipazine) 3.53.5 WAY-100635 2.5 33

2.5 22 2.5 5HT-1D 22 1.5 Metergoline short-term insomnia in cats S48 1.51.5 11 Normalized Waking ActivityWaking Normalized 11 Normalized Sleep/10 minutes Sleep/10 Normalized 5HT-2/3

0.5 0.50.5 Cinanserin increased sleep; increased 0 00 0 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 (S38, S49-52) Experimental Time (Hours) Experimental Time (Hours) Ketanserin exploration LY-53857 5HTAverage-1A Waking antagonists Activity ,3727,Nan-190 Sleep,3727,Nan-190 33 3.53.5 Norcyclobenzaprine 3 2.5 (NAN-190) 3 2.52.5 22 Reuptake 22 1.5 1.51.5 Inhibitors 1

1 1 Normalized Waking ActivityWaking Normalized

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5

0.50.5 6-Nitroquipazine improves insomnia in (S53-55) Normalized Rest Normalized 00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Fluoxetine patients; REM suppression; Experimental Time (Hours) Experimental Time (Hours) Fluvoxamine biphasic effects on SWS in rats/cats -- up short term, Paroxetine Average Waking Activity ,2503,Metergoline Phenylmethyl Ester Sleep,2503,Metergoline Phenylmethyl Ester down long term 3 5HT-1D antagonists 3.5 3 3.5 Zimelidine 3 Normalized Waking Activity Waking Normalized 2.5 (Metergoline) 3 2.52.5 22 22 1.5 1.51.5 11

Normalized Waking ActivityWaking Normalized 1

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 0.50.5

0 00 00 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) 5-HT-2/3 antagonists

Average Waking Activity ,1248,Ketanserin Tartrate Sleep,1248,Ketanserin Tartrate 33 (Ketanserin) 3.53.5

3 2.5 3 2.52.5 22 22 1.5 1.51.5 1

1 1 Normalized Waking ActivityWaking Normalized

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 0.50.5

0 00 00 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Serotonin Reuptake

Average Waking Activity ,666,Fluvoxamine Maleate Sleep,666,Fluvoxamine Maleate 33 Inhibitors (Fluvoxamine) 3.53.5 3 2.5 3

2.5 22 2.5 22 1.5 1.51.5 11

Normalized Waking ActivityWaking Normalized 1

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 0.50.5

00 0 0 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Time Time 29 Figure S10 Dopamine Signaling Waking Activity Rest Selected Agonists Wake Rest Effects in mammals D1- agonists References Average Waking Activity ,1496,Skf 83565 Hydrobromide Sleep,1496,Skf 83565 Hydrobromide 33 3.53.5 D1-Receptor (SKF-83565) 3 2.5 3 Chloro-APB 2.52.5 /- 22 22 increased waking, reduction 1.5 CY 208-243 - 1.51.5 in sleep (rat and monkeys), (S56-59) 11 1 Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized R(+)-6-Bromo-APB - and increased grooming 0.5 0.50.5 N-Allyl-SKF-38393 00 00 - 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) SKF 83565 - AverageD2 Waking- Activityagonists ,2347,R(-)Apomorphine Hydrochloride Hemihydrate Sleep,2347,R(-)Apomorphine Hydrochloride Hemihydrate 33 3.53.5

3 D2-Receptor 2.5 (Apomorphine) 3 biphasic; at low 2.52.5 22 Apomorphine concentrations, reduces 22 waking and locomotor 1.5 Bromocriptine (S60-63) 1.51.5 activity, and increases 1

1 1 Normalized Waking ActivityWaking Normalized Normalized Sleep/10 minutes Sleep/10 Normalized 1 Dipropyl-6,7-ADTN sleep. At high doses, the 0.5 0.50.5 opposite. 0 00 Pergolide 00 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) (-)-3PPP D2/3Average Waking-agonists Activity ,1399,Ropinirole Hydrochloride Sleep,1399,Ropinirole Hydrochloride 33 3.53.5

3 2.5 (Ropinirole) 3 D2/3-Receptor 2.52.5 22 Quinelorane - 22 1.5 reduced waking; no 1.5 (S42,S64) 1.5 Quinpirole - significant changes in sleep 11

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized 1 -/ 0.5 0.50.5 Ropinirole

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) D4-Receptor non-stereotyped shuffling in Experimental Time (Hours) S65 D4-receptor agonists PD 168077 rats

Average Waking Activity ,3574,Pd 168077 Sleep,3574,Pd 168077 33 (PD-168077) 3.53.5 3 Antagonists 2.5 3

2.5 22 2.5 D1-Receptor 22 decreased waking, 1.5 R(-)-SCH-12679 (S56-59, 1.51.5 increased sleep S61,S66) 11 -

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized 1 SCH-23390 0.5 0.50.5

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 D2-Receptor Experimental Time (Hours) Experimental Time (Hours) Benperidol

D1Average-antagonists Waking Activity ,1274,R(-)-Sch-12679 Maleate Sleep,1274,R(-)-Sch-12679 Maleate 3.5 extra-pyramidal effects 33 3.5 Droperidol

3 (movement disorders); (S57,S61,

2.5 (SCH-12679) Rest Normalized 3 Domperidone S66) 2.52.5 increased sedation 22 22 L-741626 1.5 1.51.5 1 1 1 short term increase in

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized

Normalized Waking Activity Waking Normalized D3-Receptor 0.5 0.50.5 waking followed by long- DS121 S65 00 00 term increases in sleep 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 -/ Experimental Time (Hours) Experimental Time (Hours) GR 103691 amount D2-antagonists

Average Waking Activity ,2913,Benperidol Sleep,2913,Benperidol 33 (Benperidol) 3.53.5

3 2.5 3 2.52.5 22 22 1.5 1.51.5 11

1 Normalized Waking ActivityWaking Normalized

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 0.50.5

0 00 0 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) D3 antagonists

Average Waking Activity ,885,S(-)-Ds 121 Hydrochloride Sleep,885,S(-)-Ds 121 Hydrochloride 33 (DS121) 3.53.5

3 2.5 3

2.5 22 2.5 22 1.5 1.51.5 1

1 1 Normalized Waking ActivityWaking Normalized

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 0.50.5

0 0 00 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Time Time

30 Figure S11 GABAergic Signaling Waking Activity Rest GABA-A agonist Selected Average Waking Activity ,3384,Gbld345 Sleep,3384,Gbld345 Effects in mammals 33 3.53.5 Agonists Wake Rest References (GBLD-345) 33 2.5 GABA-A 2.52.5 22 22 GBLD-345 - anxiolytic; sedative S68 1.5 1.51.5 1 Avermectin B1

1 11 Normalized Waking ActivityWaking Normalized Normalized Sleep/10 minutes Sleep/10 Normalized -

0.5 0.50.5 0 GABA-B 00 0 hypnotic S69 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) CGP-13501 /-

Average Waking Activity ,410,Cgp-13501 Sleep,410,Cgp-13501 GABA-B- agonist 3.5 33 3.5

3 2.5 (CGP-13501) 3 Antagonists 2.52.5 22 GABA-A 22 1.5 1.51.5 1S,9R-Hydrastine 11 1

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized Endosulfan 0.5 0.50.5 increased locomtor activity; (S70-72)

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Heptachlor induction of fighting; Experimental Time (Hours) Experimental Time (Hours) pro-convulsant GABA-A-antagonist Chlordane

Average Waking Activity ,4354,"*(S)1S,9R-Hydrastine" Heptachlor (1S,9R-Hydrastine) Sleep,4354,"*(S)1S,9R-Hydrastine" 33 3.53.5

2.5 33 Aldrin

2.5 22 2.5 anxiolytic; dose dependant 22 Mixed (+/-) Modulator 1.5 increase or decrease in (S73,74) 1.51.5 11 Tracazolate locomotor activity

Normalized Waking ActivityWaking Normalized -

11 Normalized Sleep/10 minutes Sleep/10 Normalized

0.5 0.50.5

00 Neuroactive Steroid GABA Regulators 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Allopregnanolone - GABA agonist/ stimulant; anxiolytic; ataxic; antagonist Pregnanolone depressant (S75-77)

Average Waking Activity ,3474,*(D)Tracazolate Hydrochloride Sleep,3474,*(D)Tracazolate Hydrochloride Normalized Rest Normalized 33 (Tracazolate) 3.53.5 Alfadolone hypnotic 3 2.5 3

2.5 22 2.5 22 1.5 Normalized Waking Activity Waking Normalized 1.51.5 11

Normalized Waking ActivityWaking Normalized 1

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 0.50.5

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Steroidal GABA agonist Average Waking Activity ,3397,Alfadolone Acetate 33 Sleep,3397,Alfadolone Acetate 3.53.5

2.5 (Alfadolone) 33

2.5 22 2.5

2 1.5 2 1.51.5 11

Normalized Waking ActivityWaking Normalized 1

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 0.50.5

00 0 0 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Time Time

31 Figure S12 Melatonin Signaling Selected Waking Activity Rest Agonists Wake Rest Effects in mammals References Melatonin Agonist (8-methoxy- 8-methoxy-2- propionamidotetralin increase in sleep propensity 2-Averagepropionamidotetralin Waking Activity ,1952,8-Methoxy-2-Propionamidotetralin ) Sleep,1952,8-Methoxy-2-Propionamidotetralin 33 3.5 in dirunal species; circadian (S78-81) 3.5 Melatonin - 3 2.5 3 phase shifting 2.52.5 22 IIK7 /- 22 1.5 1.51.5 11 Antagonists 1 Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized /- 0.5 0.50.5 K 185 blocks melatonin effects on S82 00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 DH 97 behavior Experimental Time (Hours) Experimental Time (Hours)

Average Waking Activity ,919,Iik7 Sleep,919,Iik7 33 Melatonin Agonist 3.53.5

3 2.5 (IIK7) 3 2.52.5 22 22 1.5 1.51.5 11 1

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized

0.5 0.50.5

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours)

Melatonin Antagonist Normalized Rest Normalized (DH 97Average) Waking Activity ,1970,DH 97 Sleep,1970,DH 97 33 3.53.5

3 2.5 3 2.52.5 22

22 Normalized Waking Activity Waking Normalized 1.5 1.51.5

1

1 11

Normalized Waking ActivityWaking Normalized Normalized Sleep/10 minutes Sleep/10 Normalized

0.5 0.50.5

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Time Time

32 Figure S13 Histamine Signaling Selected Waking Activity Rest Antagonists Wake Rest Effects in mammals References H1-receptor antagonist Average Waking Activity ,2615,Clemizole hydrochloride Sleep,2615,Clemizole hydrochloride H1-(no HERG 33 3.53.5 (Clemizole) 3 2.5 3 block)

2.5 22 2.5 Chloropyramine - sedation (less pronounced 22 (S83-86) 1.5 in loratadine) 1.51.5 Clemizole -/ 11

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized 1 Loratadine - 0.5 0.50.5

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Trimeprazine Experimental Time (Hours) Experimental Time (Hours) H1-antagonist (psychostimulant) Diphenylpyraline psychostimulant S87

Average Waking Activity ,5651,Diphenylpyraline Hydrochloride Sleep,5651,Diphenylpyraline Hydrochloride 3 (Diphenylpyraline) 3 3.53.5 H1- (HERG Blockers) 2.5 33 Astemizole 2.5 22 2.5 22 Clemastine 1.5 some sedation in 1st 1.51.5 11 Meclizine generation anti-histamines; Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized 1 /- 0.5 less pronounced in second 0.50.5 Oxatomide

00 0 generation anti-histamines 0 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Promethazine H1-antagonist(+HERG) Rest Normalized Terfenadine

(ClemastineAverage Waking Activity ,5553,Clemastine) Sleep,5553,Clemastine 33 3.53.5

3 Normalized Waking Activity Waking Normalized 2.5 3

2.5 22 2.5 22 1.5 1.51.5 11

Normalized Waking ActivityWaking Normalized 1

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 0.50.5

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Time Time

33 Figure S14

Adenosine Signaling Selected Waking Activity Rest Agonists Wake Rest Effects in mammals References Adenosine increased sleep; decreased Agonist (S88-92) Average Waking Activity ,2146,2-Chloroadenosine Sleep,2146,2-Chloroadenosine - locomotor activity 33 3.53.5 2-chloroadenosine (2-chloroadenosine) 3 2.5 3 2.52.5 22 Antagonists 22 1.5 1.51.5 A1 1 1 1 Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized 1,3-diethyl-8- (S88,93) 0.5 0.50.5 phenylxanthine 00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 locomotor stimulant Experimental Time (Hours) Experimental Time (Hours) A1-antagonist 8-cyclopentyl-1,3- dipropylxanthine (8-cyclopentyl-1,3- Average Waking Activity ,599,"8-Cyclopentyl-1,3-Dipropylxanthine" Sleep,599,"8-Cyclopentyl-1,3-Dipropylxanthine" 3 3.5 A3 3 dipropylxanthine) 3.5 unknown behavioral effects 3 2.5 3 MRS-1220 -/ 2.52.5 22 22 1.5 1.51.5 11 1

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized

0.5 0.50.5

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours)

Average Waking Activity ,2210,Mrs 1220 Sleep,2210,Mrs 1220 3 3 A3-antagonist Rest Normalized 3.53.5 2.5 (MRS-1220) 33 2.5 22 2.5 22 1.5

1.5 Normalized Waking Activity Waking Normalized 1.5 11

Normalized Waking ActivityWaking Normalized 1

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 0.50.5

00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Time Time

34 Figure S15

Glutamate Signaling Selected Waking Activity Rest Antagonists Wake Rest Effects in mammals References NMDA antagonist NMDA

(L-701252Average Waking Activity) ,3659,"L-701,252" Sleep,3659,"L-701,252" 33 3.53.5 DCQX

3 2.5 3 7-chlorokynurenic 2.52.5 22 acid 22 1.5 1.51.5 CNS-1102 11 1

Normalized Waking ActivityWaking Normalized 1

Normalized Sleep/10 minutes Sleep/10 Normalized -/

0.5 IEM 1460 0.50.5 anxiolytic; hyperlocomotion; 00 00 (S94,95) 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) L-689560 anti-convulsant mGluR 5 antagonist L-701252 (MPEP) L-701324 Average Waking Activity ,1990,2-Methyl-6-(phenylethynyl)pyridine Sleep,1990,2-Methyl-6-(phenylethynyl)pyridine 33 3.53.5

3 MK-801 2.5 3

2.5 22 2.5 SDZ 220-581 22 1.5 Normalized Rest Normalized 1.51.5 11

Normalized Waking ActivityWaking Normalized 1

Normalized Sleep/10 minutes Sleep/10 Normalized 1

0.5 mGluR5 0.50.5 MPEP anxiolytic; can block drug 00 00 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 (S96,97) Experimental Time (Hours) Experimental Time (Hours) Normalized Waking Activity Waking Normalized SIB 1757 -/ induced hyperlocomotion Time Time

35 Figure S16

A. Waking Activity Rest

OO

OO OO YSYSAverage--035035 Waking Activity ,1546,Ys-035 Hydrochloride Sleep,1546,Ys-035 Hydrochloride 55 44 4.5 OO NN 3.5 44 4 3 3.5 3 33 2.5 2.5 22 2 2 1.5

1.5 Normalized Waking ActivityWaking Normalized

Normalized Sleep/10 minutes Sleep/10 Normalized 1 11 1 0.5 0.50 00 0 0 5 10 15 20 25 30 35 40 45 50 000 55 10 15 20 2525 3030 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours)

Methoxyverapamil Sleep,2351,Verapamyl Hydrochloride MethoxyAverageAverage Waking Waking Activity Activity-verapamil ,2351,Verapamyl ,83,Methoxy Verapamil Hydrochloride Sleep,83,Methoxy Verapamil 4 555 4 4 OO 4 55 OO 3.5 4.54.5 3.5

444 3 44 OO NN O3O 3 3.53.5 3

2.5 RestNormalized 2.5 3 O 333 NN O 3 2 2.52.5 2 22 1.5 2222 1.5

1.51.5 minutes Sleep/10 Normalized Normalized Waking ActivityWaking Normalized

Normalized Waking ActivityWaking Normalized 1 Normalized Sleep/10 minutes Sleep/10 Normalized 1 1111 1 1 1 0.5 0.5 0.50.5

0 Rest Normalized 0000 0 5 0100 155 20 25 30 35 40 45 50 00 5 10 15 20 25 30 3535 4040 4545 50 0 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 0 Experimental Time (Hours) ExperimentalExperimental TimeTime (Hours)(Hours) Experimental Time (Hours)

5 Verapamil 4 Normalized Waking Activity Waking Normalized 4 O 3 O N O 3 O N 2 2 60 1 1

0 0 Time Time

50

B. YSYS-035-035 Rest C. YS-035 Waking Activity 6060 6 6 40 6 50 50 5 5

4040 4 4

3030 Day 3 30 3 Night 20 2 202

2 (seconds/minute)

(seconds/minute) minutes rest per hour per rest minutes

Average Waking Activity Activity Waking Average 1 Minutes Rest per Hour perRest Minutes

Average Waking Activity Activity Waking Average 10 1 101

0 0 20 0uM 100nM-7 300nM-7 1uM -6 3uM -6 10uM -5 30uM-5 0uM0 1x10100nM100nM-7 3x10300nM300nM-7 1x101uM1uM -6 3x103uM3uM -6 1x1010uM10uM -5 3x1030uM30uM-5 0 1x10 3x10 1x10 3x10 1x10 3x10 Concentration (Molar) Concentration (Molar) Concentration (Molar)

10

36

0 0uM 100nM 300nM 1uM 3uM 10uM 30uM Figure S17.

A. B. Glucocorticoids Glucocorticoid betamethasone Betamethasone Sleep,318,Betamethasone Average Waking Activity ,318,BetamethasoneO OH 4 5 O 4 5 HO clobetasol 4.5 O 3.5 4 4 F 33 desoxycorticosterone 3.5 O 2.5 33 dexamethasone 2.5 22 22 1.5

1.5 Normalized Waking ActivityWaking Normalized flumethasone minutes Sleep/10 Normalized 11 11 0.5 0.5

flunisolide 0 00 00 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) hydrocortisone Time Time medrysone 6α-methylprednisolone ursolic acid PDE inhibitor

Average Waking ActivityP ,4662,Fosfosal Sleep,4662,Fosfosal 5 4 5 Fosfosal O 4 4.5 3.5 OH 4 4 3 O 3 PDE inhibitors 3.5 2.5 33 diplosalsalate 2.5 22 22 1.5

1.5 Normalized Waking ActivityWaking Normalized fosfosal minutes Sleep/10 Normalized 11 11 0.5 papaverine 0.5 0 00 00 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) propentofylline Time Time Ro 20-1724 SKF-94836 NSAID

Average Waking Activity ,4460,FENOPROFEN Sleep,4460,FENOPROFEN 5 O OH 44 5 Rest Normalized Non-Steroidal Anti-Inflammatory drugs (NSAIDs) 4.5 3.5 Fenoprofen O 4 4 33 bufexamac 3.5 2.5 33 2 diflunisal ActivityWaking Normalized 2.5 2 22 1.5

1.5 Normalized Waking ActivityWaking Normalized fenoprofen minutes Sleep/10 Normalized 11 11 0.5 0.5

piroxicam 0 00 00 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Experimental Time (Hours) Experimental Time (Hours) Time Time Other Anti-inflammatory agents aminophenazone Immunomodulator capsazepine HO O O O O H Average Waking Activity ,2764,CyclosporinN A N Sleep,4664,Cyclosporine N N N 5 4 catechin tetramethyl ether 5 Cyclosporin A O 4 4.5 O N 3.5

H H 4 N N N 4 N N 3 cepharanthine H O O 3 3.5 O O O 2.5 pentamidine 33 2.5 22 2 theaflavin digallate 2 1.5

1.5 Normalized Waking ActivityWaking Normalized Normalized Sleep/10 minutes Sleep/10 Normalized 11 11 0.5 valproate sodium 0.5

0 0 00 5 10 15 20 25 30 35 40 45 50 00 5 10 15 20 25 30 35 40 45 50 5-aminopentanoic acid Experimental Time (Hours) Experimental Time (Hours) Time Time Immunomodulator (NFAT signaling) Waking Activity Rest cyclosporin A

37 Figure S18

Waking Activity Rest

F F F A. 5 Halofantrine 4 4 Cl 3 3 N Cl OH 2 2 1 1 0 0

5 4 Terfenadine OH OH 4 N 3 3 2 2 1 1

0 0 Normalized Rest Normalized

5 4 Psora-4 O

Normalized Waking Activity Waking Normalized 4 3

O 3 O O 3.5 2 2 1 1 3 0 0 Time Time 2.5

3.5 3.53.5 B. Fexofenadine 2 Citirizine 3 3 3.5 ERGLoratadine blockers Cisapride 1.5 2.5 2.52.5 Dofetilide 3 2 2 1

2.5 1.5 1.51.5 Non-ERG

0.5 blockers (seconds/minute) 1 1 Fexofenadine 2

CetirizineCitirizine Average Night Waking Activity Activity Waking Night Average 0 0.5 0.50.5 Loratadine 0 1uM 3uM 10uM 30uM Cisapride 1.5 0 0 Dofetilide 00 0 1x101uM -61uM 3x103uM -3uM6 1x1010uM 10uM-5 3x1030uM-30uM5 1 Concentration (Molar)

0.5 38

0 0 1uM 3uM 10uM 30uM Table S1

Highest Non- Toxic Dose Lowest Drug Name (30/45 µM max) Effective Dose Correlated Dose Range Clonidine 45 µM 1.5 µM 1.5-45 µM Ropinirole 45 µM 0.45 µM 0.45-45 µM NAN-190 30 µM 0.10 µM 0.10-30 µM MK-801 45 µM 0.45 µM 0.45-45 µM Betamethasone 45 µM 0.15 µM 0.15-45 µM Chloro-APB 45 µM 0.45 µM 0.45-45 µM Buspirone 45 µM 4.5 µM 4.5-45 µM Cyclosporin A 45 µM 2 µM 2-45 µM Ketanserin 15 µM 0.15 µM 0.15-45 µM Nicergoline 15 µM 1.5 µM 4.5-45 µM Quipazine 45 µM 1.5 µM 0.15-45 µM Warfarin 45 µM 4.5 µM 4.5-45 µM Fluvoxamine 45 µM 1.5 µM 0.45-45 µM Valproic Acid 45 µM 15 µM 0.45-45 µM Carvedilol 4.5 µM 0.45 µM 0.45-4.5 µM 2-Chloroadenosine 45 µM 45 µM 0.15-45 µM Phenelzine 45 µM 45 µM 4.5-15 µM Trazodone 45 µM 1.5 µM 1.5-15 µM Clozapine 15 µM 1.5 µM 1.5-15 µM Amoxapine 30 µM 3.0 µM 1.0-10 µM Cyclobenzaprine 15 µM 4.5 µM 1.5-15 µM Fluoxetine 15 µM 1.5 µM 1.5-4.5 µM Haloperidol 15 µM 1.5 µM 0.15-1.5 µM Loxapine 15 µM 0.45 µM 0.15-4.5 µM Papaverine 3.0 µM 1.0 µM 0.30-3.0 µM Pergolide 45 µM 0.45 µM 4.5-45 µM; 0.15-1.5 µM Bromocriptine 15 µM 0.15 µM 1.5-15 µM; 0.15-1.5 µM Dihydroergotamine 45 µM 0.15 µM 4.5-45 µM; 0.15-1.5 µM Metergoline 15 µM 15 µM 4.5-15 µM; 0.15-1.5 µM

39

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Supporting Online Material www.sciencemag.org Table of Contents Materials and Methods Figs. S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18 Table S1

43