Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

Summary

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Abstract

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Introduction

The overall goal of this approach is to enable researchers to quickly measure the electrogenesis of the central nervous system (CNS) in the model insect, Drosophila melanogaster. This method is reliable, quick, and cost-efficient to perform physiological and toxicological experimentation. The CNS is essential for life and therefore, the physiological pathways critical for proper neural function have been explored extensively in an effort to understand or modify neural function. Characterization of the signaling pathways within the arthropod CNS has enabled the discovery of several chemical classes of insecticides that disrupt invertebrate neural func....

Protocol

1. Equipment and Materials

1. Prepare the required components (listed in Table of Materials) of the electrophysiology rig to perform suction electrode recordings of the Drosophila CNS.
   NOTE: Prior to experimentation, it is necessary to construct chambers for dissection of the Drosophila CNS and to be used for bathing the ganglia in saline during recordings. A step-by-step outline of chamber construction is provided below.
2. Prepare the larval chamber.
   1. Melt the black wax using a hot plate.
   2. Pour 2 mL of melted wax into a chamber that has a maximum volume of 2 - 2.5 mL.
   3. Let the w....

Results

Spontaneous activity of the descending peripheral nerves arising from the Drosophila central nervous system can be recorded using extracellular suction electrodes with consistent reproducibility. Spontaneous activity of the excised and transected Drosophila CNS produces a cyclical pattern of bursting with 1-2 s of firing with approximately 1 s of near quiescent activity. For example, the CNS is near quiescent (1-2 Hz) for 0.5-1 s, followed by a burst (100-400 Hz) for app.......

Discussion

The details provided in the associated video and text have provided key steps in order to record the activity and spike discharge frequency of the Drosophila CNS ex vivo. The dissection efficacy is the most critical aspect of the method because short or few descending neurons will reduce the baseline firing rate that will result in large variances between replicates. However, once the dissection technique has been mastered, the data collected with this assay are highly reproducible and amendable for a w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drosophila melanogaster (strain OR)	Bloomington Drosophila Stock Center	2376
Vibration isolation table	Kinetic Systems	9200 series
Faraday Cage	Kinetic Systems	N/A
Dissecting Microscope on a Boom	Nikon	SMZ800N	Multiple scopes can be used; boom stand is critical
AC/DC differential amplifier	ADInstruments	AM3000H	The model 1700 can be used instead of the model 3000
audio monitor	ADInstruments	AM3300
Hum Bug Noise Eliminator	A-M Systems	726300
Data Acquisition System (PowerLab)	ADInstruments	PL3504	Multiple PowerLab models can be used.
Lab Chart Pro Software	ADInstruments	N/A - Online Download
Fiber Optic Lights	Edmund Optics	89-740	Different light sources can be used, but fiber optics are the most adaptable
Micromanipulator	World Precision Instruments	M325
Microelectrode Holder	World Precision Instruments	MEH715	Different models are acceptable
BNC cables	World Precision Instruments	multiple based on size
Glass Capillaries	World Precision Instruments	PG52151-4
Microelectrode Puller	Sutter Instruments	P-1000	Also can use Narashige PC-100
Black Wax	Carolina Biological Supply	974228
Non-coated insect pins, size #2	Bioquip	1208S2
Fince Forceps	Fine Science Tools	11254-20
Vannas Spring Scissors	Fine Science Tools	15000-03