Elsevier

Methods in Enzymology

Volume 393, 2005, Pages 759-772
Methods in Enzymology

Section X
Essentials of Sleep Recordings in Drosophila: Moving Beyond Sleep Time

https://doi.org/10.1016/S0076-6879(05)93040-1Get rights and content

Abstract

The power of Drosophila genetics can be used to facilitate the molecular dissection of sleep regulatory mechanisms. While evaluating total sleep time and homeostatic processes provides valuable information, other variables, such as sleep latency, sleep bout duration, sleep cycle length, and the time of day when the longest sleep bout is initiated, should also be used to explore the nature of a genetic lesion on sleep regulatory processes. Each of these variables requires that the recording interval used to identify periods of sleep and waking be determined accurately and empirically. This article describes the procedures for recording sleep in Drosophila and associated methodological constraints. In addition, it provides results from a normative data set of 1037 Canton-S female flies and 639 male flies to illustrate the nature and variability of sleep variables that one can extract from 24 h of data collection in Drosophila.

Introduction

Sleep is defined by periods of quiescence that are associated with a reduced responsivity to the external environment and are homeostatically regulated (Campbell 1984, Carskadon 2000, Hendricks 2000, Shaw 2000). In Drosophila melanogaster, quiescent episodes can be measured easily and reliably using an apparatus originally designed for investigating circadian rhythms in locomotor behavior (Hamblen et al., 1998). Whereas the circadian periodicity of locomotor activity is tightly regulated, average sleep time and other sleep-related parameters can display a high degree of variability. Thus, while Drosophila shows great promise for quickly identifying sleep regulatory process, care must be taken to accurately describe sleep phenotypes. This article describes the procedures for recording sleep in Drosophila and associated methodological constraints. In addition, it provides results from a normative data set of 1037 Canton-S (Cs) female flies and 639 male flies to illustrate the nature and variability of sleep phenotypes that one can expect to find when initiating sleep studies.

Section snippets

Procedure

We measure quiescence in Drosophila utilizing the activity monitoring system (AMS) developed by TriKinetics (Waltham, MA). Analysis of sleep and wake values is done on a population of flies of the same age, genotype, and sex exposed to similar environmental conditions (e.g., food, lighting, temperature, humidity). The minimum number of flies for a given genotype that need to be tested to get a reliable estimate of sleep parameters is usually 16. Repeated measurements across several generations

Constraints

The recording interval used to quantify sleep and waking parameters is constrained by the behavior of the fly within the apparatus. We have determined that 5 min is a very robust interval that can be used to accurately identify periods of sleep and waking in a wide range of experimental conditions (Shaw et al., 2000). Nonetheless, we would like to emphasize that the appropriate interval must be determined empirically and can be influenced by age, sex, and genotype. Thus, while a 5-min interval

Basic Characteristics of Sleep

We have thoroughly evaluated sleep in a normative data set of 1037 Cs flies maintained on a 12:12 LD schedule. We present these data as an example of the number and kind of sleep and wake parameters that can be extracted from 24 h of data collection. As described previously, flies are diurnal and sleep is largely confined to the dark period (Fig. 1, center). On average, female flies sleep between 40 and 50 min per hour during the night. Closer inspection of data, however, reveals that there is

Sleep Deprivation

Although the quantification of basic sleep parameters such as total sleep time and average bout duration can be quite informative, the regulation of sleep is evaluted more precisely using sleep deprivation and evaluating the subsequent homeostatic response (Shaw and Franken, 2003). The size of the homeostatic response is evaluated by constructing cumulative difference plots (Edgar 1997, Shaw 2002).

This is accomplished for each individual fly, first by subtracting the minutes of sleep during

Sexual Dimorphism

To simplify the discussion, this article has focused exclusively on female flies. However, it is important to note that there is a pronounced sexual dimorphism in average sleep time in Cs flies (Shaw et al., 2000). The difference in total sleep time is accounted for by significantly more sleep during the light period. Thus, while both male and female Cs flies tend to obtain similar amounts of sleep at night, males sleep more during the day and their sleep bouts are longer (Table I). This

Conclusions

A number of sleep parameters can be identified readily in D. melanogaster using standard off-the-shelf equipment (Hendricks 2000, Shaw 2000). The data collection interval used to accurately record sleep and waking episodes can be influenced by age, sex, and genotype and must be determined empirically. Total sleep time constitutes only one of several variables that can be used to evaluate the genetic basis of sleep regulation. Other variables, such as sleep latency, sleep bout duration, sleep

Acknowledgements

Research conducted at The Neurosciences Institute was supported by Neurosciences Research Foundation.

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