Measuring and Altering Mating Drive in Male Drosophila melanogaster

Summary

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Abstract

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Introduction

Work in Drosophila has provided deep and pioneering insight into many biological phenomena, including the nature of the gene¹, principles of embryonic development², circadian rhythms³, and the development and wiring of the nervous system^4,5,6. Motivation remains far less well understood than these phenomena, perhaps because of the limitations on the systems that have been studied thus far. Motivation in the fly is primarily studied in the context of hunger, which presents many challenges due to t....

Protocol

NOTE: This protocol describes the preparation (Sections 1 - 3), execution (Section 4), and analysis (Section 4) of 2-D satiety assays. Then, using dopaminergic stimulation as an example, Section 5 shows how to combine thermogenetic stimulation with 2-D satiety assays to induce hypersexuality. Section 6 describes 3 ways to verify the results of 2-D satiety assays. Finally, Section 7 shows how to measure the recovery of mating drive in male flies.

1. Fabricating 8- and 32-chamber Behavioral Arenas

NOTE: Each behavioral arena consists of several layers of laser-cut plastic sheets held together by hex screws and thumb ....

Results

To characterize Drosophila mating drive, 3-day-old, WT Canton-S males were tested in a 2-D satiety assay. Over the course of the assay (4.5 h), males mate an average of 4.8 ±0.3 (mean ±standard error of mean, SEM) times. Matings initiate mostly in the first 2 h (78%) (Figure 6A, 6B) and become less frequent as the assay progresses (Figure 6A, 6B). This decrease is not due to the lack of mating partners (74% females remain unmated throughout the a.......

Discussion

Motivational states can be satiated, maintained, and recovered³⁴. We present a 2-D satiety assay that quickly and robustly measures all of these aspects of mating drive in the fly. This assay opens up the possibility of using advanced fly genetic manipulations to study the molecular and circuit components of a motivated behavior.

The satiety assay relies on the male's ability to successfully court and copulate, and to terminate copulations at the appropriate time. T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1/16 inch clear acrylic	McMaster-Carr	8589K12	Used to make arenas; see Supplemental Material 1 for designs.
1/8 inch clear acrylic	McMaster-Carr	8589K42	Used to make arenas; see Supplemental Material 1 for designs.
3/16 inch clear acrylic	McMaster-Carr	8560K219	Used to make arenas; see Supplemental Material 1 for designs.
1/32 inch black delrin	McMaster-Carr	8575K132	Used to make arenas; see Supplemental Material 1 for designs.
Hex screws, 1 inch long (50x)	McMaster-Carr	92314A115	Used to make arenas. Can be replaced by 3/4 inch screws (92314A113, McMaster-Carr) for 32-chamber arenas.
Thumb nuts (25x)	McMaster-Carr	92741A100	Used to make arenas. Can be replaced by regular hex nuts (90480A005, McMaster-Carr).
Camcorder	Canon	Vixia HF R700	Can be replaced by any consumer comcorder.