Ethoscopes: An open platform for high-throughput <i>ethomics</i>

<div><p>Here, we present the use of ethoscopes, which are machines for high-throughput analysis of behavior in <i>Drosophila</i> and other animals. Ethoscopes provide a software and hardware solution that is reproducible and easily scalable. They perform, in real-time, tracking and profiling of behavior by using a supervised machine learning algorithm, are able to deliver behaviorally triggered stimuli to flies in a feedback-loop mode, and are highly customizable and open source. Ethoscopes can be built easily by using 3D printing technology and rely on Raspberry Pi microcomputers and Arduino boards to provide affordable and flexible hardware. All software and construction specifications are available at <a href="http://lab.gilest.ro/ethoscope" target="_blank">http://lab.gilest.ro/ethoscope</a>.</p></div

The ethoscope platform.

<p>(A) A diagram of the typical setup. Ethoscopes, powered through a USB adapter, are connected in an intranet mesh through an AP or a Wi-Fi router. A computer in the network acts as the node, receiving data from ethoscopes and serving a web-UI through which ethoscopes can be controlled, either locally or remotely. (B) Screenshot of the homepage of the web-UI, showing a list of running machines and some associated experimental metadata (e.g., username and location). (C) Screenshot of an ethoscope control page on the web-UI, providing metadata about the experiment and a real-time updated snapshot from the ethoscope point of view. AP, access point; GMT, Greenwich mean time; FPS, frames per second; USB, universal serial bus; web UI, web-based user interface.</p

The ethoscope.

<p>(A) Exploded drawing of an archetypal ethoscope. The machine is composed of 2 main parts: an upper case housing the rPi and its camera, and a lower case providing diffused infrared light illumination and support for the experimental arena. The 2 cases are separated by spacers maintaining a fixed focal distance (140 mm for rPi camera 1.0). (B) A rendered drawing of the assembled model, showing the actual size without cables. The presence of USB and connection cables will slightly increase the total size (cables not shown for simplicity). The arena slides in place through guides and locks into position. A webGL interactive 3D model is available as <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003026#pbio.2003026.s001" target="_blank">S1 Fig</a>. (C) The LEGOscope, a version of the ethoscope built using LEGO bricks. A detailed instruction manual is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003026#pbio.2003026.s002" target="_blank">S1 Text</a>. (D) The PAPERscope, a paper and cardboard version of the ethoscope, best assembled using 220 gsm paper and 1 mm gray board. Blueprints are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003026#pbio.2003026.s003" target="_blank">S2 Text</a>. In all cases, ethoscopes must be powered with a 5 V DC input using a common USB micro cable either connected to the main or to a portable power-pack. DC, direct current; HD, high-definition; LED, light-emitting diode; rPi, Raspberry Pi; USB, universal serial bus.</p

Tracking and validation of behavioral classification.

<p>(A) To build a statistical model of activity, we used ethoscopes to record offline 2,736 hours of video (144 hours x 19 flies) at resolution of 1,280 x 960 pixels and frame rate of 25 FPS. Video fragments of the duration of 10 seconds were sampled every hour for all 19 animals and scored by at least 3 experienced fly researchers in a randomized order. Consensual annotations—where majority of scorers agreed—were kept, resulting in a ground truth of 1,297 video fragments (116 ambiguous annotations were excluded by using this latter criteria). Scorers manually annotated both the position of the animal in the tube and the perceived behavioral state (i.e., immobile, micromoving, or walking). Ethoscope video tracking was run independently on the whole video down-sampled between 1 and 5 FPS, all realistic frame rates for real-time analysis. (B) Distribution of corrected maximal velocity (relative unit, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003026#pbio.2003026.s005" target="_blank">S4 Text</a>) for each behavior, showing the thresholds used to detect movement (1: dotted line) and walking (2, 5: dashed line). (C) Five days’ recording of activity of 10 representative flies: 5 males (cyan boxes) and 5 females (rose boxes). Flies were kept in a regime of constant climate in a 12 hour:12 hour light-dark cycle (as indicated by the lower bar alternating white and black). The yellow frame highlights the 3-hour window shown in D. (D) Detailed activity for the same individuals shown in C, during a 3-hour window spanning a light to dark transition. The black line shows the position of the animals from the food end to other extremity of the tube (bottom to top). The background colors highlight the behavioral features as detected in real-time by the ethoscope, with a definition of 10 seconds per pixel (same legend as B). FPS, frames per second, px, pixel.</p

Versatility of use with custom behavioral arenas.

<p>(A-H) Examples of 8 different behavioral arenas whose files for 3D printing are available on the ethoscope website. (A) Sleep arena. Most commonly used arena in our laboratory for sleep studies, lodging 20 individual tubes. (B) Long tubes arena. It houses 13-cm tubes and can be used for odor delivery studies or, more generally, for behaviors requiring more space. (C) Food bullet arena. Animals are placed directly on the arena and food can be replaced by pushing in a new bullet [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003026#pbio.2003026.ref011" target="_blank">11</a>]. It does not require glass tubes and can be used for quick administration of chemicals in the food. (D) Decision making arena. It can be used to study simple decision making behaviors, adapted from Hirsch [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003026#pbio.2003026.ref003" target="_blank">3</a>]. (E) Square wells arena. It can be used for courtship assay or to record activity in a bidimensional environment. (F, G) Conceptually analogous to A and I, but designed to work in high-resolution (full-HD) settings. (H) Round wells arena, modelled following specifications from Simon and Dickinson [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003026#pbio.2003026.ref022" target="_blank">22</a>]. Note that all arenas are marked with 3 visible reference points (indicated by a red circle in A) that are used by the ethoscope to automatically define regions of interest for tracking. HD, high-definition.</p

Versatility of use with behavioral feedback-loop modules.

<p>(A) Diagram and (B) detail of the AGO-delivery module. Two independent flows (blue and purple in the drawing) are fed into the module using external sources. The module features 10 LEGO pneumatic valves, each independently controlled through a servo motor. The motor switches the air source on the valve, selecting which source will be relayed to the tube containing the fly. Available positions are blue source, purple source, and closed. (C) Representative response of 3 flies subjected to CO₂ administration using the AGO module. CO₂ release lasts 5 seconds (grey bar) and it is triggered by midline crossing (red dot). The blue line indicates the fly position in the tube over the 150 second period. (D) Model and (E) detail of the rotational module. The module employs a servo motor to turn the tube hosting the fly. The direction, speed, duration, and angle of the rotation can be modulated to change the quality of the stimulus. (F) Representative response of 3 flies upon stimulation using the rotational module shown in (D, E). Rotation of the tube is triggered by 20 consecutive seconds of immobility (dashed line) and is followed by 5 seconds of masking, during which tracking is suspended to avoid motion artefacts (cyan area). The bottom panel shows traces of a dead fly. (G) Model of the optomotor module able to simultaneously stimulate single flies with rotational motion and light. (H) Detailed view of the optomotor minimal unit. Light is directed into the tube using optical fiber. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003026#pbio.2003026.s007" target="_blank">S2 Movie</a> shows the optomotor module in action. (I-P) The servo module employed for a sleep deprivation experiment. Flies shown in grey are unstimulated mock controls, never experiencing tube rotations. Flies shown in light blue experience rotation either after 20 seconds of inactivity (I-L) or after midline crossing (M-P). (J, N) Sleep profile of flies along 3 days in conditions of 12 hour:12 hour, light and dark cycles. Gray shadings indicated the stimulation period and the following sleep rebound period. (K, O) Number of tube rotations delivered during the 12-hour stimulation period. (L, P) Quantification of sleep rebound during the first 3 hours of the day following the stimulation. AGO, air/gas/odor; DC, direct current; LED, light-emitting diode; rpm, revolutions per minute; SD, secure digital; ZT, Zeitgeber time.</p

Increased PER and TIM levels rescue the behavioral phenotype associated with <i>shn</i> overexpression.

<p>(A) Representative double-plotted actograms of flies of the indicated genotypes. Behavioral experiments were carried out as detailed in the legend to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio-1001733-g001" target="_blank">Figure 1E</a>. (B) Graph shows the quantitation of period and rhythmicity for the indicated genotypes (see legend to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio-1001733-g001" target="_blank">Figure 1C</a> for more details). Statistical analysis included one-way ANOVA for period determination, and different letters indicate significant differences in Tukey comparisons, α = 0.05. Error bars represent SEM, and averages represent at least three independent experiments. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio.1001733.s007" target="_blank">Table S1</a> for details. (C) <i>per</i> and <i>tim</i> are transcriptionally modulated by <i>shn</i>. Luciferase activity from total head extracts was measured at ZT02 in transgenic flies carrying <i>per</i> (upper panel) or <i>tim</i> (bottom panel) transcriptional reporters combined with <i>shn</i> overexpression in the entire clock circuit (<i>tim</i>-G4). Three independent experiments were carried out. Data from each experiment were normalized against the mean value of all measurements to contemplate different absolute luciferase activity levels. Three independent experiments were carried out and were analyzed with Student's <i>t</i> test; <i>per</i> LUC <i>p</i> = 0.020, <i>tim</i> LUC <i>p</i> = 0.002.</p

<i>schnurri</i> deregulation modulates locomotor behavior in PDF+ cells.

<p>(A) Schematic diagram of a fly brain hemisphere displaying all clock-gene expressing neurons. lLNvs, large ventral Lateral Neurons; sLNvs, small ventral Lateral Neurons; LNds, dorsal Lateral Neurons; DN1as, Dorsal Neurons 1 anterior; DN1ps, Dorsal Neurons 1 posterior; DN2s, Dorsal Neurons 2; DN3s, Dorsal Neurons 3; LPNs, lateral posterior neurons. Modified from Muraro et al. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio.1001733-Muraro1" target="_blank">[66]</a>. PDF+ and TIM+PDF− neurons were color-coded to facilitate their identification throughout. (B) <i>shn</i> overexpression lengthens the endogenous period. Representative double-plotted actograms of individual flies of the indicated genotypes. During the experiments, flies were kept in LD for 4 d, then switched to DD (shaded gray area), and monitored for 8 additional days. (C) Graph shows the quantitation of period and rhythmicity for the indicated genotypes; bars and diamonds represent average period and percentage of rhythmicity, respectively. Statistical analysis included one-way ANOVA for period determination, and different letters indicate significant differences in Tukey comparisons, α = 0.05. Purple bars indicate the treatments in which the BMP pathway is being modulated in PDF+ neurons. (D) Schematic diagram of the <i>shn</i> locus; the four alternative transcription initiation sites, the relative position of the transposon, and the common translation initiation site (ATG) are indicated (not to scale). Black boxes, untranslated regions; white boxes, coding region. (E) <i>shn</i> knockdown leads to deconsolidation of locomotor activity. Representative double-plotted actograms of individual flies of the indicated genotypes. During the experiments, flies were kept in LD for 3 d, then switched to DD (shaded gray area), and monitored for 9 additional days. (F) Graph shows the quantitation of period and rhythmicity for the indicated genotypes (see legend to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio-1001733-g001" target="_blank">Figure 1C</a> for more details). Statistical analysis for rhythmicity data included one-way ANOVA, and different letters indicate significant differences in Tukey comparisons, α = 0.05. Error bars represent SEM and averages of at least three independent experiments. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio.1001733.s007" target="_blank">Tables S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio.1001733.s008" target="_blank">S2</a> for details.</p

A combination of BMP ligands orchestrate the function of the circadian network.

<p>(A) Representative double-plotted actograms of flies of the indicated genotypes. Behavioral experiments were carried out as detailed in the legend to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio-1001733-g001" target="_blank">Figure 1E</a>. (B) Graph shows the quantitation of rhythmicity for the indicated genotypes. Statistical analysis included one-way ANOVA; * indicates significantly different treatments in Tukey comparisons, α = 0.05. Error bars represent SEM and averages represent at least three independent experiments. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio.1001733.s009" target="_blank">Table S3</a> for details.</p

SHN down-regulates CLK protein levels through modulation of <i>Clk</i> promoter activity.

<p>(A) Representative double-plotted actograms of flies of the indicated genotypes. Behavioral experiments were carried out as detailed in the legend to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio-1001733-g001" target="_blank">Figure 1E</a>. (B) Graph shows the quantitation of period and rhythmicity for the indicated genotypes (see legend to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio-1001733-g001" target="_blank">Figure 1C</a> for more details). Statistical analysis included one-way ANOVA for period determination, and different letters indicate significant differences in Tukey comparisons, α = 0.05. Error bars represent SEM, and averages represent at least three independent experiments. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio.1001733.s007" target="_blank">Table S1</a> for details. (C) The schematic diagram depicts the <i>Clk</i> locus highlighting the existence of five alternative transcripts (according to FlyBase) along with the DNA fragment contained in the GAL4 reporter line; gray boxes represent untranslated exons, red boxes indicate translated exons, and the green box indicates the fusion between the first 18 amino acids of CLK and the transcription factor GAL4 (not to scale). Putative MAD (red) and MED (blue) binding elements and the relative position within the locus are indicated. The Clk⁸⁵⁶GAL4 reporter line contains a total of 2,334 bp <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001733#pbio.1001733-Gummadova1" target="_blank">[51]</a>. (D) Whole mount brain immunofluorescence was performed to follow PDF (upper panel) and GFP (upper and bottom panels) accumulation at ZT2 in the −856 <i>Clk</i> reporter line (<i>Clk</i>⁸⁵⁶). This time point was selected to reduce the impact of differences stemming from the period lengthening phenotype. Representative confocal images of sLNvs for the indicated genotypes are shown. Note that controls also include a second UAS-driven transgene. Scale bar, 5 µm. (E) Quantitation of GFP nuclear intensity. PDF+ staining and cell body size were used to identify sLNvs. A total of 8 to 10 brains were analyzed in each experiment; the average of 2–4 neurons was used for each determination, and the images were taken employing the same confocal settings throughout an individual experiment. Three independent experiments were carried out, and data were analyzed with Randomized Blocks ANOVA to contemplate potential differences due to the different confocal settings. Paired measurements are linked by colored lines. p_blocks = 0.04, p_genotypes = 0.02. (F) Whole mount brain immunofluorescence was performed to follow PDF (upper panel) and CLK (upper and bottom panels) accumulation at ZT14 in the indicated genotypes. This time point was selected to maximize CLK levels and reduce the impact of the period lengthening phenotype. Scale bar, 5 µm. (G) Quantitation of CLK nuclear intensity, as described in (E). Five independent experiments were carried out, and data were analyzed with Student's <i>t</i> test, <i>p</i> = 0.017.</p