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A multi-animal tracker for studying complex behaviors

View Article: PubMed Central - PubMed

ABSTRACT

Background: Animals exhibit astonishingly complex behaviors. Studying the subtle features of these behaviors requires quantitative, high-throughput, and accurate systems that can cope with the often rich perplexing data.

Results: Here, we present a Multi-Animal Tracker (MAT) that provides a user-friendly, end-to-end solution for imaging, tracking, and analyzing complex behaviors of multiple animals simultaneously. At the core of the tracker is a machine learning algorithm that provides immense flexibility to image various animals (e.g., worms, flies, zebrafish, etc.) under different experimental setups and conditions. Focusing on C. elegans worms, we demonstrate the vast advantages of using this MAT in studying complex behaviors. Beginning with chemotaxis, we show that approximately 100 animals can be tracked simultaneously, providing rich behavioral data. Interestingly, we reveal that worms’ directional changes are biased, rather than random – a strategy that significantly enhances chemotaxis performance. Next, we show that worms can integrate environmental information and that directional changes mediate the enhanced chemotaxis towards richer environments. Finally, offering high-throughput and accurate tracking, we show that the system is highly suitable for longitudinal studies of aging- and proteotoxicity-associated locomotion deficits, enabling large-scale drug and genetic screens.

Conclusions: Together, our tracker provides a powerful and simple system to study complex behaviors in a quantitative, high-throughput, and accurate manner.

Electronic supplementary material: The online version of this article (doi:10.1186/s12915-017-0363-9) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

The Multi-Animal Tracker is particularly suitable for studying complex behaviors such as chemotaxis. a An image of the experimental chemotaxis plate. Agar plugs soaked with the attractant odorant (red circle), or the control buffer (yellow circle), are placed on the plate’s lid. Neither the attractant, nor the buffer, come in contact with the agar on the plate. Approximately 150 worms are loaded at the starting point (blue circle). The chemoattractant source, the buffer source, and the starting point form an imaginary equilateral triangle with an edge of 4 cm. b A quantitative cumulative dynamics of worm position in the chemotaxis assay over the course of approximately 15 minutes. The lines indicate the number of worms in each of the regions of interest throughout the experiment (color coded). In this experiment, nearly 180 worms were loaded on the assay plate. About two-thirds of them reached the chemoattractant during the first 15 minutes of the assay. c Images were taken at the specific time points (I–IV) throughout the assay (indicated as dashed lines in b) to illustrate chemotaxis progression in the assay plate. Shown are also the trajectories (red) as identified by the tracker software. d–g The software suite includes a module to generate Attraction Fields (AF), a visualization designed to provide spatial representation of the chemotaxis process throughout the experiment. Shown here are AFs of two chemotaxis assays in which the isoamyl-alcohol attractant was used in 10−4 (d) and 10−2 (f) dilutions. The experimental field is binned to squares (35 × 25 in this case, but any binning defined by the user is possible). Arrows represent the average direction of the worms and the color code indicates the overall occupancy throughout the course of the experiment (15 minutes, ~1000 frames). e, g Chemotaxis dynamics as detailed in section b. Together, these plots provide a full spatiotemporal representation of the chemotaxis behavior of multiple animals over the course of thousands of captured frames
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Fig3: The Multi-Animal Tracker is particularly suitable for studying complex behaviors such as chemotaxis. a An image of the experimental chemotaxis plate. Agar plugs soaked with the attractant odorant (red circle), or the control buffer (yellow circle), are placed on the plate’s lid. Neither the attractant, nor the buffer, come in contact with the agar on the plate. Approximately 150 worms are loaded at the starting point (blue circle). The chemoattractant source, the buffer source, and the starting point form an imaginary equilateral triangle with an edge of 4 cm. b A quantitative cumulative dynamics of worm position in the chemotaxis assay over the course of approximately 15 minutes. The lines indicate the number of worms in each of the regions of interest throughout the experiment (color coded). In this experiment, nearly 180 worms were loaded on the assay plate. About two-thirds of them reached the chemoattractant during the first 15 minutes of the assay. c Images were taken at the specific time points (I–IV) throughout the assay (indicated as dashed lines in b) to illustrate chemotaxis progression in the assay plate. Shown are also the trajectories (red) as identified by the tracker software. d–g The software suite includes a module to generate Attraction Fields (AF), a visualization designed to provide spatial representation of the chemotaxis process throughout the experiment. Shown here are AFs of two chemotaxis assays in which the isoamyl-alcohol attractant was used in 10−4 (d) and 10−2 (f) dilutions. The experimental field is binned to squares (35 × 25 in this case, but any binning defined by the user is possible). Arrows represent the average direction of the worms and the color code indicates the overall occupancy throughout the course of the experiment (15 minutes, ~1000 frames). e, g Chemotaxis dynamics as detailed in section b. Together, these plots provide a full spatiotemporal representation of the chemotaxis behavior of multiple animals over the course of thousands of captured frames

Mentions: We used the tracking system to extract trajectories of a large number of animals tracked simultaneously during 30 minutes of chemotaxis. To study the chemotaxis performance quantitatively, we defined three regions of interest (ROIs, Fig. 3a), namely the start point where a drop containing over 150 worms is placed (Blue), an area circling the spot of the chemotactic cue (Red), and an area circling the control area typically spotted with the buffer solution used to dilute the chemical cue (Orange). The tracking software counts the number of worms entering or leaving each of these ROIs, thus providing a quick and quantitative analysis of chemotaxis kinetics that can be viewed as a temporal variation of the often used ‘chemotaxis index’ (Fig. 3b, c).Fig. 3


A multi-animal tracker for studying complex behaviors
The Multi-Animal Tracker is particularly suitable for studying complex behaviors such as chemotaxis. a An image of the experimental chemotaxis plate. Agar plugs soaked with the attractant odorant (red circle), or the control buffer (yellow circle), are placed on the plate’s lid. Neither the attractant, nor the buffer, come in contact with the agar on the plate. Approximately 150 worms are loaded at the starting point (blue circle). The chemoattractant source, the buffer source, and the starting point form an imaginary equilateral triangle with an edge of 4 cm. b A quantitative cumulative dynamics of worm position in the chemotaxis assay over the course of approximately 15 minutes. The lines indicate the number of worms in each of the regions of interest throughout the experiment (color coded). In this experiment, nearly 180 worms were loaded on the assay plate. About two-thirds of them reached the chemoattractant during the first 15 minutes of the assay. c Images were taken at the specific time points (I–IV) throughout the assay (indicated as dashed lines in b) to illustrate chemotaxis progression in the assay plate. Shown are also the trajectories (red) as identified by the tracker software. d–g The software suite includes a module to generate Attraction Fields (AF), a visualization designed to provide spatial representation of the chemotaxis process throughout the experiment. Shown here are AFs of two chemotaxis assays in which the isoamyl-alcohol attractant was used in 10−4 (d) and 10−2 (f) dilutions. The experimental field is binned to squares (35 × 25 in this case, but any binning defined by the user is possible). Arrows represent the average direction of the worms and the color code indicates the overall occupancy throughout the course of the experiment (15 minutes, ~1000 frames). e, g Chemotaxis dynamics as detailed in section b. Together, these plots provide a full spatiotemporal representation of the chemotaxis behavior of multiple animals over the course of thousands of captured frames
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Fig3: The Multi-Animal Tracker is particularly suitable for studying complex behaviors such as chemotaxis. a An image of the experimental chemotaxis plate. Agar plugs soaked with the attractant odorant (red circle), or the control buffer (yellow circle), are placed on the plate’s lid. Neither the attractant, nor the buffer, come in contact with the agar on the plate. Approximately 150 worms are loaded at the starting point (blue circle). The chemoattractant source, the buffer source, and the starting point form an imaginary equilateral triangle with an edge of 4 cm. b A quantitative cumulative dynamics of worm position in the chemotaxis assay over the course of approximately 15 minutes. The lines indicate the number of worms in each of the regions of interest throughout the experiment (color coded). In this experiment, nearly 180 worms were loaded on the assay plate. About two-thirds of them reached the chemoattractant during the first 15 minutes of the assay. c Images were taken at the specific time points (I–IV) throughout the assay (indicated as dashed lines in b) to illustrate chemotaxis progression in the assay plate. Shown are also the trajectories (red) as identified by the tracker software. d–g The software suite includes a module to generate Attraction Fields (AF), a visualization designed to provide spatial representation of the chemotaxis process throughout the experiment. Shown here are AFs of two chemotaxis assays in which the isoamyl-alcohol attractant was used in 10−4 (d) and 10−2 (f) dilutions. The experimental field is binned to squares (35 × 25 in this case, but any binning defined by the user is possible). Arrows represent the average direction of the worms and the color code indicates the overall occupancy throughout the course of the experiment (15 minutes, ~1000 frames). e, g Chemotaxis dynamics as detailed in section b. Together, these plots provide a full spatiotemporal representation of the chemotaxis behavior of multiple animals over the course of thousands of captured frames
Mentions: We used the tracking system to extract trajectories of a large number of animals tracked simultaneously during 30 minutes of chemotaxis. To study the chemotaxis performance quantitatively, we defined three regions of interest (ROIs, Fig. 3a), namely the start point where a drop containing over 150 worms is placed (Blue), an area circling the spot of the chemotactic cue (Red), and an area circling the control area typically spotted with the buffer solution used to dilute the chemical cue (Orange). The tracking software counts the number of worms entering or leaving each of these ROIs, thus providing a quick and quantitative analysis of chemotaxis kinetics that can be viewed as a temporal variation of the often used ‘chemotaxis index’ (Fig. 3b, c).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Animals exhibit astonishingly complex behaviors. Studying the subtle features of these behaviors requires quantitative, high-throughput, and accurate systems that can cope with the often rich perplexing data.

Results: Here, we present a Multi-Animal Tracker (MAT) that provides a user-friendly, end-to-end solution for imaging, tracking, and analyzing complex behaviors of multiple animals simultaneously. At the core of the tracker is a machine learning algorithm that provides immense flexibility to image various animals (e.g., worms, flies, zebrafish, etc.) under different experimental setups and conditions. Focusing on C. elegans worms, we demonstrate the vast advantages of using this MAT in studying complex behaviors. Beginning with chemotaxis, we show that approximately 100 animals can be tracked simultaneously, providing rich behavioral data. Interestingly, we reveal that worms’ directional changes are biased, rather than random – a strategy that significantly enhances chemotaxis performance. Next, we show that worms can integrate environmental information and that directional changes mediate the enhanced chemotaxis towards richer environments. Finally, offering high-throughput and accurate tracking, we show that the system is highly suitable for longitudinal studies of aging- and proteotoxicity-associated locomotion deficits, enabling large-scale drug and genetic screens.

Conclusions: Together, our tracker provides a powerful and simple system to study complex behaviors in a quantitative, high-throughput, and accurate manner.

Electronic supplementary material: The online version of this article (doi:10.1186/s12915-017-0363-9) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus