Name: Electrophysiological Recording From Drosophila Labellar Taste Sensilla (Video) | JoVE Journal
Uploaded: 2014-02-26T23:00:00.000+00:00
Duration: 6 min 32 s
Description: Learn electrophysiological recording from Drosophila labellar taste sensilla for neuroscience research. This method measures action potential responses to taste compounds and helps researchers study sensory processing in feeding behavior.

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5 minutes

This work was supported by an NRSA predoctoral grant 1F31DC012985 (to R.D.) and by NIH grants to J.C.
We would like to thank Dr. Linnea Weiss for helpful comments on the manuscript, Dr. Ryan Joseph for help compiling figures, and Dr. Frederic Marion-Poll for helpful technical advice. We would also like to acknowledge the helpful comments of four reviewers.

The following protocol complies with all the animal care guidelines of Yale University.
1. Reagents and Equipment Preparation
<ol>
	<li>Recording equipment setup (Figure 1A).
	<ol>
		<li fo:keep-together.within-page="always">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. 
		<img alt="Microscopy and spectroscopy setup for optical excitation and analysis; displays and measurement devices." fo:content-width="5in" fo:src="/files/ftp_upload/51355/51355fig1highres.jpg" src="/files/ftp_upload/51355/51355fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
		Figure 1. (A) Overview of recording rig setup. Stereomicroscope (a) is mounted on anti-vibration platform (b). Reference electrode holder (c) is mounted on platform opposite the headstage (d), via micromanipulators. An outlet plastic tube (e) delivering humidified air stream directed at the fly preparation is also mounted on the platform. The headstage is connected to the amplifier (f), which is connected to the digital acquisition system (DAS) (g), which is connected to a PC (h). (B) Configuration of electrodes and outlet tube: reference electrode on the left, recording electrode on the right, and air stream outlet tube directed at fly preparation. <a href="https://www.jove.com/files/ftp_upload/51355/51355fig1highres.jpg" target="_blank">Click here to view larger image.</a></li>
		<li>Mount stereomicroscope to center of anti-vibration table or platform.</li>
		<li>Attach micromanipulators for the reference electrode/insect preparation and headstage/recording electrode to the left and right of the microscope, respectively, using magnetic stands.</li>
		<li>Mount outlet plastic tube in a third micromanipulator to the rear of the microscope, oriented such that the tube opening is pointed toward location of fly prep (see Figure 1B).</li>
		<li>Using flexible plastic tubing, attach outlet plastic tube to a vacuum flask partially filled with water. Connect a small aquarium pump to bubble air through the water in the flask, generating a humidified air stream through the outlet plastic tube towards the fly.</li>
		<li>Mount fiber optic light source off the vibration table, orienting the outputs to illuminate the preparation by reflecting light via a piece of white card paper directly below the preparation. Ensure that the light source does not rest on the table. Note: the benefit of reflecting the light source on a paper disc is two-fold: it improves the contrast, making sensilla easier to visualize, and it prevents heating of the preparation that would result from direct light.</li>
		<li>Plug tastePROBE amplifier into the digital acquisition system (DAS), and the DAS into a personal computer, according to supplier manual. Plug foot pedal trigger in and arrange under workspace. Note: Electrically isolated wall sockets for the amplifier and DAS are highly desirable.</li>
		<li>Electrically ground microscope, micromanipulators, and light source by connecting metal components to table using alligator clips and lengths of insulated electrical wire and electrical tape. Electrically ground metal platform by connecting to building ground or DAS, which is grounded through power supply plug.</li>
		<li>Install appropriate acquisition software for the DAS of choice on the personal computer. Note: Ensure that the digital acquisition drivers are compatible with the operating system on the PC.</li>
		<li>Configure software amplification (10-100x), signal filtering (typically a Bessel bandpass filter set from 100 Hz-3,000 Hz), and sampling rate (at least 10 KHz). Note: Signal amplitudes from gustatory neurons are typically in the 0.5-2 mV range, so the display scale is set to facilitate their visualization. Note: The 100 Hz filter helps to exclude extraneous electrical noise; however, it changes the shape of spikes and can make advanced spike sorting more challenging. Alternatively, a 1 Hz filter can be used.</li>
		<li>Optionally, a Faraday cage can be set up around the whole vibration table. However, small sheets of aluminum foil are usually sufficient to reduce any noise generated by the external environment or investigator.</li>
	</ol>
	</li>
	<li fo:keep-together.within-page="always">Glass Electrode Preparation 
	<img alt="Microinjection needles, microscopy image; analysis of sharpness and tip consistency across samples." fo:content-width="5in" fo:src="/files/ftp_upload/51355/51355fig2highres.jpg" src="/files/ftp_upload/51355/51355fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
	Figure 2. Reference and recording electrodes. Photograph under magnification of glass capillaries pulled into reference electrode, with (A) and without (B) tip broken, and recording electrode (C). White bar represents 2 mm. <a href="https://www.jove.com/files/ftp_upload/51355/51355fig2highres.jpg" target="_blank">Click here to view larger image.</a>
	<ol>
		<li>Pull the reference electrode from a glass capillary using a pipette puller instrument. Note: The exact settings of the pipette puller program will vary from instrument to instrument. Try to achieve a very long gradual taper. The pore size at the tip is not crucial because the tip will be broken before fly preparation (Figures 2A and 2B); however, make sure that the diameter of the tapered length of the electrode is neither too thin, which will not allow for sufficient immobilization of the labellum, nor too large, which could damage the gustatory neurons or rupture the salivary glands.</li>
		<li>Pull recording electrode from a borosilicate glass capillary with filament using a pipette puller instrument. Try to achieve a taper that is shallower than that of the reference electrode, and a pore diameter of approximately 10-15 μm (Figure 2C)28.</li>
	</ol>
	</li>
	<li>Tastant solutions preparation
	<ol>
		<li>Use Beadle-Ephrussi Ringer solution (B&amp;E) as the reference electrode electrolyte. To make one liter of B&amp;E, dissolve 7.5 g NaCl, 0.35 g KCl, and 0.279 g CaCl2∙2H2O in one liter of ultrapure water. Store smaller aliquots at -20 °C.</li>
		<li>Use 30 mM tricholine citrate solution (TCC) as the recording electrode electrolyte and solvent for tastant solutions25, if bitter or sugar GRN responses are to be measured. Alternatively, 1-3 mM potassium chloride solution can be used if responses of the water cell are to be measured.</li>
		<li>To make tastant solutions, weigh appropriate amount of tastant in powder form and add to TCC to make an initial stock concentration. Use this to make serial dilutions from this initial stock to yield the desired concentration for testing. Note: If tastants do not readily dissolve in water, another solvent, such as ethanol, can be used to make initial stock concentration. An appropriate control solution of TCC and solvent without tastant should be used in this case.</li>
		<li>Store aliquots long term at -20 °C. Store one working aliquot of a tastant solution at 4 °C for recording use for up to a week, depending on chemical properties of tastant.</li>
	</ol>
	</li>
</ol>
2. Drosophila Preparation
<img alt="Drosophila phenotypic analysis; microscope images focus on red-eyed fruit fly morphology and anatomy." fo:content-width="5in" fo:src="/files/ftp_upload/51355/51355fig3highres.jpg" src="/files/ftp_upload/51355/51355fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Preparation of fly for recording. (A) Insertion position of reference electrode into dorsal thorax of fly. The white arrow indicates the reference electrode. (B) Intermediate position of reference electrode: advanced through neck and head, proboscis not yet extended. (C,D) Fly with reference electrode in final position with tip of electrode inside labellum, and proboscis fully extended. <a href="https://www.jove.com/files/ftp_upload/51355/51355fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<ol>
	<li>Collect newly eclosed flies for recording from well-maintained fly cultures, grown under temperature- and humidity-controlled conditions, and age them 5-10 days in fresh culture vials before recording.</li>
	<li>Chill microscope plate on ice for 15-30 min before preparing fly.</li>
	<li>Backfill glass reference electrode with B&amp;E solution using a long, thin plastic needle of 0.5 mm diameter, such as a spinal needle and 1 ml syringe and gently tap out any bubbles. Break small amount of tip off using forceps and use capillary action to draw out all remaining bubbles with a tissue, observing under dissecting microscope.</li>
	<li>Slide B&amp;E-filled reference electrode onto wire of reference electrode holder, taking care not to introduce air bubbles.</li>
	<li>Aspirate fly into a P200 pipette tip, using fly aspirator built from tubing, mesh, and pipette tip;29 place in ice bucket and chill for 30-60 sec.</li>
	<li>Remove microscope plate from ice, wipe off any moisture, and position underneath microscope. Gently tap fly out of pipette tip onto microscope plate. 
	Note: the fly should be sufficiently immobilized to manipulate easily.</li>
	<li>Under low magnification, gently remove the forelegs with one pair of forceps, while holding the thorax stable with the other pair of forceps. Position the fly on its ventral side, dorsal side facing up. Note: Always be careful to avoid touching the labellum with the forceps at all times during the preparation process to minimize mechanical damage.</li>
	<li>While holding the fly in place with one pair of forceps, insert the reference electrode at the midline of the posterior dorsal thorax. A suggested angle of entry is approximately forty-five degrees, in the direction of the head (Figure 3A).</li>
	<li>Secure the reference electrode holder with modeling clay such that the fly is visible underneath the microscope at high magnification. Maneuver and angle the glass electrode through the neck and head, by sliding the fly towards the reference electrode holder using two pairs of forceps. Note: Work quickly but smoothly; it is easier to complete this step while the fly is still immobilized from the cold (Figure 3B).</li>
	<li>Gently extend the proboscis with one pair of forceps, while sliding the fly further down the glass reference electrode, until the tip of the electrode is inside the labellum and the proboscis is fully extended (Figures 3C and 3D). Note: Take care not to puncture any part of the proboscis tissue or distend the edge of the labellum with the reference electrode, as this may damage the fly and/or taste neurons and affect the recording quality.</li>
</ol>
3. Recording from Labellar Sensilla
<img alt="Insect leg dissection, microscopic image, experiment setup, biological structure examination." fo:content-width="5in" fo:src="/files/ftp_upload/51355/51355fig4highres.jpg" src="/files/ftp_upload/51355/51355fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. Recording from fly. (A) labellum of fly preparation on left with recording electrode aligned for contact on right, under high magnification. (B) recording electrode and single sensillum on labellum in contact, under high magnification. <a href="https://www.jove.com/files/ftp_upload/51355/51355fig4highres.jpg" target="_blank">Click here to view larger image.</a>
<ol>
	<li>Always ground yourself by touching the metal surface of the anti-vibration table or platform prior to touching any equipment during recording process! Note: It is extremely important not to deliver a static charge to the headstage as that can damage the circuitry.</li>
	<li>Secure reference electrode holder to micromanipulator mounted on air table of recording rig. Position one lobe of labellum in microscope field of view, under high magnification (typically at least 140X), and in line with humidified air stream.</li>
	<li>Turn on humidified air stream, computer, DAS, and amplifier. Open acquisition software.</li>
	<li>Rinse and fill glass recording electrode with desired tastant.
	<ol>
		<li>Rinse glass recording electrode with ultrapure water by using a syringe and plastic tubing28 to pull small amounts of water through the tube at least ten times.</li>
		<li>Rinse recording electrode with tastant at least five times. Fill recording electrode approximately one-third to halfway full with tastant and remove from tubing. If there are air bubbles, tap to release or simply refill the electrode.</li>
		<li>Slide electrode onto silver wire of the headstage quickly and smoothly so as not to introduce air bubbles.</li>
	</ol>
	</li>
	<li>Stimulate single sensillum with tastant-filled recording electrode.
	<ol>
		<li>Use the micromanipulator to bring the recording electrode aligned with sensillum of interest.</li>
		<li>Press foot pedal to trigger acquisition mode of the amplifier.</li>
		<li>Advance the recording electrode with the fine control knob of micromanipulator carefully until it makes contact with tip of sensillum and recording commences.</li>
		<li>Remove electrode after 1-2 sec.</li>
		<li>Repeat step 3.5 with other sensilla, if desired. Note: Wait at least 1 min in between presentations to the same sensillum. If recording with a single tastant for a prolonged period of time, the tastant solution may dry out and the solution in the tip may become more concentrated. This can be remedied by gently contacting the tip of the glass electrode with smooth paper to remove a small amount of liquid by capillary action.</li>
	</ol>
	</li>
	<li>To record responses to another tastant, rinse and load recording electrode with new tastant and repeat step 3.4. Note: Thoroughly rinsing the electrode between tastants is absolutely crucial to avoid cross-contamination.</li>
	<li>Save data files periodically with identifying information, such as date, genotype, and tastants. Note: It is important to keep a written record of the tastant and sensillum identity of each presentation during recording session for data analysis.</li>
</ol>

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<li>Benton, R., Dahanukar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+recording+from+Drosophila+taste+sensilla%5BTitle%5D)+AND+%22Cold+Spring+Harbor+protocols%22%5BJournal%5D)">Electrophysiological recording from Drosophila taste sensilla.</a> Cold Spring Harbor protocols. 2011, 839-850 (2011).</li>
<li>Pellegrino, M., Nakagawa, T., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+sensillum+recordings+in+the+insects+Drosophila+melanogaster+and+Anopheles+gambiae%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Single sensillum recordings in the insects Drosophila melanogaster and Anopheles gambiae.</a> J. Vis. Exp. , e1725(2010).</li>
<li>Axon Instruments. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22The+Axon+Guide+for+Electrophysiology+%26+Biophysics+Laboratory+Techniques%22">The Axon Guide for Electrophysiology & Biophysics Laboratory Techniques</a>. , (1993).</li>
<li>Fujishiro, N., Kijima, H., Morita, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impulse+frequency+and+action+potential+amplitude+in+labellar+chemosensory+neurones+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Journal+of+insect+physiology%22%5BJournal%5D)">Impulse frequency and action potential amplitude in labellar chemosensory neurones of Drosophila melanogaster.</a> Journal of insect physiology. 30, 317-325 (1984).</li>
<li>Marion-Poll, F., Tobin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Software+filter+for+detecting+spikes+superimposed+on+a+fluctuating+baseline%5BTitle%5D)+AND+%22Journal+of+neuroscience%22%5BJournal%5D)">Software filter for detecting spikes superimposed on a fluctuating baseline.</a> Journal of neuroscience. 37, 1-6 (1991).</li>
<li>Meunier, N., Marion-Poll, F., Lansky, P., Rospars, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimation+of+the+individual+firing+frequencies+of+two+neurons+recorded+with+a+single+electrode%5BTitle%5D)+AND+%22Chem+Senses%22%5BJournal%5D)">Estimation of the individual firing frequencies of two neurons recorded with a single electrode.</a> Chem Senses. 28, 671-679 (2003).</li>
<li>Marion-Poll Lab Website. , Available from: 
 
 	 <a target="_blank" href="http://taste.versailles.inra.fr/fred">http://taste.versailles.inra.fr/fred</a> (2013).</li>
<li>Meunier, N., Marion-Poll, F., Rospars, J. P., Tanimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+coding+of+bitter+taste+in+Drosophila%5BTitle%5D)+AND+%22Journal+of+neurobiology%22%5BJournal%5D)">Peripheral coding of bitter taste in Drosophila.</a> Journal of neurobiology. 56, 139-152 (2003).</li>
</ol>

The authors have nothing to disclose.

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....

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 pheromones1-5. 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 its tip2,6. Sensilla are found on the labellum, the legs, the wing margin, and the pharynx6.&#160;On the labellum, the number and location of sensilla is stereotyped. There are three morphological classes of sensilla based on length: the long (L), intermediate (I), and short (S) sensilla7,8. Each sensillum contains either two (I-type) or four (L- and S- type) gustatory receptor neurons (GRNs)9. Different GRNs respond to different categories of taste stimuli: bitter, sugar, salt and osmolarity7,10 and express different subsets of gustatory receptors8,11-13. Only I and S-type sensilla contain bitter-responsive GRNs8,10. The GRNs project to the subesophageal ganglion (SOG) and their activation by taste molecules is relayed to the higher central nervous system for decoding, resulting in a behavioral response6. The relatively small number of neurons and the amenability to molecular and behavioral analysis make the Drosophila taste system an excellent model for the investigation of gustatory systems in general. The relative ease with which the system can be manipulated via genetic mutation or the GAL4-UAS expression system also serves as a valuable tool14,15.
Because these sensilla protrude from the surface of the labellum, they make excellent targets for electrophysiology. The firing of the GRNs can be monitored using extracellular recording. Historically, the side-wall recording method, which uses a glass electrode inserted into the sensillum to record neuronal activity,26 has been used. However, this method is technically challenging to perform, and it is difficult to record for long from each preparation. The tip-recording method, which measures the response of the neurons with an electrode that simultaneously delivers a tastant, has since become the method of choice9,16. It has been utilized to investigate the taste system of Drosophila melanogaster8,10,17,18 as well as a number of other insect species19-23. It has been greatly facilitated by the development of the tastePROBE amplifier, which overcame one of the major drawbacks of the tip-recording method by compensating for the large potential difference between the reference electrode and the insect sensillum, allowing the GRN action potentials to be recorded without excessive amplification or filtering24. Another important development was the use of tricholine citrate as the recording electrolyte25. TCC suppresses responses from the osmolarity-sensitive GRN and does not stimulate the salt-sensitive GRN, making responses generated by bitter and sugar tastants much easier to analyze25.
Here we describe how tip recording of Drosophila labellar sensilla is currently performed in the Carlson laboratory. This protocol will explain how to establish a suitable electrophysiology rig, how to prepare the fly, and how to perform taste recordings. We also present some representative data obtained by recording from subsets of Drosophila sensilla, as well as some common issues and potential solutions that may be encountered when using this technique.

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

electrophysiological recording from drosophila labellar taste sensilla

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

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.

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...

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

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

The overall goal of this procedure is to measure responses of individual taste sensa on the labum of drosophila. This is accomplished by first building an electrophysiology rig and setting up all the necessary equipment. The second step is to prepare the fly for recording.Next, an individual lum is stimulated with a taste compound while the neuronal response is simultaneously recorded. Ultimately, electrophysiological tip recording from individual label silla is used to show the responses of taste illa to different taste compounds. Visual demonstration of this method is critical as the fly preparation steps can be technically challenging and difficult to learn.In this procedure, collect the newly ELOs flies from the well-maintained fly cultures. Then age them five to 10 days in fresh culture vials before recording, before the fly preparation, chill the microscope plate on ice for 15 to 30 minutes. Next, backfill the glass reference electrode with B and e solution.And gently tap out any bubbles under the dissecting microscope. Use the forceps to break off a small section of the tip. Subsequently use capillary action to draw out any remaining bubbles with the tissue.Then slide the B and E filled reference electrode over the wire of the electrode holder, and to be careful not to introduce any air bubbles after that, aspirate a fly into a P 200 pipette tip. Place it in an ice bucket and chill for 30 seconds to one minute. Next, remove the microscope plate from ice and position it under the microscope after wiping off the moisture.Then gently tap the fly out of the pipette tip onto the microscope plate under low magnification. Remove the four legs with one pair of forceps while holding the Forex stable with the other pair of forceps. Position the fly on its ventral side with the dorsal side facing up.Always be careful to avoid touching the label beum with the forceps during the preparation process to minimize mechanical damage while holding the fly in place with one pair of forceps, insert the reference electrode at the midline of the posterior dorsal thorax at approximately 45 degrees in the direction of the head. Then secure the reference electrode holder with modeling clay such that the fly is visible underneath the microscope at high magnification. Maneuver and angle the glass electrode at its neck and head, and slide the fly towards the reference electrode holder using two pairs of forceps.Now, gently extend the probos with one pair of forceps and slide the fly further down the glass reference electrode until the tip of the electrode is inside the label beum and the probos is fully extended. In this step, secure the reference electrode holder to the micro manipulator mounted on the air table. Position one lobe of label labum in the microscope field of view under high magnification and in line with the humidified airstream.After that, turn on the Humidified Airstream Computer Digital Acquisition System or DAS and Amplifier. Next, use a syringe and plastic tubing to draw a small amount of ultrapure water through the tube at least 10 times. To rinse the glass recording electrode, subsequently rinse the recording electrode with TAST at least five times.Then fill the recording electrode approximately one third to halfway full with taste and remove it from the tubing. If there are air bubbles, tap to release them or simply refill the electrode. Now slide the electrode onto the silver wire of the head stage quickly and smoothly.In order to avoid introducing air bubbles to stimulate the single send, first, use the micro manipulator to bring the recording electrode into alignment with the sens of interest. Next, press the foot pedal to trigger acquisition mode of the amplifier. Then advance the taste and filled recording electrode with the fine control knob of the micro manipulator carefully until it makes contact with the tip of the sens and start recording.Remove the electrode after one to two seconds. Repeat the procedure again with other illa and wait at least one minute in between presentations to the same lum. If recording with a single taste and for a prolonged period of time, the taste and solution may dry out and the solution in the tip may become more concentrated.This can be remedied by gently contacting the tip of the glass electrode with smooth paper to remove a small amount of liquid by capillary action to record the responses to another taste and rinse and load the recording electrode with new taste and repeat the procedure. Save data files periodically with identifying information such as date, genotype, and taste. This figure shows the response of an centum to a sugar sucrose.The same centum does not respond to a bitter compound berberine, and this figure shows that an eye type sens, which contains a bitter responsive neuron, displays larger amplitude spikes in response to berberine and smaller amplitude spikes in response to sucrose. Shown here, a representative suboptimal electrophysiological results. This trace represents a complete lack of signal.This is the 50 or 60 hertz noise from external electrical sources, and this is the stochastic noise. This trace represents that the Meno sensory neuron was firing alone while both bitter gustatory receptor neuron and the Meno sensory neuron were firing. After watching this video, you should have a good understanding of how to record neuronal responses from Oph Atory Silla on the labum.

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Research

JoVE Journal

Neuroscience

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Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

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Electrophysiological Recording in the Drosophila Embryo

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

This video outlines the protocol for electrophysiological recording of photoreceptor activity in the eye of an immobilized Drosophila, enabling precise measurement of voltage changes in response to light stimuli.

Electrophysiological Recording of Voltage Responses of Drosophila Retinal Photoreceptors to Light Stimuli

In this video, a live Drosophila fly is mounted in a tube for electrophysiological studies. Electrodes are positioned at the base of the sensillum to record specific neuronal responses to pheromones with the fly's olfactory receptor neurons.

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The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

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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.

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

A rarely used method of electrophysiological recording, base recording, allows analysis of features of taste coding that cannot be examined by conventional recording methods. Base recording also allows the analysis of taste responses to hydrophobic stimuli that cannot be studied using traditional electrophysiological methods.

Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila

A calcium imaging approach enables the recording of taste-induced responses in the brains of awake flies while a solution is applied to the labellum. Primary gustatory responses in Drosophila melanogaster are used as an example, but this protocol can be adapted to study downstream neurons or other species.

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Learn electrophysiological recording from Drosophila labellar taste sensilla for neuroscience research. This method measures action potential responses to taste compounds and helps researchers study sensory processing in feeding behavior.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

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