Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila

Abstract

Age-related behavioral declines are common manifestations of aging in animals. Negative geotaxis, an innate escape response during which flies ascend the wall of a cylinder after being tapped to its bottom, is one of the behaviors that senesces in Drosophila. Many laboratories, including ours, have used a variety of negative geotaxis assays based on the performance of single flies. To circumvent limitations of single-fly assays, we developed a new method for assessing negative geotaxis called rapid iterative negative geotaxis (RING). In RING assays, digital photography is used to document negative geotaxis in multiple groups of animals simultaneously. We show that performance in RING assays is not influenced by the density of flies being tested, the time of day, or repeated testing. We used the RING assay to demonstrate that negative geotaxis declines with the age of animals as previously shown in single fly studies and that senescence of negative geotaxis is sensitive to genetic background. Finally, we used RING assays to show that long-lived Indy and chico mutants exhibit delayed senescence of negative geotaxis. Our results demonstrate that RING is a powerful method for assessing negative geotaxis that should facilitate the search for manipulations that influence behavioral aging in Drosophila.

Introduction

Aging is the result of a complex, inter-related collection of changes in physiological status that culminate in death (Kirkwood and Austad, 2000, Kenyon, 2001). Among the changes that occur in animals as they grow older, age-related functional declines are arguably the most distressing. Locomotor abilities (Forster et al., 1996, Joseph et al., 1999, Le Bourg and Minois, 1999, Cook-Wiens and Grotewiel, 2002, Goddeeris et al., 2003), sensory system function (Nusbaum, 1999, Winkler et al., 1999, Cook-Wiens and Grotewiel, 2002), and cognitive processes (Zyzak et al., 1995, Grady, 1998, Foster et al., 2001) all senesce in animals. The search for the factors underlying these changes would be greatly facilitated by high-throughput assays capable of rapidly assessing the effects of various manipulations on functional senescence.

The influences of genetic and other factors on aging and age-related functional changes are being resolved through research in model organisms such as Drosophila. The combination of a comprehensive set of genetic tools, relatively short life span and inexpensive housing makes flies well suited for studies aimed at understanding the genetic basis of aging. Importantly, flies perform a number of behaviors that can be quantitatively assessed in the laboratory (Connolly and Tully, 1998) and some of these decline with age (Arking and Wells, 1990, Le Bourg and Minois, 1999, Cook-Wiens and Grotewiel, 2002, Goddeeris et al., 2003). Flies are therefore a powerful model system for exploring the connections between genes, behavior and senescence.

Geotaxis is a commonly assessed type of behavior in Drosophila that can be quantitated in two different assays. One type of assay measures ‘negative geotaxis’, an innate escape response in which flies ascend the wall of a container after being tapped to its bottom. Negative geotaxis is elicited by mechanical stimulation of the flies. The other type of assay measures ‘geotaxis’. In this assay, groups of flies are placed in a bifurcating array of tubes (Hirsch, 1959, Hirsch and Erlenmeyer-Kimling, 1962, Hostetter and Hirsch, 1967, Ricker and Hirsch, 1988, Toma et al., 2002). Flies are not mechanically stimulated in this type of assay, but instead can choose to walk up or down by way of moving toward a distal light cue. Although wild type flies principally move away from the force of gravity in both assays, the relationship between the two types of behaviors they measure remains unclear. Here, we focus on negative geotaxis since this behavior has been reproducibly shown to decline with age in Drosophila (Arking and Wells, 1990, Orr and Sohal, 1994, Benguria et al., 1996, Le Bourg and Minois, 1999, Minois et al., 2001, Cook-Wiens and Grotewiel, 2002, Kang et al., 2002, Goddeeris et al., 2003).

A number of laboratories, including ours, have previously measured negative geotaxis using a variety of single-fly assays. In our experience, single-fly assays are rather tedious and time-consuming especially for studies on age-related decline in negative geotaxis. These limitations make single-fly assays somewhat problematic for small experiments and wholly unsuitable for large high-throughput studies such as forward genetic screens. Toward establishing an improved negative geotaxis assay, we developed a method called rapid iterative negative geotaxis (RING). The method is based on digital imaging of flies and requires a nominal amount of equipment and space. After validating the method, we used it to assess the effects of genetic background and two life span-extending mutations on senescence of negative geotaxis. Our studies demonstrate that the RING assay is well suited for assessing negative geotaxis in addition to age-related decline in this behavior. We suggest that the RING method should be suitable for studies that require efficient high-throughput assessment of negative geotaxis in Drosophila.