We thank Zina Berman for support, Lisa Baik for comments on the manuscript, and other members of the Carlson laboratory for discussion. This work was supported NIH grant K01 DC020145 to H.K.M.D; and NIH grants R01 DC02174, R01 DC04729, and R01 DC011697 to J.R.C.

The following protocol complies with all the animal care guidelines of Yale University.
1. Flies
<ol>
	<li>Place 10-15 newly emerged flies in fresh standard culture vials at 25 &#176;C and 60% relative humidity in a 12:12 h light-dark cycle.</li>
	<li>Use flies when 3-7 days old.</li>
</ol>
2. Chemosensory stimuli
<ol>
	<li>Obtain chemosensory stimuli of the highest available purity. Store them as recommended by the vendor until use.</li>
	<li>Dissolve chemosensory stimuli and dilute to the desired concentrations in water or another desired, non-toxic solvent such as paraffin oil. Stir the prepared solutions for a minimum of 1 h in the case of dissolved solid compounds.</li>
</ol>
3. Glass stimulus capillary
<ol>
	<li>Pull a glass capillary to hold the stimulus from a borosilicate glass capillary (100 mm length, 1 mm outer diameter, 0.58 mm inner diameter) using a pipette puller instrument. Aim to achieve a tip diameter between 3 &#181;m and 10 &#181;m.</li>
	<li>Fill the glass capillary with the preferred stimulus solution using a microloader pipette tip. Take care to avoid bubbles, which can be removed by gentle tapping.</li>
	<li>If the stimulus crystallizes at the tip, clean or replace the glass stimulus capillary.</li>
</ol>
4. Reference and recording electrodes
<ol>
	<li>Use tungsten rods (127 &#181;m diameter and 76.2 mm length) for both reference and recording electrodes. Sharpen the reference and recording electrodes to approximately 1 &#181;m diameter at the tip (the shapes of these electrodes are depicted in Delventhal et al.36) by dipping them repeatedly for several seconds in either a 10% KNO3 (~1 M) solution or a 10% KOH (1.8 M) solution. 
	NOTE: This solution requires current (0.3-3 mA) to facilitate this process.</li>
</ol>
5. Preparation of the fly for base recording
<ol>
	<li>Draw a single fly from the vial into an aspirator. Withdraw the aspirator and trap the animal by placing a finger over the end.</li>
	<li>Expel the fly into a 200 &#181;L plastic pipette tip. Keeping the end of the aspirator in the pipette tip, use the end to push the fly forward, headfirst, toward the narrow end of the pipette tip.</li>
	<li>Trim the pipette tip at each end (i.e., anterior and posterior to the animal) using a razor blade.</li>
	<li>Use either clay or a small piece of cotton to push the fly forward further, until half of the head protrudes from the end of the trimmed pipette tip. Use forceps to gently push until the labellum at the front of the head is exposed.</li>
	<li>Fix the trimmed pipette tip onto a glass microscope slide using clay (Figure 1).</li>
	<li>Under the stereomicroscope, position the labellum laterally on a cover slip so that one lobe, along with its 31 taste sensilla, is exposed (Figure 1). The cover slip keeps the labellum in place.</li>
</ol>
6. Electrophysiology rig
<ol>
	<li>Select a room for the rig setup that has stable temperature and relative humidity (&#60;70%) and is isolated from sources of electrical and mechanical noise, such as refrigerators and centrifuges.</li>
	<li>Place the microscope on the center of an antivibration table.</li>
	<li>Secure a manual micromanipulator to the antivibration table (Figure 2).</li>
	<li>Attach a stainless-steel shaft that holds the tungsten reference electrode to the manual micromanipulator (Figure 2).</li>
	<li>Connect motorized manipulators-one with a holder for the recording electrode probe and a second one with a holder connected to a stainless-steel shaft for the glass stimulus capillary-to the same table using stands (Figure 2).</li>
	<li>Connect the recording electrode probe to an Intelligent Data Acquisition Controller (IDAC) system or another amplifier/digitizer system.</li>
	<li>Link this IDAC system to the computer at the workstation.</li>
	<li>Ground the manual and motorized manipulators to the same location within the rig.</li>
	<li>Install appropriate acquisition software for the IDAC system on the computer. Ensure that the digital acquisition drivers are compatible with the operating system (e.g., Windows XP-7, -8, or -10) on the computer.</li>
</ol>
7. Recording from taste sensilla
<ol>
	<li>Place the preparation slide on the microscope stage with a low magnification (e.g.,&#160;10x) objective in position. Move the stage until the labellum is in focus at the center of the field of view at both low-magnification and high-magnification (e.g., 50x) objectives.</li>
	<li>Insert the reference electrode into the eye using the low magnification objective. To insert the reference electrode, target the eye on the side of the fly opposite the side with the recording electrode, for example, if the recording electrode is approaching from the right, place the reference electrode in the left eye. Utilize a manual micromanipulator for precise insertion.</li>
	<li>Bring the tip of the glass stimulus capillary into focus at the center of the field of view of both low-magnification and high-magnification objectives using a motorized micromanipulator (Figure 3).</li>
	<li>Under low magnification, bring the recording electrode close to the labellum using a second motorized micromanipulator.</li>
	<li>Under high magnification, insert the recording electrode into the base of a taste sensillum using the motorized micromanipulator until the sound of neuronal firing activity from the audio output of the IDAC system is heard.</li>
	<li>Once a stable signal has been established, start recording the signal using the software that comes with the IDAC system (Figure 4A-D). To start recording, press the Start recording button.</li>
	<li>Bring the tip of the stimulus glass capillary to cover the tip of the taste sensillum using the motorized manipulator.</li>
	<li>To end the stimulus, remove the glass stimulus capillary from the sensillum using the motorized manipulator.</li>
	<li>Mark the start and end of the stimulation manually using a pedal. The pedal is connected to the IDAC, and its communication with the software is facilitated through the IDAC to mark the start/end of the stimulus.</li>
</ol>
8. Analysis
<ol>
	<li>Use the different functions of the software that comes with the IDAC system to sort spike populations by amplitude (when possible) and analyze response dynamics.
	<ol>
		<li>To count spikes, left click on the recording of interest, bringing up a window to select from. Choose To Spikes, initiating another window named Convert waves to spikes. Enter a name in the New field and press the OK button.</li>
		<li>Entering the name in the New field in step 8.1.1 leads to the Amplitude Histogram view. Choose the amplitude to count, then close this view. Left click to add a counter.</li>
		<li>Examine the spikes manually to confirm conclusions based on analysis with software. 
		NOTE: The software also allows the exportation of data in different formats for further analysis.</li>
	</ol>
	</li>
</ol>

A Novel Method for Enhanced Analysis of Insect Taste Neuron Activity

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The authors have no conflicts of interest to disclose.

In recordings from some types of sensilla, it can be challenging to differentiate the spikes of different neurons. For example, the sugar neurons and mechanosensory neurons of S and I sensilla produce spikes of similar amplitudes, making it difficult to distinguish them4,14. We find that the use of a very sharp tungsten recording electrode reduces the firing of the mechanosensory neuron, as does the judicious placement of the recording electrode. Insertion of the...

Insects, including drosophilid flies, are endowed with a sophisticated taste system that enables them to extract complex chemical information from their surroundings. This system allows them to discern the chemical composition of various substances, distinguishing between those that are nutritious and those that are harmful1,2.
At the core of this system are specialized structures known as taste hairs or sensilla, strategically located on various body parts. In drosophilid flies, these sensilla are located on the labellum, which is the major taste organ of the fly head1,2,3,4, as well as on the legs and wings1,2,5,6. The labellum is located at the tip of the proboscis and contains two lobes4,7,8. Each lobe is covered with 31 taste sensilla categorized as short, long, and intermediate4,7,8. These sensilla each house 2-4 taste neurons1,2,9,10. These taste neurons express members of at least four different gene families, namely the Gustatory receptor (Gr), Ionotropic receptor (Ir), Pickpocket (Ppk), and Transient receptor potential (Trp) genes1,2,11,12,13. This diversity of receptors and channels equips insects with the ability to recognize a wide array of chemical compounds, including both nonvolatile and volatile cues1,2,14.
For over 50 years, scientists have quantified the response of taste neurons and their receptors using a technique called tip recording3,4,6,8,13,15,16,17,18,19,20,21,22,23,24,25,26,27,28, 
29,30,31,32,33,34,35. However, this method has major limitations. First, neural activity can be measured only during contact with the stimulus, and not before or after contact. This limitation precludes the measurement of spontaneous spiking activity and prevents the measurement of OFF responses. Second, only tastants that are soluble in aqueous solutions can be tested.
These limitations can be overcome by a rarely used alternative electrophysiological technique called &#34;base recording.&#34; Here we describe this technique, which we have adapted from a method used by Marion-Poll and colleagues24, and show the crucial taste coding features that it can now conveniently measure14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>equipped with a 50X objective (LMPLFLN 50X, Olympus) and 10X eyepieces.&#160;</td>
		</tr>
		<tr>
			<td>Antivibration Table</td>
			<td>TMC</td>
			<td>63-7590E</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized Micromanipulators</td>
			<td>Harvard Apparatus and M&#228;rzh&#228;user Micromanipulators</td>
			<td>Micromanipulator PM 10 Piezo Micromanipulator</td>
			<td></td>
		</tr>
		<tr>
			<td>manual Micromanipulators</td>
			<td>M&#228;rzh&#228;user Micromanipulators</td>
			<td>MM33 Micromanipulator</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stands</td>
			<td>ENCO</td>
			<td>Model #625-0930</td>
			<td></td>
		</tr>
		<tr>
			<td>Reference&#160; and recording Electrode Holder</td>
			<td>Ockenfels Syntech GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulus glass capillary Holder</td>
			<td>Ockenfels Syntech GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Single Ended Probe</td>
			<td>Ockenfels Syntech GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-CHANNEL USB ACQUISITION CONTROLLER , IDAC-4</td>
			<td>Ockenfels Syntech GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulus Controllers</td>
			<td>Ockenfels Syntech GmbH</td>
			<td>Stimulus Controller CS 55</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>Tungsten Rod</td>
			<td>A-M Systems</td>
			<td>Cat#716000</td>
			<td></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-97 Flaming/Brown Micropipette Puller</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>VMZ 1x-4x</td>
			<td>For fly preparation</td>
		</tr>
		<tr>
			<td>p200 Pipette Tips</td>
			<td></td>
			<td></td>
			<td>Generic</td>
		</tr>
		<tr>
			<td>Microloader tips&#160;</td>
			<td>Eppendorf</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml Syringe</td>
			<td></td>
			<td></td>
			<td>Generic</td>
		</tr>
		<tr>
			<td>Crocodile clips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Power Transformers</td>
			<td>STACO ENERGY PRODUCTS</td>
			<td>STACO 3PN221B</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></td>
			<td></td>
			<td>Generic</td>
		</tr>
		<tr>
			<td>Plastic Tubing</td>
			<td>Saint Gobain</td>
			<td>Tygon S3&#8482; E-3603</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard culture vials</td>
			<td>Archon Scientific</td>
			<td></td>
			<td>Narrow 1-oz polystyrene vails, each with 10 mL of glucose medium, preloaded with cellulose acetate plugs</td>
		</tr>
		<tr>
			<td>Berberine chloride (BER)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# Y0001149</td>
			<td></td>
		</tr>
		<tr>
			<td>Denatonium benzoate (DEN)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# D5765</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diethyl-m- toluamide (DEET)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# 36542</td>
			<td></td>
		</tr>
	</tbody>
</table>

base recording: a technique for analyzing responses of taste neurons in drosophila

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential food source, compounds from the food source enter through the pore and activate neurons within. For over 50 years, these responses have been recorded using a technique called tip recording. However, this method has major limitations, including the inability to measure neural activity before or after stimulus contact and the requirement for tastants to be soluble in aqueous solutions. We describe here a technique that we call base recording, which overcomes these limitations. Base recording allows the measurement of taste neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It can be used to study hydrophobic compounds such as long-chain pheromones that have very low solubility in water. In summary, base recording offers the advantages of single-sensillum electrophysiology as a means of measuring neuronal activity - high spatial and temporal resolution, without the need for genetic tools - and overcomes key limitations of the traditional tip recording technique.

Author Spotlight: Advances in Chemoreception &amp;#8211; From Insect Odor Receptors to Non-Coding RNAs

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

Our lab investigates how animals detect and respond to odors, taste, and some pheromone. We've made significant strides in understanding the molecular basis of chemoreception. We've made several advances in the field, including the discovery of the first insect odor receptors, the discovery of the first insect taste receptors, and the elucidation of basic principles of the logic of odor and taste coding.This technique allows the measurement of test neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It also allows the measurement of taste neuron activity to the river of volatile compounds.The classical technique for taste electrophysiology was unable to measure taste responses to hydrophobic compounds such as long-chain pheromones that have very low solubility in water. However, with the help of this technique, we can overcome this limitation by separating the recording electrode from the glass stimulus capillary. Our research explores the functioning of the chemosensory systems in animals, focusing on their role in feeding and mating choices.We're currently examining non-coding RNAs and micro peptides in fruit flies'olfactory systems, and how mosquitoes and tsetse flies use these systems to find human hosts.

Figure 4A shows spontaneous spikes that arise from a sensillum. They fall into two classes based on amplitude, with the larger spikes deriving from the neuron that is sensitive to bitter compounds and the smaller spikes from the neuron that responds to sugars. The relationship between spike amplitude and functional specificity has been corroborated by genetic experiments4,14,37,<sup class...

Read this Scientific Article Protocol about Author Spotlight: Advances in Chemoreception at JoVE.com

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.

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential ...

base-recording-technique-for-analyzing-responses-taste-neurons

Research

JoVE Journal

Neuroscience

Preparing the Recording Electrodes and Drosophila for Analyzing Responses of Taste Neurons

Base Recording from Taste Sensilla of Drosophila for Analyzing Responses of Taste Neurons

730 Views.  Yale University.  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.   

A Novel Method for Enhanced Analysis of Insect Taste Neuron Activity

概要