WO2016187416A1

WO2016187416A1 - A high-throughput assay for quantifying appetite and digestive dynamics

Info

Publication number: WO2016187416A1
Application number: PCT/US2016/033252
Authority: WO
Inventors: Josua JORDI; Florian Engert
Original assignee: Jordi Josua; Florian Engert
Priority date: 2015-05-19
Filing date: 2016-05-19
Publication date: 2016-11-24

Abstract

A high throughput method to determine compounds that alter appetite, satiation or digestion in vertebrates is provided. In one embodiment a method to assay a test compound in a teleost larvae or adult is disclosed, comprising: providing a plurality of teleost larvae or adults in wells of a multi-well substrate having a food source comprising an optically detectable, ingestible, lipophilic molecule and a test compound, wherein at least one well has an individual larvae or adult; simultaneously exposing the plurality of larvae or adults to a non- laser infrared light source; and simultaneously detecting or quantifying emissions from the molecule while the teleost larvae or adult is alive and freely moving. Also disclosed, in one embodiment, is a method to detect nutrient homeostasis in a zebrafish larvae or adult, comprising: providing a plurality of live zebrafish larvae or adults in individual wells of a multi-well substrate having a food source comprising an optically detectable, ingestable, lipohilic molecule; and detecting simultaneously with an infrared macroscope the presence and/or amount of the optically detectable, lipophilic molecule in the intestine of the live, freely moving zebrafish larvae or adult, thereby detecting or quantifying intake and/or digestion rates.

Description

A HIGH-THROUGHPUT ASSAY FOR QUANTIFYING

APPETITE AND DIGESTIVE DYNAMICS

Cross -Reference to Related Applications

This application claims the benefit of the filing date of U.S. application Serial No. 62/163,711 , filed on May 19, 2015, and U.S. application Serial No. 62/331 ,730, filed on May 4, 2016, the disclosures of which are incorporated by reference herein.

Statement of Government Rights

This invention was made with government support under NIH U01 NS090449-01 , NIH DP1 NS082121 , and NIH 5R01 DA030304-02 awarded by the National Institutes of Health. The government has certain rights in the invention.

Background

Food intake and digestion are key physiological processes that provide nutrients to drive all bodily functions. Nutrient intake is matched to nutritional needs by the brain-a process termed nutrient homeostasis-using an intertwined organism-wide array of extrinsic and intrinsic cues coding food availability and demand (Williams and Elmquist, 2012; Begg and Woods, 2013). Dysfunction of feeding and digestive behavior is at the root of global health problems such as obesity, malnutrition and type 2 diabetes, among many others. Therefore, a deeper understanding of feeding and digestive behavior is of high importance (Morten et al., 2014).

Drugs or genetic manipulations that alter food intake or digestion are highly desirable remedies for food-related disorders. To screen for genes or small molecules with clinically desirable impact, the technology of choice needs to support the analyses of hundreds to thousands of individual animals. Large scale studies of feeding behavior have solely been feasible in small invertebrates such as Drosophila; studies with invertebrates were constrained to 20-50 conditions, thereby making large scale screens elusive (Gasque et al., 2013; Solan-Biet et al., 2014; Jordi et al., 2014). Technically even more demanding than measuring food intake is the quantification of nutrient digestion, as there is no direct access to the gastrointestinal tract in most species. More specialized technologies such as bioluminescence, MRI or computed tomography have been valuable to generate insights into in vivo dynamics of digestive function (Fuhrmann and Leroux, 2011 ; Schwizer et al., 2013). However, all these methods employ immobilization of the experimental subject to reduce motion artifacts, thereby complicating concurrent behavioral observations. Consequently, quantifying the dynamics of food intake and its digestion with high- throughput is a formidable technological challenge in any freely behaving animal.

Summary

The invention provides a high throughout method for freely moving lower vertebrates. In one embodiment, the invention provides a method to assay an agent or the effect of a compound in a teleost larvae, juvenile or adult. The method includes providing a plurality of teleost larvae or adults in wells of a multi-well substrate having an agent such as a food source linked to an optically detectable, e.g., fluorescently detectable, ingestible molecule, wherein at least one well has an individual larvae, juvenile or adult; simultaneously exposing the plurality of larvae or adult to a non-laser light source; and simultaneously detecting or quantifying emissions from the molecule while the teleost larvae, juvenile or adult is alive and freely moving. In one embodiment, the teleost is a zebrafish. Compounds that may be screened in the assay may be from a library, e.g., Prestwick Library - http:/A«ww.prestwichchemical.∞r^ Spectrum Library - http^/vmw.msdiscovery.corn/spectrum.html, Biomol Neurotransmitter Library - http://Vvww.enzolifesciences.com/BML-281 O/screen-well-neurotransmitter-library-10-plate-set/, and Chembridge Library - http://www.chembridge.com/screening libraries/diversity libraries/index.php. In one embodiment, the teleost is fasted prior to providing the agent and/or molecule. In one embodiment, the teleost is fed prior to providing the agent and/or molecule. In one embodiment, the molecule is excitable by infrared light, is lipophilic, is photostable. e.g., bleaches at a slow rate and/or persists for 1 , 2, 5, 10 or for more than 24 hours, is detectable at low concentrations, or any combination thereof. In one embodiment, the molecule is linked to a naturally occurring food source. In one embodiment, ingestion and digestion rates are quantified. In one embodiment the light source is a non-laser infrared light source. In one embodiment, a macroscope is employed to detect or quantify emissions. In one embodiment, the molecule is photostable. In one embodiment, ingestion and digestion rates are quantified. In one embodiment, the agent is a food source such as a dry food pellet or a protozoa. In one embodiment, the optically detectable molecule is a fluorescent protein, dye, biotin, or a substrate of a luminescent protein. For example, the food source comprises a protozoa that expresses a luciferase which ingests a molecule comprising a luciferase substrate. In one embodiment, the food source is linked to biotin and streptavidin is linked to an optically detectable dye. In one embodiment, the detection captures at least 120 images in a time window of 120 minutes.

Exemplary dyes include but are not limited to 1-Anilinonaphthalene-8-Sulfonic Acid, 2- Anilinonaphthalene-6-Sulfonic Acid, 4-(4-(Didecylamino)styryl)-N-Methylpyridinium Iodide, 4-(4- (Dilinoleylamino)styryl)-N-Methylpyridinium 4-Chlorobenzenesulfonate, FAST Dil™ oil; DilA9,12-C18(3), CI04 (1 ,1'-Dilinoleyl-3,3,3\3'-Tetramethylindocarbocyanine Perchlorate, 1 ,1'-Dilinoleyl-3,3,3',3'-

Tetramethylindocarbocyanine, 4-Chlorobenzenesulfonate, 3,3'-Dilinoleyloxacarbocyanine Perchlorate, 9- Amino-6-Chloro-2-Methoxyacridine, 8-Aminonaphthalene-1 ,3,6-Trisulfonic Acid, Disodium Salt, alkaline phosphatase, DapoxyW Sulfonic Acid, 4-(4-(Dihexadecylamino)styryl)-N-Methylpyridinium Iodide, 1 ,1'- Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine Perchlorate, nile red, Neurotrace®, Vybrant®, 1 ,1'- Dioctadecyl-e,e'-Di(4-Sulf0phenyl)-3,3,3\3'-Tetramethylindoca(1}ocyanine.Streptavidin may be linked to gold particles, linked to HRP, AP, DyLight, rhodamine, RPE, or Qdot.

In one embodiment, the invention provides a method to detect nutrient homeostasis in a zebrafish larvae, juvenile (e.g., up to 30 dpf) or adult. The method includes providing a plurality of live zebrafish larvae, juvenile or adults in individual wells of a multi-well substrate having a food source comprising an optically detectable, ingestable, lipophilic molecule; and detecting simultaneously with a macroscope the presence and/or amount of the optically detectable, lipophilic molecule in the intestine of the live, freely moving zebrafish larvae, juvenile or adult, thereby detecting or quantifying intake and/or digestion rates, e.g., in the presence of one or more test compounds. In one embodiment, the zebrafish is fasted prior to providing the molecule. In one embodiment, the zebrafish is fed prior to providing the molecule. In one embodiment, the molecule is photostable. In one embodiment, ingestion and digestion rates are quantified. In one embodiment, the zebrafish is exposed to a test compound prior to providing the molecule. In one embodiment, the zebrafish is exposed to a test compound after providing the molecule. In one embodiment, the method further comprises identifying whether the compound induces or inhibits feeding behavior. In one embodiment, the food source is a dry food pellet or a protozoa. In one embodiment, the molecule is a fluorescent protein, dye, biotin, or a substrate of a luminescent protein. For example, the food source comprises a protozoa that expresses a luciferase which ingests a luciferase substrate. In one embodiment, the food source is linked to biotin and streptavidin is linked to an optically detectable dye. In one embodiment, the detection captures at least 120 images in a time window of 120 minutes.

In one embodiment, the invention provides a method to measure kinematics in a zebrafish larvae, juvenile (e.g., up to 30 dpf) or adult. The method includes providing a plurality of live zebrafish larvae, juvenile or adults in individual wells of a multi-well substrate having a food source comprising an optically detectable, ingestable, lipophilic molecule; and detecting simultaneously with a macroscope the presence and/or amount of the optically detectable, lipophilic molecule in the intestine of the live, freely moving zebrafish larvae, juvenile or adult, thereby quantifying food intake, feeding rate and/or digestion rate.

In one embodiment, the invention provides a method to detect nutrient homeostasis in a zebrafish larvae. The method includes providing a plurality of live zebrafish larvae in individual wells of a multi-well substrate having media containing a food source linked to an optically detectable, e.g., a substrate for a luciferase, biotin or streptavidin, ingestable molecule; and detecting simultaneously with a macroscope, e.g., an infrared macroscope, the presence and/or amount of the optically detectable, ingestable molecule in the intestine of the live, freely moving zebrafish larvae, thereby detecting or quantifying intake and/or digestion rates. In one embodiment, the zebrafish is fasted prior to providing the molecule. In one embodiment, the zebrafish is fed prior to providing the molecule. In one embodiment, the molecule is excitable by infrared light, is lipophilic, is photostable, e.g., bleaches at a slow rate and/or persists for 1 , 2. 5, 10 or for more than 24 hours, is detectable at low concentrations, or any combination thereof. In one embodiment, the molecule is linked to a naturally occurring food source. In one embodiment, ingestion and digestion rates are quantified. In one embodiment, the zebrafish is exposed to a test compound prior to providing the molecule and/or agent. In one embodiment, the zebrafish, e.g., larvae or juvenile, is exposed to a test compound after providing the molecule and/or agent. In one embodiment, after the test compound is contacted with the zebrafish larvae, juvenile or adult, optical emissions from the optically detectable molecule in the intestine are measured to determine the effect, or degree of effect, of the test compound on feed intake, feeding rate, digestive rate, and/or swimming behavior.

Brief Description of Figures

Figure 1. Performance of the infrared macroscope. (A) A camera was situated above a transparent behavioral platform enabling imaging of a multi-well plate. The camera was protected by a long-pass filter (LP) with a cutoff at 780 nm. The behavioral platform was illuminated by three distinct light sources: 1) a white light to mimic day light; 2) a far-red light refined by a bandpass filter (BP; 670 - 750 nm) for fluorescent excitation (FL); 3) a 940 nm light array (IR) to enable transmitted light macroscopy. The light sources were alternated to capture either fluorescent (2, white dashes represent border of a single well in a 96 well-plate, diameter 6.8 mm) or transmitted (3) light images. The latter allowed for online tracking of zebrafish larvae swimming displacement, and the former for intestinal dye content measurement. (B) Different doses of DiR' dye dissolved in DMSO, oil or water were excited with the FL light and the fluorescence emission was recorded for 100 ms. Mean ± SD, n = 3. DMSO: y = 0.094 ± 0.002 x + 0.3 ± 0.2, R² = 0.99; oil: y = 0.016 1 0.001 x + 1.2 ± 0.3, R² = 0.93. (C) The light-stability of the DiR¹ dye was assessed by exposing different doses of the dye in DMSO to 2 hours continuous IR and white light, followed by 1 hour continuous FL (760 lux) and white light illumination. The fluorescent emission was recorded every 5 minutes for 100 milliseconds. Mean ± SD, n = 3.

Figure 2. Characteristics of the infrared labeled paramecia. (A, B) A standardized number of paramecia were stained with different doses of DiR' dye, heat-fixed, and fluorescence and brightfield images were captured with a conventional microscope (see representative images in A). Cell number was counted in both channels and compared to estimate the efficacy of paramecia staining. Mean i SD; n = 3 (each >100 cells). (C) Paramecia were stained with different doses of DiR' dye, washed, maintained in standard conditions and their optical density (OD) was measured before and after staining for several hours to test for an impact of staining on paramecia viability. As a control, one group of paramecia was heated to 90°C, which caused cell lysis. Mean i SD; n = 3 (each >100 cells). (D) Paramecia were stained with different doses of DiR' dye, washed, maintained in standard conditions and heat fixed after defined time-intervals post-staining to test the time-stability of the paramecia staining. The emission signal of individual paramecia was measured with the infrared macroscope (100 millisecond exposure time), and paramecia were bleached post-experiment using FL light (760 lux, 2 hours) as a positive control. Mean ± SD; n = 3 (each >100 cells). (EE) Paramecia were exposed for 2 hours continuously to IR and white light followed by 1 hour continuous exposure to FL (340 lux) and white light. The fluorescence emission was recorded every 5 minutes. These light intensities were used for all future experiments. Mean ± SD; n = 3 (each >100 cells).

Figure 3. High-resolution images of the ingested DiR' dye. Fasted zebrafish larvae were exposed to none (A), non-labeled (B) or labeled (C) paramecia for 30 minutes before euthanasia in iced water, agar embedding and imaging with the infrared macroscope using the transmitted (1) or FL (2) light mode, and a Zeiss microscope using brightfield (3) and fluorescence (4) microscopy. Fish were euthanized to minimize digestion. Scale bar reflects 1 mm in both images.

Figure 4. Monitoring feeding and kinematic behavior of freely swimming zebrafish larvae. (A)

EExample fluorescence recording of a 7-dpf zebrafish larvae offered 20 stained paramecia in a single well of a 96 well-plate (white dashes, diameter 6,8 mm). (B, C) Individual zebrafish larvae were fasted for 2 hours prior exposure to the indicated number of labeled paramecia to estimate the sensitivity of the macroscope. Fluorescence images were captured every 10-seconds for28-minutes and the fluorescence emission was quantified. The area under the curve (AUC) is plotted in C. Mean ± SEM; n = 36. (D) Raw image sequence of a 7-dpf zebrafish larva swimming in a 96-well plate well acquired by the CMOS camera during transmitted light mode. Machine vision enables detection of the zebrafish larvae and extraction of its center of mass as x-y coordinates. (E, F) 96 individual zebrafish larvae were exposed to no light flashes, then to repetitive FL light flashes and finally to repetitive dark flashes (300 millisecond each) to test for the sensitivity of motion detection. The mean speed of all fish was plotted at a resolution of 20 Hz (EE). The triggered average speed of all flashes is plotted in (F). Bars represent flash timing. Mean l SEM; n = 9.

Figure 5. Zebrafish larvae actively control nutrient homeostasis and digestion. (A, B) Individual zebrafish larvae were fasted for different time-intervals or constantly fed prior exposure to an excess of labeled paramecia. Their subsequent ingestion and digestion of labeled paramecia was quantified simultaneously, as well as their swimming behavior, using the infrared macroscope. An example fluorescence image sequence of two distinctly fed zebrafish larvae hunting live paramecia is shown in (A) (white dashes - border of a single well of a 96 well plate, diameter 6.8 mm). Mean ± SEM; n = 36. (C) Individual zebrafish larvae were fed different meal sizes. Subsequently, all available paramecia were removed by washing, thereby enabling the recording solely of the digestion of a given meal and the swimming behavior of the fish. A linear regression was fitted into the data (first 40 minutes, dashed line). Mean ± SEM; n = 36. (D) The rate of fluorescence signal change in the larval intestine reflects an interplay of two biological processes, ingestion rate of labeled paramecia (Fimake) and digestion rate (Faction) of previously ingested paramecia (F). Panel C suggested a linear form for the digestion function, and machine learning was used to determine the functional form for the ingestion function. This model can be fit to all experimental data acquired by the infrared macroscope. (Di) Here, an example fit is shown of the model to the raw data in panel B (dimmed). (Dii) The extracted parameters correspond to the saturation level (a) and initial intake rate (α * β I A) of the ingestion function, and the linear slope of the digestion function (v). (Dii) The ingestion (solid line) and digestion function (dashed line) can be plotted using these parameters.

Figure 6. Feeding, digestive and kinematic behavior of short and long-termed starved zebrafish larvae. (A, B) Individual zebrafish larvae were fasted for different time-intervals or constantly fed prior exposure to an excess of labeled paramecia. Their subsequent ingestion and digestion of labeled paramecia was quantified simultaneously with their swimming behavior using the infrared macroscope. (C, D) The model established in Figure 5 can be fit to the above data (dimmed - original data, solid line - model fit) and the experimental outcome parameterized accordingly. Mean ± SEM; n = 36.

Figure 7. Zebrafish larvae behavior upon exposure to an excess of paramecia. Individual zebrafish larvae (A - nac; B - TL strain; 7 dpf) were fasted for 2 hours prior exposure to an approximate number of labeled paramecia per well. The fed group was continuously fed and exposed to > 140 paramecia/well. Their subsequent ingestion and digestion of labeled paramecia was quantified simultaneously with their swimming behavior using the infrared macroscope. Mean ± SEM; n = 36.

Figure 8. Zebrafish larvae actively control nutrient homeostasis and digestion. Analogous experiments as presented in Fig. 4 for the nac larval strain were conducted here with 7 dpf old TL larval zebrafish (distinct wild type strain). (A) Individual zebrafish larvae were fasted for different time-intervals or constantly fed prior exposure to an excess of labeled paramecia. Their subsequent ingestion and digestion of labeled paramecia was quantified simultaneously, as well as their swimming behavior, using the infrared macroscope. Mean i SEM; n = 24. (B) Individual zebrafish larvae were fed different meal sizes. Subsequently, all available paramecia were removed by washing and thereby enabled to record solely the digestion of a given meal and the swimming behavior of the fish. A linear regression was fitted into the data (first 50 minutes, dashed line). Mean ± SEM; n = 24. (C) The model established in Figure 5 was applied here to the TL strain. The model fit to the experimental data acquired in panel A (dimmed data) is shown as a solid line and the parameters were extracted (Ci).

Figure Θ. Distinct environmental, pharmacological and physiological interventions modulate feeding and swimming behavior. (A) Individual zebrafish larvae were fasted for 2 hours or constantly fed prior exposure to an excess of labeled paramecia. White light illumination was shut off 12 minutes post- paramecia exposure (pointed line). Zebrafish ingestion and digestion of labeled paramecia was quantified simultaneously with their swimming behavior. Mean ± SEM; n = 24. (B-F) Individual zebrafish larvae were fasted for 2 hours, exposed for 30 minutes to a specific compound prior being given access to an excess of labeled paramecia. Zebrafish ingestion, digestion and swimming behavior was monitored with the infrared macroscope. Distinct compounds were used: sedating pharmaceuticals (B), common daily drugs

(C) , individual nutrients (D), and physiological peptides (E-F). Mean l SEM; n = 32 (B, C, D), 36 (E, F). All the experimental data acquired were fit to the model established in Figure 4 and the parameters describing the ingestion (G) and the digestion (H) function were extracted.

Figure 10. Developmental and strain specific impact on feeding, digestive and kinematic behavior. Individual zebrafish larvae were fed or fasted for 2 hours prior exposure to an excess of labeled paramecia. Their subsequent ingestion and digestion of labeled paramecia was quantified simultaneously with their swimming behavior using the infrared macroscope. Distinct zebrafish reference strains, (A) nac- little pigmentation; (B) TL-wikJ type; (C) GCaMP6f (brain-wide neuronal activity reporter)-AB background;

(D) TLAB-wild type; (E) EK-wild type; were tested at five, six and seven dpf as indicated. Mean ± SEM; n = 36.

Figure 11. Use of a multi-behavioral screening assay to identify specific appetite or satiation stimulants.

Figure 12. Behaviors that may be measured in the assay.

Figure 13. In an exemplary embodiment, the assay includes a food source (Paramecium) and a larval zebrafish.

Figure 14. Once fed, zebrafish go through a satiation phase which in turn is followed by digestion.

Figures 15A-B. A) Time line for one exemplary assay. B) Data overtime for larvae that were fed before exposure to Paramecium, were fasted for two hours before exposure to Paramecium, fasted for four hours before exposure to Paramecium, or fasted for six hours before exposure to Paramecium. Top graph is Paramecium content in intestine overtime, and lower graph is speed of movement overtime.

Figure 16. Zebrafish larvae physiology.

Figure 17. A schematic of a behavioral screening assay to identify specific appetite or satiation stimulants using measures of feeding and activity or alertness and habitation.

Figures 18A-B. A) Activity (spontaneous) and intestinal Paramecium content in zebrafish fasted for 2 hours or fed before exposure to Paramecium. B) Activity after a dark flash and after a tap (noise).

Figures 19A-B. A) Breakdown of compounds screened in one library and percent of animals employed for treated and control. The library to be a screened may include known compounds such as rapamycin and rimonabant. B) Results from the library. Figure 20. Assay results using anorectic drugs including 1 ,4-dinithphenol, sibutramine, ephedrine, rimonabant, bupropion, benfluorex, fenfluramine, pf-03246799, nicotine, phenyl

propanolamine, melformine and naltrexone. Sibutramine, ephedrine and metformine show a preferential effect on feeding behavior over other behaviors.

Figure 21. Profile of results for 353 primary hits and 27 candidates using multi-behavioral assay.

Figure 22. GPCR binding results for specific appetite and satiation stimulants.

Figure 23. Plot of chemical properties of drug "likeness" applied to the 27 hits based on molecular weight and hydrogen bond donor vs. acceptor.

Figure 24. Drug likeness applied to 27 hits based on topological polar surface area.

Figure 25. Results depicted by behaviors that were modified.

Figure 26. Calculation for SSMD.

Figure 27. Feeding behavior presents a challenge for identifying drugs with specificity for feeding behavior.

Figure 28. Odds ratio for rimonabant.

Figure 29. Components of the Zebrafish model.

Food intake and digestion are vital functions and their deregulation is fundamental for many human diseases. Current methods do not support their dynamic quantification on large scales in unrestrained vertebrates. As described herein, an infrared macroscope was combined with fiuorescently labeled food to quantify feeding behavior and intestinal nutrient metabolism with high temporal resolution, sensitivity and throughput in naturally behaving zebrafish larvae. Using this method and rate-based modeling, it is demonstrated that zebrafish larvae match nutrient intake to their bodily demand and that larvae adjust their digestion rate according to the ingested meal size. Such adaptive feedback mechanisms make this model system amenable to identify potential chemical modulators. For example nicotine, L-lysine, ghrelin, and insulin had an analogous impact on food intake in zebrafish larvae as in mammals. Consequently, the method disclosed herein promotes large-scale translational research of food intake and digestive function in a naturally behaving vertebrate.

Zebrafish (Danio rerio) larvae gained popularity as a vertebrate model system in biomedical research, because their genetic, anatomical and physiological homology to mammals coupled with their small size (about 4 mm) and short generation time make them desirable for large scale screening (Pannekemp and Peterson, 2014; Stewart et al., 2014). Most organs, including the digestive system, are already formed within four to six days post fertilization (dpf) (Lohr and Hammerschmidt, 2011 ; Schlegel and Stainier, 2007). At that age, the larval yolk sac is depleted and they start to depend on external food sources (Gut et al., 2013). One of their natural feeds are live paramecia, a unicellular protozoan, which they hunt with characteristic kinematic behaviors (Gantan et al., 2005; Bianco et al., 2011 ; Trivedi and Bollmann, 2013). Whether these complex paramecia capture maneuvers are simply triggered by a moving prey or actively regulated by a nutrient homeostatic state, i.e., hunger and satiation ,is unknown. A strategy to track food intake and its digestion is to stain the food source with fluorescent-labeled macrospheres, phospholipids or membrane dyes in these transparent vertebrates. Past-efforts implemented such strategies and generated key insights into digestive physiology (Farber et al., 2001 ; Ho et al., 2006; Hama et al., 2009; Bernier et al., 2014; Shimada et al., 2012). However, all of these were based on euthanized or anesthetized larvae, which makes dynamic measurements of intestinal food content difficult. Yet, nutrient homeostasis clearly depends not only on food intake but also on food digestion, absorption and storage.

The infrared macroscope (as well as macroscopes adapted with filters and light sources specific to an optically detectable molecule) can overcome those limitations through three technological innovations. First, it enables high temporal resolution studies of the dynamics in intestinal fluorescent dye content in freely behaving animals. To facilitate the interpretation of these fluorescent traces, analytical tools were developed to extract separate metrics for intake and digestive rates respectively. Second, it supports the concurrent observation of a plurality of, e.g., 96 or more, individual zebrafish larvae. Third, it allows for the simultaneous measurement of fish kinematics using infrared light. The infrared macroscope was used to demonstrate that zebrafish larvae actively regulate nutrient homeostasis and intestinal nutrient metabolism even at a very young age, and that anorectic and orexigenic compounds altered feeding behavior in fish in a similar way as they do in mammals. Overall, the infrared macroscope can quantify feeding and digestive behavior with high temporal resolution, sensitivity and throughput in an unrestrained vertebrate.

The present invention relates to a method useful to identify a compound that alters food intake rates, food digestion rates, or both using fluorescently-tagged or other optically detectable molecules. For example, a fluorescently-tagged molecule that is ingested results in an increase in intestinal fluorescence intensity thus allowing ingestion or digestive metabolism to be followed in vivo. In one embodiment of the instant invention, optically transparent zebrafish larvae are exposed to the fluorescently-tagged molecule. When used in the context of a screen, these reagents provide a high throughput readout of digestive physiology that cannot be assessed using standard screening strategies. The optically-detectable molecules allow assaying of physiological processes, e.g., feeding behavior, in vivo. The reagents are generally non-toxic, lipophilic and/or photostable.

The fluorescently-tagged molecules are simpler to use since it can be administered to and assayed in a wide range of organisms. By providing a visual assay of metabolic processes, the tagged molecules can be used to determine or quantify feeding behavior, or identify compounds that alter behavior.

For example, the following molecules may be linked to a food source: 1 ,8-ANS (1- Anilinonaphthalene-8-Sulfonic Acid), high purity, , 2,6-ANS (2-Anilinonaphthalene-6-Sulfonic Acid), 4-Di- 10-ASP (4-(4-(Didecylamino)styryl)-N-Methylpyridinium Iodide). FAST DiA™ solid; DLA9,12-C18ASP. CBS (4-(4-(Dilinoleylamino)styryl)-N-Methylpyridinium 4-Chlorobenzenesurfonate), FAST Dil™ oil;

DilA9,12-C18(3), CI04 (1 ,1'-Dilinoleyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate), FAST Dil™ solid; DilA9,12-C18(3), CBS (I .V-Dilinoleyl-S.S.S'.a'-Tetramethylindocarbocyanine, 4- Chlorobenzenesulfonate), FAST DiO™ Solid; DiOA9,12-C18(3), CI04 (3,3'-Dilinoleyloxacarbocyanine Perchlorate), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), ANTS (8-Aminonaphthalene-1 ,3,6-Trisulfonic Acid, Disodium Salt), Alkaline Phosphatase Live Stain, CellTracker™ CM-Dil Dye, CellTracker™ CM-Dil Dye, DapoxyKS) Sulfonic Acid, Sodium Salt, Di-2-ANEPEQ (JPW 1114), Di-4-ANEPPS, Di-8-ANEPPS, DiA; 4-Di-ie-ASP (4-(4-(Dihexadecylamino)styryl)-N-Methylpyridinium Iodide), DiD' oil; DilC18(5) oil (1 ,1'- Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine Perchlorate), DiD' solid; DilC18(5) solid (1 ,1 - Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt), DilC12(3) (1 ,1 - Didodecyl-3,3,3',3'-Tetramethylindocart)ocyanine Perchlorate), DilC18(3) (1 ,1'-Dihexadecyl-3,3,3',3'- Tetramethylindocarbocyanine Perchlorate), DilC18(3)-DS) 1 ,1'-Dioctadecyl-3,3,3',3'-

Tetramethylindocarbocyanine-5,5'-Disulfbnic Acid), DilC18(5)-DS (1 ,1'-Dioctadecyl-3,3,3',3'- Tetramethylindodicarbocyanine-5,5'-Disulfonic Acid), DiO'; DiOC18(3) (3,3'-Dioctadecyloxacarbocyanine Perchlorate), DiOC6(3) (3,3'-Dihexyloxacarbocyanine Iodide), DiOC16(3) (3,3- Dihexadecyloxacarbocyanine Perchlorate), DiOC2(3) (3,3'-Diethyloxacarbocyanine Iodide), DiR';

DilC18(7) (l.l'-DioctadecykS.S.S'.S'-Tetramethylindotricarbocyanine Iodide), DiSC3(5) (3,3-

Dipropylthiadicarbocyanine Iodide), Dil Stain (1 ,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate ('Dil'; DilC18(3))), Dil Stain (1 ,1'-Dioctadecyk3,3,3',3'-Tetramethylindocarbocyanine

Perchlorate ('Dil'; DilC18(3))), FluoVolt™ Membrane Potential Kit, Fluorescein bis-(5-Carboxymethoxy-2- Nitrobenzyl) Ether, Dipotassium Salt (CMNB-Caged Fluorescein), Fluorescein-5-(and-6)-Sutfonic Acid, Trisodium Salt, HCS LipidTOX™ Deep Red Neutral Lipid Stain, for cellular imaging, HCS LipidTOX™ Green Neutral Lipid Stain, for cellular imaging, Laurdan (6-Dodecanoyl-2-Dimethylaminonaphthalene), Lipophilic Tracer Sampler Kit, Merocyanine 540, NBD-X (6-(N-(7-Nitrobenz-2-Oxa-1 ,3-Diazol-4- yl)amino)Hexanoic Acid), NeuroTrace® CM-Dil Tissue-Labeling Paste, NeuroTrace® Dil Tissue-Labeling Paste, NeuroTrace® DiO Tissue-Labeling Paste, NeuroTrace® Multicolor Tissue-Labeling Kit - DiO, Dil, DiD pastes, Nile Red, SP-DilC18(3) (1 ,1'-Dioctadecyl-e,e'-Di(4-Sulfbphenyl)-3,3,3^,,3'-

Tetramethylindocarbocyanine), Vybrant® CM-Dil Cell-Labeling Solution, Vybrant® DiD Cell-Labeling Solution, Vybrant® Dil Cell-Labeling Solution, Vybrant® DiO Cell-Labeling Solution

Animal Models

The methods of the present invention, which are directed to screening compounds for activities, are generally applicable for use in a various animals, including vertebrate animals, such as fish. Various species offish are suitable, including teleosts. Suitable teleosts include, for example, zebrafish (Danio rerio), Medaka, Giant rerio, and puffer fish. Animal models are fish that are, in one embodiment, transparent or translucent (i.e., optically clear) in at least one of the following stages: the embryonic, larval, juvenile, or adult stage.

Certain teleosts, including the zebrafish, Medaka, Giant rerio, and puffer fish, offer important advantages over other animal model systems for use in methods of the present invention. First, these teleosts are vertebrates whose genetic makeup is more closely related to that of man than are other models, such as the Drosophila and nematode. All essential components of human form and organ development are mimicked in these teleosts and the morphological and molecular bases of tissue and organ development are either identical or similar to other vertebrates, including man. As a result, these teleosts serve as an excellent model for the study of vertebrate development and human disease states.

Second, these teleosts provide advantageous animal models because some developmental stages are very transparent. Given the transparency, activity in for example the gut can be detected much more rapidly than in non-transparent animals. These activities can be detected in the more mature larval and juvenile forms of the zebrafish. These activities can also be detected in vivo in all three forms or in cells thereof in vitro. By contrast, the mouse, which is commonly used as an animal model system, is an opaque animal and does not allow a similar rapid or in vivo assessment of phenotypic or developmental changes in whole animal or whole organs or tissues.

In general, the body plan, organs, tissues, and other systems of teleosts develop much more rapidly than do such components in other vertebrate model systems (e.g., the mouse). The entire vertebrate body plan of the zebrafish, for example, is typically established within 24 hours. The remaining organs of the zebrafish, including the gut, liver, kidney, and vasculature, are established within 48 hours. The hatched zebrafish embryo nearly completes morphogenesis within 120 hours, thereby making it highly accessible to manipulation and observation and amenable to high-throughput automated observation and detection procedures.

The activity of a compound and responses indicating these activities can be monitored in whole teleosts and/or in vivo or in cells thereof in vitro overtime— a procedure not possible or readily practiced with other animal embryos which develop in utero, such as the mouse. Moreover, the effects of a compound on the whole teleost or on more than one system, organ, or tissue can be detected simultaneously using transparent teleosts. The persistence of such effects can be monitored by using simple visualization methods and over selected time intervals. By comparison, it is extremely difficult to detect and assess developmental and phenotypic changes in organs, tissues, and systems overtime in animals which develop in utero. Mouse embryos, for example, must be removed from the mother— a labor intensive procedure— before an assay can be performed.

Teleosts also offer the advantage that compounds to be evaluated can be administered directly to the developing teleost. Direct introduction of candidate compounds is hindered in animals which develop in utero, such as the mouse embryo. Further, the teleost embryo is an intact, self-sustaining organism. It is different from a mouse embryo, for example, which because it is physically removed from its mother's womb, rt is not self-sustaining or intact; a mouse embryo would function more as an "organ^* culture or the like.

Another significant advantage is cost. Mouse assays are expensive, primarily due to the cost of breeding and maintenance and the need to manually perform injections and subsequent analysis. In contrast, teleosts, such as zebrafish, are comparatively inexpensive to generate and maintain. A single mating of a zebrafish produces 100-200 eggs. Inbred strains are available and thousands of zebrafish can be raised inexpensively in a small room of aquaria. Moreover, teleost eggs, including those of the zebrafish, are externally fertilized. Teleost embryos (such as zebrafish) can survive by diffusion of oxygen from the water and nutrients from the yolk and thus even the absence of the entire circulatory system is well tolerated during early development.

Additionally, single whole teleosts can be maintained in vivo in fluid volumes as small as, for example, 10 to 100 microliters for the first six days of development. Intact live embryos can be kept in culture in individual microtiter wells or multi-well plates. Test compounds can be added directly to the solution in which the fish is immersed. A multi-well format is particularly attractive for high through-put and automated compound screening. The activities of a compound can be assayed in the fish simultaneously in vivo. Wild-type or mutant strains of teleosts may be employed. The mutation can be a heritable mutation, including, e.g., a heritable mutation associated with a developmental defect. The teleost can also be transgenic.

Compounds to be Screened

A variety of compounds from various sources can be screened for enhancing or inhibiting feeding behavior by using the methods of the present invention. Compounds to be screened can be naturally occurring or synthetic molecules. Compounds to be screened can also obtained from natural sources, such as, e.g., marine microorganisms, algae, plants, fungi, etc. Alternatively, a compound to be screened can be from combinatorial libraries of Compounds, including peptides or small molecules, or from existing repertories of chemical compounds synthesized in industry, e.g.. by the chemical, pharmaceutical, environmental, agricultural, marine, cosmeceutical, drug, and biotechnological industries. Compounds can include, e.g., pharmaceuticals, therapeutics, environmental, agricultural, or industrial agents, pollutants, cosmeceuticals, drugs, organic compounds, lipids, glucocorticoids, antibiotics, peptides, proteins, sugars, carbohydrates, chimeric molecules, etc.

Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step fashion. Such compounds include polypeptides, proteins, nucleic acids, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substrtuted glycines and oligocarbamates. Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in Affymax, WO 95/12608, Affymax WO 93/08121 , Columbia University, WO 94/08051 , Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which is incorporated herein by reference in its entirety for all purposes). Peptide libraries can also be generated by phage display methods. See, e.g., Devlin, WO 91/18980. Compounds to be screened can also be obtained from governmental or private sources, including, e.g., the National Cancer Institute's (NCI) Natural Product Repository, Bethesda, Md., the NCI Open Synthetic Compound Collection, Bethesda, Md., NCI's Developmental Therapeutics Program, or the like.

Administration of Compounds

Compounds to be screened can be administered to the teleost by adding the compound, and the molecule, directly to the media containing the live teleost. Alternatively, the compound, and molecule can first be dissolved in the media and the live teleost submerged in the media subsequently. Such approaches have been used to introduce anesthetics and other chemicals to fish embryos. See, e.g., M. WesterfieW, THE ZEBRAFISH BOOK: A GUIDE FOR THE LABORATORY USE OF ZEBRAFISH (3d. ed. 1995), which is incorporated herein in its entirety for all purposes. Compounds can also be administered to the teleost by using microinjection techniques in which the compound is injected directly into the live teleost. For example, sompounds can be injected into either the yolk or body of a teleost embryo or both.

Compounds can also be administered to teleosts by electroporation, lipofection, or ingestion or by using biolistic cell loading technology in which particles coated with the biological molecule are

"biolistically" shot into the cell or tissue of interest using a high-pressure gun. Such techniques are well known to those of ordinary skill in the art. See, e.g., Sambrook et al., supra; Chow et al., Amer. J. Pathol. 2(6):1667-1679 (1998). Compounds can be administered alone, in conjunction with a variety of solvents (e.g., dimethylsulfoxide or the like) or carriers (including, e.g., peptide, lipid or solvent carriers), or in conjunction with other compounds.

Compounds can be administered to the teleost before, at the same time as, or after administration of a dye used for detection of the response in the animal indicating a specific activity.

Detecting Activity and Responses in Teleosts

A variety of techniques can be used together or separately to generate a signal and to detect and assess the effect of a compound on activity. Signals can be generated by, for example, in situ hybridization, antibody staining of specific proteins. Responses indicating activity can be detected by, e.g., visual inspection, macroscope.colorimetry. fluorescence microscopy, light microscopy,

chemiluminescence, digital image analyzing, standard microplate reader techniques, fluorometry, including time-resolved fluorometry, visual inspection, CCD cameras, video cameras, photographic film, or the use of current instrumentation such as laser scanning devices, fluorometers, photodiodes, quantum counters, plate readers, epifluorescence microscopes, scanning microscopes, confocal microscopes, flow cytometers, capillary electrophoresis detectors, or by means for amplifying the signal such as a photomultipliertube, etc. Responses can be discriminated and/or analyzed by using pattern recognition software. Automated Methods

The present invention provides methods for rapid screening of teleosts and compounds for activities, using automated procedures. Multi-well formats are particularly attractive for high through-put and automated compound screening. Screening methods can be performed, for example, using a standard microplate well format, with a whole zebrafish larvae in each well of the microplate. This format permits screening assays to be automated using standard microplate procedures and microplate readers to detect enhancement or inhibition of feeding activity in the zebrafish in the wells. A microplate reader includes any device that is able to read a signal from a microplate (e.g., 96-well plate), including macroscopy, fluorometry (standard or time-resolved), luminometry, or photometry in either endpoint or kinetic assays. Using such techniques, the effect of a specific compound on a large number of teleosts in vivo can be ascertained rapidly. In addition, with such an arrangement, a wide variety of compounds can be rapidly and efficiently screened for their respective effects on the cells of teleosts contained in the wells.

Sample handling and detection procedures can be automated using commercially available instrumentation and software systems for rapid reproducible application of dyes and compounds, fluid changing, and automated screening of target compounds. To increase the throughput of a compound administration, currently available robotic systems, most of which use the multi-well culture plate format, can be used. Screening Methods

The present invention provides methods of screening a compound that enhances, inhibits, or blocks feeding ingestion or digestion activity in a teleost in response to the administration of a dose of a compound to the teleost. Activity is assessed relative to contemporaneous and/or historical control teleosts (or tissues, organs, or cells thereof) to which the compound has not been administered. Activity can be monitored overtime in the teleost to which a compound has been administered as well as in control teleosts. Responses can be detected in a whole teleost or in one or more organs or tissues of a teleost, either simultaneously or separately. Responses can be detected overtime and at predetermined time intervals. These responses can also be detected in cells of a teleost. The methods of the present invention are useful in identifying compounds that would be effective in therapeutic or prophylactic treatment of a variety of digestive diseases. In one aspect, the methods comprise administering the compound to be screened to a teleost larvae by submerging the larvae in culture media in which the compound has been dissolved and which has an optically detectable molecule linked to a food source. After a suitable period, the larvae are analyzed.

Activity can also be detected by techniques indicated previously, including, e.g., a device having a macroscope that captures images overtime and includes a light source and filters, colorimetry, fluorescence microscopy (including, e.g., time-resolved fluorometry), chemiluminescence, digital image analyzing, standard microplate reader techniques, pattern recognition software for response

discrimination and analysis, etc.

The invention will be further described by the following non-limiting example.

.Example

Methods

Animals. Adult and larval zebrafish fish (Danio rerio) were maintained on a 14:10 hour light:dark cycle at 28°C. Larvae were raised at a density of >1 mL/fish in blue water (pH 7.0 sodium-bicarbonate buffer, 1 g/L methylene blue, 0.3 mL/L instant ocean salt, exchanged daily). If not stated directly, fish from the rritfa-'- (nacre) strain were used in all experiments at 7 dpf (Lister et al., 1999). They lack body pigmentation and are therefore significantly more translucent than wild type strains. To minimize clutch specific effects, embryos of at least two independent clutches were pooled for experiments. All protocols and procedures involving zebrafish were approved by the Harvard University/Faculty of Arts & Sciences Standing Committee on the Use of Animals in Research and Teaching (IACUC).

Infrared macroscope. The infrared macroscope (see Figure 1 A) was assembled and aligned using standard structural framing and optomechanic components (Thoriabs, USA) within a light-proof enclosure. The large behavioral platform was machined from clear acrylic and situated above an array of 280 infrared LED's (940 nm, Pinecomputers, USA). The platform was illuminated from above with one cool white light (LZ4, LED engine, USA) and with eight far-red LED's (740 nm, LZ4, LED engine) mounted behind a band pass filter (ET710/75, Chroma, USA). Images of the platform were captured with a CMOS camera (Ul- 3370CP-NIR, USB 3.0, 4MP - 2048 x 2048, 12 bit, full well capacity 13500 β", quantum efficiency 43% at 800 nm, IDS, Germany) protected with a long pass filter (ET 780lp, Chroma). A low F-number lens (EF 50mm f/1.4 USM. Canon, Japan) was used for emission light collection. Custom-written Labview code was used to control the illumination settings and image capture. The white light was always turned on to mimic daylight unless stated. The far-red light served as the excitation light source for fluorescent imaging and the LED array for transmitted macroscopy. For behavioral imaging, fluorescent images were captured every minute for 2 hours with 100 millisecond of exposure time (except Figure 3); and transmitted images were used to trace the swimming distance of all zebrafish larvae at a rate of 20 Hz in real time.

Paramecia staining. Paramecia cultures were grown under standard laboratory conditions. The culture media was filtered through a fine mesh (pore size about 20 μπι), which retained the paramecia and thereby enabled purification. Paramecia were collected into blue water, concentrated by centrifugation for 2 minutes at 1500 rcf, and stained with 0.5 % (vol./vol.) DiR' dye (2.5 mg/mL, 1 ,1'-Dioctadecyl-3,3,3',3'- Tetramethylindotricarbocyanine Iodide, dissolved in DMSO, Molecular Probes, USA) for 20 minutes. Post- staining, paramecia were centrifuged as above, washed and suspended in the desired volume. This protocol was followed throughout the study. The amount of DiR' dye varied for Figure 2 only. Paramecia were quantified by two distinct methods: A) Exact quantification (for Figures 3B-C) was performed manually by aspirating single paramecia into a pipette under a dissecting macroscope and placing them into single wells. B) Approximate paramecia numbers were measured by optical density (OD, 490 nm). OD (x) correlates linearly with paramecia number (y * 10³ / mL) as follows: y = 37 x - 2, R² = 0.81. The paramecia number per mL for this correlation was determined at different ODs using a hemocytometer. For direct imaging purposes, paramecia were heat-fixed by placing 40 pL of a defined paramecia culture onto a 90°C preheated 25 x 75 mm macroscope glass slide (VWR, USA) for 5 minutes. Subsequently paramecia were imaged with the infrared macroscope using 100 millisecond exposure time and/or a Zeiss SteREO Discovery. V12 microscope (AchromatS 1x FWD 60 mm objective, AxioCam HRC camera).

Behavioral experiments. Zebrafish larvae were continuously fed with unlabeled paramecia starting at 4 dpf and were held in petri-dishes prior to every experiment. Both feeding and digestive experiments were conducted. For all experiments larvae were randomly allocated to treatment groups and positioned on the plate.

For feeding experiments, zebrafish larvae were either continuously fed by exposure to an excess of unlabeled paramecia, or were fasted by washing and transferring zebrafish larvae to a new petri-dish containing no paramecia for a given time-interval. Prior exposure to labeled paramecia and behavioral imaging, zebrafish larvae were washed and subsequently placed individually into 160 pL of blue water in a single well of a 96-well plate (Falcon, VWR). Within 1 minute prior to the start of behavioral imaging, 40 pL of labeled paramecia (> 140 paramecia unless stated) was added to each well and the plate imaged with the infrared macroscope as described above. If stimuli were used, zebrafish larvae were fasted for 2 hours, randomly placed into one of the 96 wells, and then 50 pL of drugs or vehicle were added to each well. The zebrafish larvae were pre-exposed to the drug for 30 minutes prior to the addition of 40 pL of labeled paramecia (>140 individual paramecia) and subsequent behavioral imaging. All drugs were dissolved in blue water and the drug concentration per well was as follows: carvedilol (about 10 μΜ, Sigma, USA), melatonin (about 10 μΜ, Sigma), tricaine mesylate or MS-222 (about 10 mM, Sigma), nicotine (about 10 pM, Sigma), caffeine (about 10 pM, Sigma). D-glucose (about 10 mM, Fluka, USA), L- alanine (about 10 mM, Fluka), L-lysine (about 10 mM, Fluka), human ghrelin (about 0.03 or 0.3 pM H- 4864, Bachem, Switzerland), and human insulin (about 0.3. 3, or 30 pM Sigma). For digestive experiments, zebrafish larvae were fasted for 240, 255, 270 and 285 minutes, and then exposed to an excess of labeled paramecia in a petri-dish for 60, 45, 30 and 15 minutes, respectively. Subsequently, zebrafish larvae were washed, placed individually into 200 pL blue water within a single well of an empty 96-well plate and imaged with the infrared macroscope for 2 hours.

Image analysis. Fluorescence emission (AF) in the zebrafish larval intestine was quantified in each well using cell-profiler 2.1.1 (Carpenter et al., 2006). Acquired images were all treated and analyzed equally. First, images were corrected for background and uneven illumination as follows:

Here, AFn* is the raw image; AFbK*eround is the raw image captured at t = 0 minutes; and AFiiummation is a normalization image acquired to correct for uneven illumination. AFnummation was acquired as follows: 200 μΙ_ DiR' dye (125 ng/mL, dissolved in DMSO) was added into each well of a 96-well plate, subsequently twenty fluorescence images were captured of the dye plate under standard conditions prior to calculation of a maximal intensity projection. The mean signal intensity of each well was measured followed by normalization by the maximum intensity well. The resulting quotient was used to define each well's illumination correction. After correction, each image was segmented as follows. Pixels with a gray-value larger than 1 were grouped to adjacent pixels. All pixel groups position with a diameter of 220 pm or larger (single paramecia major and minor ferret diameter are 181 ± 28 pm and 124 ± 18 pm) were localized to each well. Within wells, the pixel group with the maximal integrated intensity (group area * mean intensity) was identified, the integrated signal intensity extracted and plotted as AF. A similar strategy was used to quantify heat fixed paramecia (Figure 2) with an adapted correction and segmentation approach.

Transmitted macroscopy was used to trace the swimming dynamics of zebrafish larvae in all wells. Images were recorded at 20 fps and analyzed online using Labview 13.0 F1 (National Instruments, USA) as follows: The software followed a real time loop, in which each image was subtracted from the next frame's image to obtain pixels that were altered in the frame interval. The resulting delta-pixel image was thresholded based on size (> 605 pm) to record x-y coordinates and a time stamp for each delta-pixel groups, presumably corresponding to a moving single fish. Speed was binned for one minute time intervals, expect for the darkflash experiment (Figures 3D-F)

Modeling. The rate of fluorescence emission (dF/dt) in the zebrafish larval intestinal cavity reflects a signal composed of at least two biological processes: ingestion and digestion of labeled paramecia (given th data showing the lack of significance of the photo-bleaching process), and can be described as a function of resulting fluorescence (F) as follows:

To obtain the exact analytical form of the ingestion and digestion functions a two-pronged approach was taken: First, it was assumed linearity in the digestion rate based on the data shown in Figure 5C, and then a machine-learning based fitting algorithm within Matlab was used to fit the data to several different models with a linear digestion function and a number of different sigmoid-like ingestion functions. The software used the fmincon function to perform a lightly constrained minimization of a function calculating the Root Mean Squared (RMS) error between the data and the model. Leave-one-out cross validation was used to select the most predictive model based on the lowest cross validation RMS error value, which turns out to be a two parameter sigmoid based on the generalized logistic function as shown below:

Here, a is the asymptote of the curve, representing the level at which the function saturates, and β is the relative rate of the function, representing the rate of ingestion before saturation. To calculate the quality of the fit, a bootstrap- based approach was used to calculate the error associated with fitting each of the model parameters.

Results

Infrared macroscope-based quantification of dynamics in intestinal dve content and swimming kinetics of freely behaving zebrafish larvae.

The aim was to measure the intake and digestion of stained paramecia by unrestrained zebrafish larvae, and simultaneously trace their swimming speed with high-throughput. To that end, a macroscope that was able to excite and detect DiR' dye within an area large enough to situate a multi-well plate was employed (Figure 1A). Three major factors were considered when selecting the DiR' dye for paramecia staining, (i) First, the dye was excitable with infrared light, because zebrafish behavior is light sensitive and therefore visible excitation light might induce unwanted behavioral responses (Burgess and Granato, 2007). (ii) Second, the DiR' dye did not stain the fish water due its lipophilic structure. Fluorescent emission was not detectable from 1 μΜ DiR' dye in water, but correlated linearly with dye concentration dissolved in DMSO and oil at detection limits of 7.8 nM (Figure 1 B, signal at least three standard deviations above noise level). The emission signal noise floor was 6.6 ± 0.4 % in DMSO and 17 1 1 % in oil, respectively, (iii) Third, the dye has photostability under the infrared array used for tracking zebrafish kinematic behavior, and under the white light required to mimic day light (Figure 1A). DiR' dye fluorescence emission was not affected by continuous exposure to the 940 nm array and white light, whereas one hour continuous exposure to the DiR' dye excitation light bleached the emission signal by 18.2 1 0.3 % (Figure 1C). Subsequently, a protocol was developed to stain paramecia with the lipophilic DiR' dye. This labeling protocol stained paramecia with high efficiency (Figures 2A-B), had no detectable impact on paramecia viability (Figure 2C) and labeling persisted for several hours (Figure 2C). Stained paramecia had a consistent major and minor feret diameter of 181 1 28 \im and 124 ± 18 \im (mean ± SD), respectively. Similar to the DiR' dye in DMSO, paramecia staining was highly photo stable and did not bleach under the imaging conditions established here (Figure 2D). These illumination settings were used throughout the rest of the study. In sum, paramecia stained with DiR' dye were viable, photo stable and detectable for at least several hours by the infrared macroscope described here.

Next, the performance of the infrared macroscope was evaluated for in vivo detection of intestinal dye content and larval swimming behavior: (i) First, it was determined whether the infrared macroscope is able to detect ingested paramecia in cold-euthanized larval zebrafish (nao-strain) and compared it to a conventional microscope. Euthanized larvae were used to minimize paramecia digestion. Solely DiR' dye labeled paramecia were detectable with both scopes and DiR' dye signal was mainly localized to the intestine of zebrafish larvae (Figure 3). (ii) Second, the sensitivity of the infrared macroscope was tested for quantitative in vivo detection of food intake. Images were segmented using a size threshold to exclude single paramecia detection. This allowed for distinguishing fluorescent emission of freely moving paramecia from paramecia within the intestinal track of a larva. To test the sensitivity of this methodology, zebrafish larvae were fed a defined number of individual paramecia and their intestinal dye content was measured with high temporal resolution (Figure 4A). Intestinal dye content increased overtime relative to the number of paramecia available to each fish (Figure 4B). Analogously, the integrated signal correlated linearly with paramecia ingestion (Figure 4C). The infrared macroscope detection limit was two ingested paramecia, and above this threshold increments of single ingested paramecia were distinguishable. As expected, paramecia availability affected the amplitude of intestinal fluorescence signal, as well as the intake rate (Figure 4B). Hence, free paramecia can be distinguished from intestinal paramecia. (iii) Third, it was tested if the infrared light used for fluorescent excitation affected fish swimming behavior. Dark flashes increased swimming activity in zebrafish larvae and therefore served as positive control (Burgess and Granato, 2007). Zebrafish larvae were exposed to constant white light illumination, then to a set of excitation light flashes and finally to a set of dark flashes while online tracking their swimming behavior with 20 Hz resolution. Excitation light flashes did not alter swimming behavior, whereas dark flashes repeatedly induced swim bursts (Figure 4E). Specifically, dark flashes elicited an increase in swimming displacement for approximately 250 milliseconds after the stimulus, comparable to previous reports

(Figure 4F) (Burgess and Granato, 2007). In sum, the presented infrared macroscope can simultaneously detect intestinal paramecia content and swimming behavior in 96 undisturbed, live zebrafish larvae, Zebrafish larvae actively control nutrient homweostasis and d igestion

Zebrafish larvae execute complex maneuvers for prey capture. However, whether this behavior is a simple motor command triggered by moving prey or actively regulated by nutrient homeostatic state is unknown. To trigger a nutrient demand, zebrafish larvae were fasted for different lengths of time and recorded their intestinal dye content and swimming behavior upon exposure to an excess of labeled paramecia. Shorter periods of fasting (0.5-6 hours) increased intestinal dye content acutely and incrementally with fasting times (Figures 5A-B, Figure 6A), suggesting that zebrafish do in fact modulate their feeding behavior based on hunger state. Longer periods of fasting (>12 hours) did trigger an acute paramecia intake and stimulated a second phase of paramecia ingestion (Figure 6B). Paramecia intake by zebrafish larvae starved for the preceding 24 hours was even lower than the fed control group suggesting that food deprivation spanning a significant portion of larval maturation (> 14 % of total life span at the time) is a major homeostatic burden. Swimming dynamics did not show dramatic changes related to the fasting periods. Occasionally, an initial period of slightly higher swim activity was observed which is likely caused by adaptation to the novel environment (see Figure 6B). In all interventions intestinal fluorescent signals decayed, highlighting the need for dynamic measurements. This decay may be caused by a combination of photo-bleaching, lack of paramecia and/or digestion, (i) Photo-bleaching can be excluded as the illumination settings did not cause significant paramecia bleaching even when continuously exposed for one hour to the excitation light (Figure 2E). In the behavioral protocol utilized here, larvae and paramecia were exposed to the excitation light for a total of 12 seconds (100 milliseconds every minute for 2 hours), (ii) Second, an excess of paramecia (>140 paramecia/fish) was provided to decrease the possibility of paramecia availability becoming a limiting factor for food intake throughout the study. Once >140 paramecia were offered to an individual fish, intestinal fluorescence content dynamics showed minor alterations (Figure 7). (iii) Consequently, intestinal fluorescent signal decay is likely mainly due to paramecia digestion. The DiR' dye gets dissolved in water upon plasma membrane lysis causing its fluorescence emission to vanish (Figure 1 B). To test this specifically, larval zebrafish were fed different meal sizes, then washed to remove all non-ingested paramecia and subsequently their intestinal dye content and swimming behavior were measured (Figure 6C). The intestinal fluorescence signal decayed over time and was fit with a linear-regression. Slopes increased with meal size suggesting that larger meals stimulate larger intestinal digestive activity. Analogous experiments were conducted in another wild type strain (TL) and showed consistent outcomes across strains (Figures 8A-B). Hence, intestinal dye content reflects a signal composed of at least two biological processes, ingestion and digestion of labeled paramecia.

In an effort to describe the complex interplay of paramecia intake and their concurrent digestion, a rate-based model was developed. The basic model structure attributed the temporal changes in intestinal fluorescence to an additive effect between the rate of paramecia ingestion and the rate of paramecia digestion. Paramecia digestion is directly dependent on the intestinal paramecia content and was modelled as a linear decay based on experimental results (Figure 5C). To identify the functional form that most closely reflects the ingestion function, machine learning was used to fit numerous asymptotic functions to the data via root mean square error minimization and selected a modified logistic function using leave-one-out cross validation. The model parameterized the intestinal fluorescence into an ingestion saturation level (a), its corresponding asymptotic rate (β), and the linear slope of the digestion function (v) (Figure 5D). These parameters reflect three biologically relevant functions - total paramecia intake (a); the initial rate of paramecia intake (α* β/4) and the digestion rate (v). Using these parameters, the ingestive and digestive processes responsible for the intestinal fluorescence measured in Figure 5B (Figure 5Diii) was reconstructed. The model suggested that an increase in starvation time stimulated a larger initial paramecia intake rate and increased the total number of ingested paramecia (Figure 5Dii). Importantly, the model predicted that digestion rate increased with meal size in agreement with independent experimental observations (Figure 5C). This rate-based model was applicable to another wild type strain showing the broader feasibility of the concept (Figure 7). The model's predictive power was limited when intestinal fluorescence had multiple or unclear peak structure (Figure 8D, Figure 5-nicotine, D-glucose or L-lysine). Hence, this model simplified the distinction of ingestive and digestive processes, and condensed the temporal data into three discrete biologically relevant parameters.

Finally, to firmly establish that zebrafish larvae, regardless of genetic background, regulate nutrient homeostasis at different stages of maturation, numerous wild type stains at different ages were tested. Notably, four out of the five strains tested here show standard pigmentation patterns (in contrast to the translucent nacre strain used mainly in this work), which demonstrates that this technique is not limited to translucent strains. All larvae increased intestinal fluorescent content post-fasting compared to fed animals at five, six and seven dpf regardless of the strain (Figure 9). Swimming activity increased from five to seven dpf larvae and showed variation between strains. Overall, it was shown that active control of nutrient homeostasis and intestinal nutrient digestion is a general principal seen across otherwise behaviorally and genetically diverse populations of zebrafish larvae.

Distinct environmental, pharmacological and physiological interventions regulate zebrafish larvae feeding and digestive behavior. Zebrafish larvae actively control food intake and its digestion. Active control enables modulation of these key metabolic functions by using the appropriate modulators, distinct known and unknown stimuli were tested for an impact on larval feeding and digestive behavior thereby taking advantage of the high- throughput capacity of the infrared macroscope. Initially, interventions known to modulate zebrafish kinematic behavior (but unknown impact on feeding behavior) were tested, thereby further validating the method, (i) Paramecia hunting behavior was proposed to be primarily mediated by vision (Gahtan et al., 2005). To confirm this finding, white light illumination was turned off 12 minutes post-paramecia exposure in the behavioral experiment. Intestinal dye content stopped rising immediately upon the onset of darkness in the starved group, but did not decline to zero indicating that fish were still able to hunt, albeit with lower efficiency (Figure 10A). Additionally, fish displacement increased acutely upon onset of darkness and settled to a more quiescent state 50 minutes after the onset of darkness. Both observations were in-line with previous fish motion studies (Gahtan et al., 2005; Burgess and Granato, 2007). (ii) Next, three sedative drugs were tested. Two of them, carvedilol and melatonin, were identified in a large-scale kinematic screen in larval zebrafish (Rihel et al., 2010), and the third, MS-222, is a standard anesthetic used in zebrafish research. Non-surprisingly these sedative drugs immobilized the larva to different extents and reduced paramecia intake accordingly (Figure 10B). Total paramecia intake, initial paramecia intake rate and digestion rate were all reduced in line with the impact of sedating drugs on distinct aspects of animal physiology (Figures 10G-H). This data highlights the importance of measuring feeding and digestive behavior simultaneously with kinematic parameters when assessing the behavioral impact of unknown compounds with potential anesthetic properties.

Subsequently, interventions known to alter feeding behavior in rodents and humans, but of unknown impact in zebrafish larvae, were tested: (i) First, common daily drugs-caffeine and nicotine- were tested. Both are popular due to their arousing effect and other properties. Smokers report using cigarettes as a means to control body weight and nicotine inhibits feeding in rodent models, whereas caffeine has no general accepted impact on appetite (Voorhees et al., 2002, Mineur et al., 2011).

Interestingly, caffeine and nicotine induced a faster initial paramecia intake rate in zebrafish larvae likely due to their arousing properties (Figures 10C, G). Caffeine had no long term impact on paramecia intake, whereas nicotine inhibited food intake extensively, (ii) Second, high protein diets are more satiating than high-fat or high-carbohydrate diets in rodents and humans, an effect likely driven by specific amino acids (Morrison et al., 2012; Morrison et al., 2015). L-lysine was shown to be particularly anorectic compared to other isomolar amino acids or glucose (Jordi et al., 2013). Analogous to rodents, L-lysine had an acute impact on paramecia intake and swimming in zebrafish larvae, whereas isomolar concentrations of L- alanine or D-glucose had no apparent behavioral impact (Figure 8D). (iii) Third, numerous hormones contribute to the control of nutrient intake and maintenance of nutrient homeostasis. Two hormones of opposing behavioral effect are the orexigenic hormone ghrelin and the anorectic peptide insulin (Wren et al., 2001 ; Wren et al., 2000; Air et al., 2002; Woods et al., 1979). Comparable to other vertebrates, ghrelin stimulated paramecia intake and insulin inhibited food intake in zebrafish larvae (Figures 8E-F).

Interestingly, the highest ghrelin dose (0.3 μΜ) reduced fish displacement slightly but stimulated paramecia intake, indicating no link between both behaviors. Altogether the impact of previously identified kinematic modulators of zebrafish swimming behavior was reproduced confirming the reproducibility of these effects. Importantly, pharmacological, dietary and endocrinal interventions had an analogous impact on feeding and digestive behavior in zebrafish larvae as previously reported for rodents and humans. Consequently, real time and parallel monitoring of zebrafish larvae feeding and digestive behavior holds promise as an appropriate assay for high-throughput translational research.

Discussion

The zebrafish larvae's appeal for biomedical research comes from their applicability in large-scale whole-animal screens (Rennekamp and Peterson, 2014; Lohr and Hammerschmidt, 2011). Eating and digestion disorders are of major clinical interest and therefore there is a need for technology to make zebrafish larvae available to these fields. The infrared macroscope described here can trace 96 larvae's swimming behavior and simultaneously quantify their food intake and digestion with a sensitivity of single paramecia (Figures 2-4). All measurements are feasible with infrared light thereby not disturbing natural fish behavior. Consequently feeding behavior and intestinal nutrient metabolism are now accessible targets for high-throughput genetic- and drug screens in vertebrates. Such unbiased whole-organism phenotype screens yield significant higher-success rate in identifying first-in-class small molecules compared to target-driven cellular or in vitro approaches thereby contributing to the appeal of the here present technology (Rennekamp and Peterson, 2014; Swinney and Anthony, 2011). Additionally, the use of a vertebrate model such a zebrafish is desirable to exploit physiological, genetic and anatomical homologies to humans. In confirmation of this, interventions linked to human behavioral changes were shown to induce similar phenotypes in fish (Figure 10). Hence, zebrafish larvae hold promise for the identification of conserved mechanisms underlying feeding behavior and intestinal nutrient metabolism.

A major utility of the technique is its ability to answer questions about nutrient homeostasis. It was shown that nutrient deprivation, generally known as hunger, caused an acute increase in nutrient intake (Figure 5). When this nutrient need was met, nutrient intake dropped and larvae were satiated. This behavior occurred on a two-hour timescale, and likely involved higher-order neuronal processing distinct but likely in control of the visual-motor programs required for hunting behavior (Semmelhack et al., 2015; Preuss et al., 2014; Bianco et al., 2015). Upon meal ingestion, food is digested within the gastrointestinal tract. Here, we revealed that digestive activity is matched to the meal size in zebrafish larvae (Figure 5). This adaptable response to the feed size indicates an active control over digestive function and represents a conserved functional feature of the gastrointestinal tract (Fuhrmann and Leroux, 2011 ; Hunt et al., 1985; Camillen, 2006). To resolve the dynamic interaction between intake and the co-occurring digestion of stained paramecia, both processes were parameterized using a rate-based model. The model successfully predicted active regulation of nutrient digestion (Figure 5) and revealed that sedating drugs inhibit digestion (Figure 10) analogous to previous observations in rodents (Tarjman et al., 2005). The model is powerful under controlled experimental conditions (abundant food, known food deprivation times), but may underrepresent other biological parameters. For instance, the ingestive function depends also on paramecia availability (Figure 4B), hunting success rate, and food preference among others; and the digestive function results from the synergistic action of gastric acid and pancreatic digestive enzyme secretion, peristaltic intestinal muscle contractions, epithelial nutrient absorption, microbiota nutrient breakdown, etc. (Hama et al., 2009; Seiler et al., 2012; Rawls et al., 2006; Wallace and Pack, 2003). As larval zebrafish share these features with higher vertebrates, the simultaneous experimental readout of these metrics and integration into the model represents abundant ground for future efforts. Thus zebrafish larvae actively regulate nutrient homeostasis and digestion.

Overall, an infrared macroscope was developed to pioneer feeding and digestive research in zebrafish larvae, and to support future high-throughput discovery endeavors. This opens the avenue for zebrafish larvae research to contribute to the neuronal understanding of nutrient homeostasis, digestive physiology and the underlying crosstalk of the autonomic and central nervous systems.

All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLA!MED IS:

1 . A method to assay a test compound in a te!eost larvae or adult, comprising:

providing a plurality of teleost larvae or adults in wells of a multi-well substrate having a food source comprising an optically detectable, ingestible, lipophilic moiecu!e and a test compound, wherein at least one well has an individual larvae or adult;

simultaneously exposing the plurality of iaa¾e or adults to a non-laser infrared light source; and simultaneously detecting or quantifying emissions from the molecule while the te!eost larvae or adult is alive and freely moving,

2. A method to detect nutrient homeostasis in a zebrafish larvae or adult, comprising:

providing a plurality of live zebrafish larvae or adults In Individual wells of a multi-well substrate having a food source comprising an optically detectable, ingestable, lipohilic molecule; and

detecting simultaneously with an infrared macroscope the presence and/or amount of the optically detectable, lipophilic molecule in the intestine of the live, freeiy moving zebrafish larvae or adult, thereby detecting or quantifying intake and/or digestion rates.

3. The method of claim 1 wherein the teleost is a zebrafish ,

4. The method of claim 1 or 2 wherein the teleost or zebrafish is fasted prior to providing the molecule.

5. The method of claim 1 or 2 wherein the teleost or zebrafish is fed prior to providing the molecule,

6, The method of any one of ciaims 1 to 5 wherein the molecule is photosfable,

7. The method of any one of claims 1 to 8 wherein Ingestion and digestion rates are quantified.

8. The method of any one of ciaims 2 to 7 wherein the zebrafish is exposed to a test compound prior to providing the molecule,

9. The method of claim 8 further comprising identifying whether the compound induces feeding behavior.

10. The method of any one of claims 1 to 9 wherein the food source is a dry food pellet or a protozoa.

1 1 . The method of any one of claims 1 to 10 wherein the molecule is a fluorescent protein, dye, biotin, or a substrate of a luminescent protein.

12. The method of any one of claims 1 to 10 wherein the molecule comprises DiR' iodide, IR-820, Qtracker®800 or a compound that binds biotin.

13. The method of any one of claims 1 or 3 to 12 further comprising identifying a test compound that selectively alters feeding behavior over spontaneous activity, visual response, acoustic response or habituation.

14. The method of any one of claims 1 or 3 to 13 wherein the detecting or quantifying emissions in wells having the test compound while the teleost larvae or adult is alive and freely moving is compared to emissions in a well having a teleost larvae or adult having a food source comprising the optically detectable, ingestible, lipophilic molecule in the absence of the test compound.