Electrophysiological Recording From Drosophila Labellar Taste Sensilla

Summary

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Abstract

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Introduction

The sense of taste allows an insect to detect a vast range of soluble chemicals and plays an important role in the acceptance of a nutritious substance, or the rejection of a noxious or toxic one. Taste is also thought to play a role in mate selection, through the detection of pheromones^1-5. These important and diverse functions have made the insect taste system a compelling target of investigation into how sensory systems translate environmental cues into relevant behavioral outputs.

The primary unit of the Drosophila melanogaster taste system is the taste hair, or sensillum. Molecules enter the sensillum via a pore at ....

Protocol

The following protocol complies with all the animal care guidelines of Yale University.

1. Reagents and Equipment Preparation

1. Recording equipment setup (Figure 1A).
   1. Choose a room for rig setup that is free of large variations in temperature or humidity and also isolated from sources of electrical and mechanical noise, such as refrigerators and centrifuges.
      

Results

Figure 5A shows the response of an L sensillum to a sugar, sucrose. The same sensillum does not respond to a bitter compound, berberine. Figure 5B shows that an I type sensillum, which contains a bitter responsive neuron, displays larger amplitude spikes in response to berberine, and smaller amplitude spikes in response to sucrose. L sensilla display a minimal background response to the solvent control, TCC, while I sensilla display virtually no response to TCC (Figure 5.......

Discussion

Labellar sensilla vary in the ease of recording due to differences in morphology and anatomical organization. Sometimes a sensillum does not respond to any tastants, even one that is known to elicit a positive response. The frequency with which this occurs varies depending on sensillum type. L sensilla are most consistently responsive and are relatively easy to access due to their length. In general, S sensilla are consistently responsive, but their short length and position on the labellum make good contact challenging........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereo Zoom Microscope	Olympus	SZX12 DFPLFL1.6x PF eyepieces: WHN10x-H/22	capable of ~150X magnification with long working distance table mount stand
Antivibration Table	Kinetic Systems	BenchMate2210
Micromanipulators	Narishige	NMN-21
Magnetic stands	ENCO	Model #625-0930
Reference Electrode Holder	Harvard Apparatus	ESP/W-F10N	Can be mounted on 5 ml serological pipette for extended range
Silver Wire	World Precision Instruments	AGW1510	0.3-0.5 mm diameter
Retort Stand			generic
Outlet Plastic Tube			generic, 1 cm diameter
Flexible Plastic Tubing	Nalgene	8000-0060	VI grade 1/4 in internal diameter
500 ml Conical Flask			generic, with side arm
Aquarium Pump	Aquatic Gardens	Airpump 2000
Fiber Optic Light Source	Dolan-Jenner Industries	Fiber-Lite 2100
White Card/Paper	Whatman	1001-110
Digital Acquisition System	Syntech	IDAC-4	Alternative: National Instruments NI-6251
Headstage	Syntech	DTP-1	Tasteprobe
Tasteprobe Amplifier	Syntech	DTP-1	Tasteprobe
Alligator Clips	Grainger	1XWN7	Any brand is fine
Insulated Electrical Wire			Generic
Gold Connector Pins	World Precision Instruments	5482
Personal Computer	Dell	Vostro	Check for compatibility with digital acquisition system and software
Acquisition Software	Syntech	Autospike	Autospike works with IDAC-4; alternatively, use LabView with NI-6251
Aluminum Foil and/or Faraday Cage			Electromagnetic noise shielding
Borosilicate Glass Capillaries	World Precision Instruments	1B100F-4
Pipette Puller	Sutter Instrument Company	Model P-87 Flaming/Brown Micropipette Puller
Beadle and Ephrussi Ringer Solution			See recipe in protocol section
Tricholine citrate, 65%	Sigma	T0252-100G
Stereomicroscope	Olympus	VMZ 1x-4x	Capable of 10-40X magnification
Ice Bucket			Generic
p200 Pipette Tips			Generic
Spinal Needle	Terumo	SN*2590
1 ml Syringe	Beckton-Dickenson	301025
Fly Aspirator			Assembled from P1000 pipette tips, flexible plastic tubing, and mesh
Modeling Clay			Generic
Forceps	Fine Science Tools By Dumont	11252-00	#5SF (super-fine tips)
10 ml Syringe	Beckton-Dickinson	301029
Plastic Tubing	Tygon	R-3603