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="Figure 1" fo:content-width="5in" fo:src="/...

<ol>
	<li>Glendinning, J. I., Jerud, A., Reinherz, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hungry+caterpillar:+an+analysis+of+how+carbohydrates+stimulate+feeding+in+Manduca+sexta.">The hungry caterpillar: an analysis of how carbohydrates stimulate feeding in Manduca sexta.</a> The Journal of experimental biology. 210, 3054-3067 (2007).</li><li>Yarmolinsky, D. A., Zuker, C. S., Ryba, N. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+female+aphrodisiac+pheromone+activates+ppk23(+)+sensory+neurons+to+elicit+male+courtship+behavior.">The Drosophila female aphrodisiac pheromone activates ppk23(+) sensory neurons to elicit male courtship behavior.</a> Cell reports. 1, 599-607 (2012).</li><li>Lu, B., LaMora, A., Sun, Y., Welsh, M. J., Ben-Shahar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppk23-Dependent+chemosensory+functions+contribute+to+courtship+behavior+in+Drosophila+melanogaster.">ppk23-Dependent chemosensory functions contribute to courtship behavior in Drosophila melanogaster.</a> PLoS Genet. 8, e1002587 (2012).</li><li>Stocker, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+the+chemosensory+system+in+Drosophila+melanogaster:+a+review.">The organization of the chemosensory system in Drosophila melanogaster: a review.</a> Cell and tissue research. 275, 3-26 (1994).</li><li>Hiroi, M., Marion-Poll, F., Tanimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+response+to+sugars+among+labellar+chemosensilla+in+Drosophila.">Differentiated response to sugars among labellar chemosensilla in Drosophila.</a> Zoological Science. 19, 1009-1018 (2002).</li><li>Weiss, L. A., Dahanukar, A., Kwon, J. Y., Banerjee, D., Carlson, J. 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J., Warr, C. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidate+taste+receptors+in+Drosophila.">Candidate taste receptors in Drosophila.</a> Science (New York, N.Y.). 287, 1830-1834 (2000).</li><li>Cameron, P., Hiroi, M., Ngai, J., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+for+water+taste+in+Drosophila.">The molecular basis for water taste in Drosophila.</a> Nature. 465, 91-95 (2010).</li><li>Croset, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancient+protostome+origin+of+chemosensory+ionotropic+glutamate+receptors+and+the+evolution+of+insect+taste+and+olfaction.">Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction.</a> PLoS Genet. 6, e1001064 (2010).</li><li>Brand, A. 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H., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+DEET+through+insect+gustatory+receptors.">Avoiding DEET through insect gustatory receptors.</a> Neuron. 67, 555-561 (2010).</li><li>Descoins, C., Marion-Poll, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+responses+of+gustatory+sensilla+of+Mamestra+brassicae+(Lepidoptera,+Noctuidae)+larvae+to+three+ecdysteroids:+ecdysone,+20-hydroxyecdysone+and+ponasterone.">Electrophysiological responses of gustatory sensilla of Mamestra brassicae (Lepidoptera, Noctuidae) larvae to three ecdysteroids: ecdysone, 20-hydroxyecdysone and ponasterone.</a> A. J Insect Physiol. 45, 871-876 (1999).</li><li>Glendinning, J. 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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 border="0" cellpadding="0" cellspacing="0" width="783">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:168px;">Stereo Zoom Microscope</td>
			<td style="width:187px;">Olympus&#160;</td>
			<td style="width:211px;">SZX12 DFPLFL1.6x PF eyepieces: WHN10x-H/22</td>
			<td style="width:219px;">capable of ~150x magnification with long working distance table mount stand</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Anti-vibration Table</td>
			<td style="width:187px;">Kinetic Systems</td>
			<td>BenchMate2210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Micromanipulators</td>
			<td style="width:187px;">Narishige</td>
			<td>NMN-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Magnetic stands</td>
			<td style="width:187px;">ENCO</td>
			<td>Model #625-0930</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Reference Electrode Holder</td>
			<td style="width:187px;">Harvard Apparatus</td>
			<td style="width:211px;">ESP/W-F10N</td>
			<td>Can be mounted on 5ml serological pipette for extended range</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">&#160;Silver Wire</td>
			<td style="width:187px;">World Precision Instruments</td>
			<td style="width:211px;">AGW1510</td>
			<td>0.3-0.5mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Retort Stand</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>generic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Outlet Plastic Tube</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>generic, 1cm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Flexible Plastic Tubing</td>
			<td style="width:187px;">Nalgene&#160;</td>
			<td>8000-0060</td>
			<td style="width:219px;">VI grade 1/4 in internal diameter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">500 ml Conical Flask</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>generic,&#160; with side arm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Aquarium Pump</td>
			<td style="width:187px;">Aquatic Gardens</td>
			<td style="width:211px;">Airpump 2000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Fiber Optic Light Source</td>
			<td style="width:187px;">Dolan-Jenner Industries</td>
			<td>Fiber-Lite 2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">White Card/Paper</td>
			<td style="width:187px;">Whatman</td>
			<td style="width:211px;">1001-110</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Digital Acquisition System</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">IDAC-4</td>
			<td>Alternative: National Instruments NI-6251 &#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Headstage</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">DTP-1</td>
			<td>Tasteprobe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Tasteprobe Amplifier</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">DTP-1</td>
			<td>Tasteprobe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Alligator Clips</td>
			<td style="width:187px;">Grainger</td>
			<td style="width:211px;">1XWN7</td>
			<td>Any brand is fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Insulated Electrical Wire</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Gold Connector Pins</td>
			<td style="width:187px;">World Precision Instruments</td>
			<td align="right" style="width:211px;">5482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Personal Computer</td>
			<td style="width:187px;">Dell&#160;</td>
			<td style="width:211px;">Vostro</td>
			<td>Check for compatibility with digital acquisition system and software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Acquisition Software</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">Autospike</td>
			<td>Autospike works with IDAC-4; alternatively, use Labview with NI-6251</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Aluminum Foil and/or Faraday Cage</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Electro-magnetic noise shielding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;"></td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Borosilicate Glass Capillaries</td>
			<td style="width:187px;">World Precision Instruments</td>
			<td style="width:211px;">1B100F-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Pipette Puller</td>
			<td style="width:187px;">Sutter Instrument Company</td>
			<td style="width:211px;">Model P-87 Flaming/Brown Micropipette Puller</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;"></td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Beadle and Ephrussi Ringer Solution</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>See recipe in protocol section</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Tricholine citrate, 65%&#160;</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:211px;">T0252-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;"></td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Stereo Microscope</td>
			<td style="width:187px;">Olympus</td>
			<td style="width:211px;">VMZ 1x-4x</td>
			<td style="width:219px;">Capable of 10x-40x magnification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Ice Bucket</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">p200 Pipette Tips</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Spinal Needle</td>
			<td style="width:187px;">Terumo</td>
			<td style="width:211px;">SN*2590</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">1ml Syringe</td>
			<td style="width:187px;">Beckton-Dickenson</td>
			<td align="right" style="width:211px;">301025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Fly Aspirator</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Assembled from P1000 pipette tips, flexible plastic tubing, and mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Modeling Clay</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Forceps</td>
			<td style="width:187px;">Fine Science Tools By Dumont</td>
			<td style="width:211px;">11252-00</td>
			<td>#5SF (super-fine tips)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">10ml Syringe&#160;</td>
			<td style="width:187px;">Beckton-Dickinson</td>
			<td align="right" style="width:211px;">301029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Plastic Tubing</td>
			<td style="width:187px;">Tygon</td>
			<td style="width:211px;">R-3603</td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Electrophysiological Recording From Drosophila Labellar Taste Sensilla at JoVE.com

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

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

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 ...

electrophysiological-recording-from-drosophila-labellar-taste-sensilla

Research

JoVE Journal

Neuroscience

16.1K Görüntüleme.  Yale University.  Bu protokol Drosophila labellar tat nöronlar tarafından ateş aksiyon potansiyel yanıtları hücre dışı kayıt açıklar. 