Locomotor Activity Tracking System

We conducted two separate experiments for this paper, the first using Canton S (CS) wild type males (N = 8) and the second using females from both a CASK-β null (CASK^P18) (DAM CASK-β N = 30, Track CASK-β N = 30) and a precise excision strain as a wild type genetic control line (CASK^P33) (DAM Control N = 30, Track Control N = 29) [19] (link). All flies were raised on a cornmeal-sucrose-agar food in a 25°C incubator with a 12-hr Light/Dark cycle and were 3–5 days old at the start of each experiment. Flies were loaded under CO₂ anesthesia into individual glass tubes. Each tube contained an agar/sucrose food plug sufficient to sustain the fly for the duration of the experiment. The tubes were sealed with parafilm at both ends to allow for the fly to be tracked all the way to the end of the tube without visual obstruction. For tracking, the tubes were taped to a piece of white office paper, providing a high visual contrast field against the dark fly and transparent glass tube (Figure 1B). The paper was positioned inside of an incubator under a USB video camera (Logitech, Quickcam for Notebooks). A red compact fluorescent bulb and red LEDs, emitting a wavelength of light not detected well by the fly visual system [4] (link) and incapable of entraining per⁰¹ flies (N.D. unpublished observations), were placed into the incubator to provide enough light for the camera to maintain an image when the white lights were off during the night. While Gilestro and Cirelli [20] (link) used infrared (IR) lighting to follow flies in the dark, we achieved better contrast and illumination with red LEDs while also avoiding the excessive heat that we found was generated from the IR emitters. Flies were also loaded into DAM boards as previously described for collecting beam-cross data [21] (link), and run in parallel to the Tracker flies in the same incubator. Data were collected following three days of light∶dark (LD) entrainment to a 12 h∶12 h cycle.