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Pharmacology - Arousal

Study of Sleep Patterns in Fruit Flies

Executive Summary

• Project background and objective

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, as shown below.

It has been shown that 5 minutes or more of inactivity is c orrelated 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:

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.

•  Sponsor's Initial Solution

            Dr. Joiner and his group had built an apparatus to perturb the flies by dropping them at different heights to create various levels of impact. The apparatus (as seen on the video on the right) also included rubber rings within the attachment to induce oscillatory motion. Unfortunately, this solution neither provided consistent nor repeatable motion. Furthermore, the apparatus did not effectively wake a broad range of flies.  From their apparatus, it was concluded that some type of oscillatory motion was effective in waking the flies us, rather than pure impact force.

• Final design

    Our apparatus produces  linear oscillatory motion of the carriage, and in turn of the activity monitors containing fruit flies, which has proven to be an effective stimulus. The system is comprised of a carriage attached to a crank and slider mechanism. The carriage platform features four shafts, each of which have ball bearings acting as wheels. The ball bearings roll along two parallel rails, limiting the carriage to a horizontal motion. The carriage can hold up to eight activity monitors, which are the devices that house and sense the activity of the specimens. The crank slider mechanism is controlled by an Arduino microcontroller. The Arduino is programmed in MATLAB; which a

llows the user to control the speed and duration of motor operation. 

The program acquires a

nd quantifies output data taken from the Arduin

o, as well as an accelerometer. The data indicates the fre

 quency and intensity of the stimulus experienced by the test subjects. Operation of the system will be done with a graphical user interface (GUI) also developed in MATLAB. The range of frequency that can be used to perturb the flies are 0.05 to 2.84 Hz. This corresponded to a maximum frequency of 1.5 g’s.

• • Summary of performance results

It was vital to have the same frequency of the mechanism to be consistent regardless of the number of monitors that were placed in it.The acceleration data seen below essentially lie on top of each other. This confirmed that the frequency input to the stepper motor was unaffected by the  number of monitors placed in the mechanism.

                    The results of from using the mechanism for testing arousal thresholds of flies can be seen in the following two figures: 

As can be seen from the above figure, the percentage of awakened flies is proportional to the frequency (acceleration increases as frequency increases). 

     The below figure suggests that the sponsor still need to research the correct protocol to wake the full range of flies. The lowest frequency in the experiment was 0.5 Hz and the highest was 2.5 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.