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

Stephanie Graham; Ryan P Rogers; Richard H Alper

doi:10.1016/j.vascn.2015.10.004

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

J Pharmacol Toxicol Methods. 2016 Jan-Feb:77:76-80. doi: 10.1016/j.vascn.2015.10.004. Epub 2015 Nov 7.

Authors

Stephanie Graham¹, Ryan P Rogers², Richard H Alper³

Affiliations

¹ Department of Pharmaceutical Sciences, University of Saint Joseph School of Pharmacy, Hartford, CT, United States; Department of Biology, University of Saint Joseph, West Hartford, CT, United States.
² Department of Biology, University of Saint Joseph, West Hartford, CT, United States; Department of Sciences, Wentworth Institute of Technology, Boston, MA, United States.
³ Department of Pharmaceutical Sciences, University of Saint Joseph School of Pharmacy, Hartford, CT, United States. Electronic address: ralper@usj.edu.

PMID: 26554339
DOI: 10.1016/j.vascn.2015.10.004

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 GABAA 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 GABAA 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 (0.03-3mM). After incubation of 2-24h, 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 1mM PTX for 4 or 24h decreased activity; whereas, a 2h 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 3.0mM: 0.3mM 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, 3.0mM.

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 GABAA 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.

Keywords: Drosophila melanogaster; GABA(A) receptors; Locomotor activity; Methods; Picrotoxin; Picrotoxin (PubChem CID: 442292); Rdl; Seizures.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Biological Assay / methods
Drosophila melanogaster / drug effects
Drosophila melanogaster / physiology*
Female
GABA Antagonists / pharmacology
Larva / drug effects
Larva / physiology*
Male
Motor Activity / drug effects
Motor Activity / physiology*
Picrotoxin / pharmacology
gamma-Aminobutyric Acid / metabolism

Substances

GABA Antagonists
Picrotoxin
gamma-Aminobutyric Acid