Introduction

In insects, such as Drosophila melanogaster, locomotor activity is implicated either directly or indirectly in activities such as courtship behavior, foraging for food, and exploration of the environment. It is important to realize that in all these cases, locomotor activity as such is the overt manifestation of a chain of events that are coordinated by the central nervous system of the fly (2). Indeed it is known that after decapitation, Drosophila maintains a normal

From: Methods in Molecular Biology, vol. 362: Circadian Rhythms: Methods and Protocols Edited by: E. Rosato © Humana Press Inc., Totowa, NJ

standing posture, shows spontaneous grooming, and can also display locomotor activity (3). The fact that a fly can walk at all without a head raises the obvious question of what role the head (and of course the brain within) plays in this phenomenon. The answer to this fascinating question is beyond the scope of this chapter, the point being, however, that locomotor activity constitutes one of the most important behavioral readouts in the study of the genetics and molecular biology of circadian rhythms in D. melanogaster. What matters in this specific instance is that wild-type flies show regular circadian (daily) "sleep-wake" cycles (4) that are paralleled, in particular, by overt cycles of locomotor activity and inactivity.

On average, fruit flies "wake up" just before sunrise, at which time they begin to move about, foraging for food, exploring their environs, interacting with other individuals, and perhaps also engaging in courtship behavior with the other sex. This state of affairs usually continues till about midday, when the flies reduce their activity and take an early afternoonnap. In the late afternoon, before sunset, they once again animate themselves until just before nightfall, at which time they reduce their activity in preparation for the night's rest. In this simplified description of a typical day in the life of a fruit fly, the central issue is that the distinction between moments of activity and those of inactivity can be further simplified by stating whether the fly is "moving about" at a certain moment in time or not. In this case, the circadian patterns of locomotor activity are taken as representing the circadian patterns of the fly's overall activity. Researchers in circadian biology have put this behavioral paradigm to good use as a handy means to screen for Drosophila circadian clock gene mutants (e.g., refs. 5-9). To this end the imperative becomes devising an approach that would allow automatic monitoring of the locomotor activity of single flies "247." To anyone with an electronic twist, this definitely sounds like a problem requiring a "triggerable event counter." This sort of device is normally employed in industrial production lines, where large numbers of small parts often need to be counted automatically. This can be done by conveying the objects into a narrow passage that will allow only one object at a time to pass through. The passage is equipped with an infrared light-emitting diode (LED) and a phototransistor receiver, facing each other on opposing sides of the passage. Interruption of the infrared beam by a passing object can thus be detected and the event scored as a count by appropriate circuitry, which is interfaced to a data-collecting device, such as a computer.

The same principle, applied to the "Drosophila circadian locomotor activity" problem, led to the conception of a glass tube container of a diameter just sufficient to house a single fly and long enough to allow adequate space for the fly to "walk" back and forth freely. The container is sealed at both ends; one

Fig. 1. General structure of a locomotor activity monitoring device. (A) A glass tube (length and diameter, not to scale), containing a single fly, ready to be loaded into the activity monitor. The infrared emitter/detector is also shown. (B) Typical organization of the glass tubes on a circuit board and the schematic connection through a digital counter.

Fig. 1. General structure of a locomotor activity monitoring device. (A) A glass tube (length and diameter, not to scale), containing a single fly, ready to be loaded into the activity monitor. The infrared emitter/detector is also shown. (B) Typical organization of the glass tubes on a circuit board and the schematic connection through a digital counter.

harbors a source of food and water and the other is plugged with foam rubber or cotton wool. Each unit so prepared can be mounted on a circuit board between a pair of infrared emitter/detectors (Fig. 1). Every time the fly passes in front of the emitter/detector pair during its meanderings, the infrared beam will be interrupted; this will be scored as a single event by an electronic counter. A computer is programmed to "interrogate" the event counter at a set frequency (usually once every 5-30 min), after which the counter is reset. It is apparent that what we are scoring in this way is a further simplification of what we had set out to score in the first place because we are not scoring locomotor activity per se, which would entail also gathering information concerning, for example, the speed and direction of the fly's motion during its bouts of activity. However, apart from the consideration that this would entail the use of an altogether different activity monitoring technology (see ref. 2), one also needs to keep in mind that the more information one wishes to gather, the more one is then confronted by the problem of data storage: typical circadian rhythm experiments consist of up to a few hundred flies monitored individually "around the clock" for several days at a time (typically, at least 7 d). Commercial versions of devices based on the above logic are currently available (for example, Trikinetics, Waltham, MA; www.trikinetics.com).

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