An automated method to assay locomotor activity in third instar Drosophila melanogaster larvae

doi:10.1016/j.vascn.2015.10.004

Journal of Pharmacological and Toxicological Methods

Volume 77, January–February 2016, Pages 76-80

https://doi.org/10.1016/j.vascn.2015.10.004 Get rights and content

Abstract

Introduction

The purpose of these studies was to describe a novel application of an automated data acquisition/data reduction system, DanioVision™ by Noldus. DanioVision™ has the ability to detect changes in locomotor activity in third instar Drosophila melanogaster larvae. The noncompetitive GABA_A receptor antagonist picrotoxin (PTX), was used as a pharmacologic agent to decrease locomotor activity.

Methods

Two strains of Drosophila were used in these studies; wild-type flies and flies with a mutation in the Rdl gene (Rdl^MD-RR). Rdl^MD-RR Drosophila are naturally occurring mutants that express an aberrant form of the GABA_A receptor, which has a lower affinity for PTX, but not GABA itself. Larvae, extracted from food in 20% sucrose, were randomly placed into vials containing vehicle or PTX (0.03–3 mM). After incubation of 2–24 h, individual larvae were put in each well of a 6-well culture plate previously coated with 2% agar, the plate was then placed in the DanioVision™ apparatus. The activity of individual larva was recorded for 5 min, digitized and analyzed using Ethovision® XT software.

Results

Incubation of third instar wild-type larvae in 1 mM PTX for 4 or 24 h decreased activity; whereas, a 2 h incubation in PTX was without effect. PTX caused a concentration-dependent decrease in activity as demonstrated by consistently reduced locomotor activity with 1.0 and 3.0 mM: 0.3 mM resulted in variable decreases in locomotor activity and 0.03 mM yielded no effect. By contrast, PTX did not affect activity in Rdl^MD-RR larvae even at the highest concentration, 3.0 mM.

Discussion

Using an automated data acquisition system, it was found that PTX decreases activity in third instar Drosophila larvae due to a selective blockade of the GABA_A receptor. The method will reduce the likelihood of human error and bias, as well as increase the speed and ease of data collection and analysis.

Introduction

Several methods currently exist to track and quantify the activity of Drosophila melanogaster larvae. Some of the most commonly used methods include grid-based approaches coupled with manual counting of grid passage per minute (Nichols, Becnel, & Pandey, 2012), video recording and digitizing followed by export and analysis into Photoshop (Stilwell, Saraswati, Littleton, & Chouinard, 2006), image enhancement to improve the low contrast of the translucent larvae against the background (Khurana, Li, & Atkinson, 2010), and most recently, the use of the TriKinetics Drosophila activity monitor (McParland, Follansbee, & Ganter, 2015). All of these methods have proven to be successful in the context of mobility assessment; however, each has caveats related to length of assay and time needed set-up and/or data analysis.

In order to enhance the speed of an assay and eliminate the need for image enhancement by adding dye to the food, or by post-hoc image processing, we employed DanioVision™. Originally designed to track the activity and behavior of zebrafish larvae (Danio rerio) in multi-well plates, we adapted it to video track, digitize, and quantify the activity of third instar Drosophila larvae in 6-well culture plates with no image enhancement using Ethovision® XT software.

To validate this novel application of DanioVision™, third instar Drosophila were exposed to the non-competitive GABA_A receptor antagonist picrotoxin, PTX, a known chemical convulsant. We were able to reproduce the report of Stilwell et al. (2006), which showed PTX decreasing activity in wild-type larvae, but not Rdl^MD-RR larvae. Rdl^MD-RR are a mutant strain of fruit flies with a point mutation in the Rdl gene, rendering them 10–50-fold less sensitive to PTX (Buckingham et al., 1996, ffrench-Constant et al., 1991, Lees et al., 2014). Thus, we conclude that DanioVision™, in conjunction with Ethovision® XT software, can be employed to accurately detect changes in the locomotor activity of Drosophila larvae. In addition to the logistic benefits of this method, we are also able to decrease the likelihood of human error and potential for bias, thereby improving the validity, objectivity and reproducibility of larval locomotor activity assessments.

Section snippets

Fly stocks and husbandry

Rdl^MD-RR mutant Drosophila, stock number 35492, were purchased from Bloomington Drosophila Stock Center (Bloomington, IN, USA). Wild-type flies were obtained from Carolina Biologic Supply (Burlington, NC, USA). Flies were housed in 28.5 × 95 mm vials and fed with blue Formula 4–24® Fly Food prepared according to the instructions (Carolina Biologic Supply, Burlington, NC, USA). Flies were housed in an incubator at 25 °C on a 12 h light/dark cycle.

Larval isolation

Adult male and female Drosophila were transferred into

PTX reduces locomotor activity independent of time

Locomotor activity in Drosophila larvae can be used to analyze the effects of anti-epileptic drugs. Studies were conducted to demonstrate that the DanioVision™ Observation Chamber can be employed to accurately assess locomotor activity in 3rd instar larvae, while simultaneously reducing experimenter bias. To that extent we chose to conduct experiments analogous to those of Stilwell et al. (2006) with slight variation in PTX concentration from 5 mg/mL (0.8 mM) to 1.0 mM PTX. In order to determine

Discussion

There are a variety of methods to assess the effects of pharmacological, toxicological, and genetic manipulations on behavior in Drosophila. One simple, yet effective method is the quantification of locomotor activity in fly larvae. Current methods for activity assays include, but are not limited to, researcher observation and manual recordings (Nichols et al., 2012), or direct video capture with digitizing software and Photoshop enhancements for analysis (Stilwell et al., 2006). In the current

Disclosures

The authors have no disclosures to declare. They have not received any assistance from Noldus Information Technology beyond standard training on the use of the hardware and software.

Authorship

Ms. Graham, the first author, conducted most of the experiments and wrote the initial draft of this manuscript in partial fulfillment of her B.S. in Biology at the University of Saint Joseph. She also was awarded a University of Saint Joseph Senior Research Award to defray the cost of the supplies that she used. Dr. Rogers contributed her knowledge of fruit-fly husbandry and genetics, and played a major role in editing the manuscript during and after she had an adjunct appointment in the

Acknowledgments

The authors are grateful for support from the University of Saint Joseph for the funds used to purchase all equipment and supplies used in these studies.

References (10)

S.D. Buckingham et al.
Wild-type and insecticide-resistant homo-oligomeric GABA receptors of Drosophila melanogaster stably expressed in a Drosophila cell line
Neuropharmacology
(1996)
W. Löscher
Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs
Seizure
(2011)
J. Song et al.
From bench to drug: Human seizure modeling using Drosophila
Progress in Neurobiology
(2008)
R.H. ffrench-Constant et al.
Molecular cloning and transformation of cyclodiene resistance in Drosophila: An invertebrate gamma-aminobutyric acid subtype A receptor locus
Proceedings of the National Academy of Sciences of the United States of America
(1991)
S. Khurana et al.
Image enhancement for tracking the translucent larvae of Drosophila melanogaster
PloS One
(2010)

There are more references available in the full text version of this article.

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Journal of Pharmacological and Toxicological Methods

Abstract

Introduction

Methods

Results

Discussion

Introduction

Section snippets

Fly stocks and husbandry

Larval isolation

PTX reduces locomotor activity independent of time

Discussion

Disclosures

Authorship

Acknowledgments

References (10)

Wild-type and insecticide-resistant homo-oligomeric GABA receptors of Drosophila melanogaster stably expressed in a Drosophila cell line

Neuropharmacology

Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs

Seizure

From bench to drug: Human seizure modeling using Drosophila

Progress in Neurobiology

Molecular cloning and transformation of cyclodiene resistance in Drosophila: An invertebrate gamma-aminobutyric acid subtype A receptor locus

Proceedings of the National Academy of Sciences of the United States of America

Image enhancement for tracking the translucent larvae of Drosophila melanogaster

PloS One

Cited by (6)

GABAergic regulation of locomotion before and during an ethanol exposure in Drosophila melanogaster

Drosophila

Lactobacillus reuteri's multifaceted role in mitigating ionizing radiation-induced injury in Drosophila melanogaster

The pursuit of precision mitochondrial medicine: Harnessing preclinical cellular and animal models to optimize mitochondrial disease therapeutic discovery

Characterization of seizure induction methods in drosophila

Evidence of learning and memory in the juvenile dwarf cuttlefish Sepia bandensis

Original articleAn automated method to assay locomotor activity in third instar Drosophila melanogaster larvae

Abstract

Introduction

Methods

Results

Discussion

Introduction

Section snippets

Fly stocks and husbandry

Larval isolation

PTX reduces locomotor activity independent of time

Discussion

Disclosures

Authorship

Acknowledgments

Neuropharmacology

Seizure

Progress in Neurobiology

Molecular cloning and transformation of cyclodiene resistance in Drosophila: An invertebrate gamma-aminobutyric acid subtype A receptor locus

Proceedings of the National Academy of Sciences of the United States of America

Image enhancement for tracking the translucent larvae of Drosophila melanogaster

PloS One

Original article
An automated method to assay locomotor activity in third instar Drosophila melanogaster larvae