Executive Summary

    Dr. William Joiner’s laboratory in the Pharmacology Department at UCSD and others study the genetic basis of sleep using fruit flies as a model organism. Due to their small size, fruit flies cannot be assayed for sleep by EEG, like humans can. Instead behavioral criteria are employed to determine sleep/wake states. Flies are placed in small glass tubes bisected by infrared beams to detect locomotor activity (Fig 1). It has been shown that 5 minutes or more of inactivity is correlated with an increased arousal threshold, which is a decreased responsiveness to environmental stimuli and is a hallmark of sleep. Therefore, sleep is defined in flies as 5 minutes or more of inactivity. However, this definition has not been rigorously tested, and alone it cannot differentiate between different arousal states such as quiet waking, sleep, injury and seizure. Our project has been to create a device that can be used to distinguish these behavioral states due to their inherent differences in arousal threshold. Our goal was to develop an automated, programmable device capable of delivering reproducible mechanical stimuli of various intensities.

    The apparatus we constructed can be loaded with up to eight activity monitors, each containing 32 flies in individual activity tubes, all placed inside an incubator under controlled environmental conditions such as fixed temperature and light/dark cycles (Fig.2). An accelerometer was incorporated into the apparatus to record the force at which animals are shaken during experiments. This measurement allows for determination of waking probability as a function of stimulus intensity. The resulting sigmoidal relationship yields a numerical value for arousal threshold, which is defined as the force at which 50% of animals move in response to stimulation.

In addition to designing and building the apparatus, team members developed software to operate it, including a graphical user interface (GUI). This feature allows the user to adjust the stimulus intensity, stimulus duration and time at which the experiment is initiated.

   Figure 3 shows how the device consists of the following main components, with their functions listed in parenthesis: A stepper motor (actuator), a crank mechanism (power transmission: conversion of rotational into linear oscillatory motion),  the carriage (holds up to 8 monitors in place), a rail system with ball bearings (constraining the carriage to 1 Degree of Freedom, allowing it to glide horizontally along the rails), a Arduino microcontroller (controls the motor via its integrated encoder), and an accelerometer (collects displacement data for feedback).

    In short, the microcontroller, located outside the incubator, controls the stepper motor, which rotates the crank mechanism attached to the carriage, causing it oscillate over a given interval of time, with a given frequency, as input by the user via the graphical user interface (GUI).  All components, except for the power supply and the microcontroller, are mounted on a base plate, which has a rubber mat on its underside to prevent sliding and eliminate unwanted vibrations.

    From several trial runs, the operating range of the apparatus was determined to be 0.05 Hz to 2.84 Hz. The number of rotations can also be increased to disturb the flies even more and wake the full percentage of flies. The fly results obtained are very promising, and confirm the mechanism provides enough variables in which the research can be conducted so as to alter the frequency to obtain the desired percentage of flies awakened.