An automated, high-throughput climbing assay for behavioral screening in Drosophila
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1
Janelia Farm Research Center, Howard Hughes Medical Institute, Fly Olympiad Project Team, United States
We developed a robust, quantitative screen for climbing behavior in Drosophila melanogaster. When mechanically startled, wild-type flies show a strong tendency to move against the direction of gravitational pull, a behavior termed “negative gravitaxis.” A typical assay for this behavior consists of shaking flies to the bottom of a vial and observing subsequent fly locomotion. In addition to the ability to use gravity as an axis for orientation, this tests for general walking capabilities, startle responses, and fly interactions. Within the fly community, a number of methods for measuring related behaviors are suitable for high-throughput screens, including the gravitaxis maze [1], a counter-current [2], and more recently the RING assay [3]. In our assay, flies are shaken to the bottom of a vial through a series of accelerations and decelerations (drops) produced by a linear motor. We capture detailed information and achieve high throughput by using a standardized protocol with a highly repeatable drop; video acquisition of entire experiments; tightly controlled and monitored rearing and experimental conditions; and running all post-processing, video analysis, and auditing via automated scripts.
By dropping 6 vials simultaneously and performing 10 drops per trial, we achieve a throughput of 15 lines per hour. Video data is then automatically analyzed and the distribution of flies is detected by image background subtraction. We can discriminate between flies with general walking deficits, slow climbers, flies that walk upwards at first but then descend later in the experiment, and normal behavior. We have screened over 600 unique Gal4 lines from the Rubin collection using temperature-sensitive shibire to inhibit neural activity and we observe a total hit rate of 15.8% across phenotype categories. In parallel, we are testing the influence of fly age on climbing behavior to compare our assay with previously published work [3].
This assay contributes to the Fly Olympiad Project at Janelia Farm, an attempt to create a functional, circuit-level map of the Drosophila brain by assaying thousands of genetic lines in the Rubin GAL4 collection in a variety of behavioral neurogenetic experiments. Behavioral phenotypes, including our hits, will be correlated with neural expression patterns obtained by the Janelia Fly Light Project, which images lines of the Rubin collection through light microscopy. Because the assay has high throughput, is simple to run and has very consistent results, it can also be used as a screen for normal fly behavior, for example to test the strength and leakiness of different effectors, or modified to perform secondary experiments on behavioral hits.
References
[1] D.P. Toma, K.P. White, J. Hirsch and R.J. Greenspan. Drosophila melanogaster geotaxis, a complex behavioral trait. Nature Genetics. 31 (2002), pp. 349-353
[2] H.K. Inagaki, A. Kamikouchi and K. Ito. Methods for quantifying simple gravity sensing in Drosophila melanogaster. Nature Protocols. 5 (2010), pp. 20-25
[3] J.W. Gargano, I. Martin, P. Bhandari and M.S. Grotewiel. Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila. Exp. Gerontol. 40 (2005), pp. 386-395
Keywords:
Behavior,
behavioral neurogenetics,
Drosophila,
Fly Olympiad,
Locomotion,
screening
Conference:
Tenth International Congress of Neuroethology, College Park. Maryland USA, United States, 5 Aug - 10 Aug, 2012.
Presentation Type:
Poster Presentation (see alternatives below as well)
Topic:
Sensorimotor Integration
Citation:
Phillips
M,
Roberts
S,
Kladt
N,
Reiser
MB and
Korff
W
(2012). An automated, high-throughput climbing assay for behavioral screening in Drosophila.
Conference Abstract:
Tenth International Congress of Neuroethology.
doi: 10.3389/conf.fnbeh.2012.27.00357
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Received:
30 Apr 2012;
Published Online:
07 Jul 2012.
*
Correspondence:
Ms. Mary Phillips, Janelia Farm Research Center, Howard Hughes Medical Institute, Fly Olympiad Project Team, Ashburn, Virginia, 20147, United States, phillipsm10@janelia.hhmi.org
Dr. Wyatt Korff, Janelia Farm Research Center, Howard Hughes Medical Institute, Fly Olympiad Project Team, Ashburn, Virginia, 20147, United States, korffw@janelia.hhmi.org