Name: Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility (Video) | JoVE Journal
Uploaded: 2017-07-27T15:00:00.000+00:00
Duration: 7 min 49 s
Description: Learn single-sensillum recording in Drosophila for studying low-volatility pheromone detection using odor delivery cartridges. This method enables precise electrophysiological analysis of olfactory receptor neuron responses.

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5 minutes

We thank Ye Zhang for the help with the sample traces and Tin Ki Tsang for the help with the pictures. This work was supported by a Ray Thomas Edwards Foundation Early Career Award and an NIH grant (R01DC015519) to C.-Y.S. and NIH grants (R01DC009597 and R01DK092640) to J.W.W.

1. Preparation of the Hardware for at4 Recording
<ol>
	<li>Use a pipette puller instrument to prepare electrodes with aluminosilicate glass capillaries (O.D 1.0 mm, I.D. 0.64 mm). Blunt the tip of the reference electrode slightly with a pair of fine forceps to facilitate insertion into the clypeus of the fly (i.e.&#160;a rounded plate at the front of the fly head, above the mouthparts). 
	NOTE: 7-day-old WT males (Berlin) were used in this study. Use AHL saline solution14 as the electrolyte for both electrodes.</li>
	<li>Prepare 1 L of AHL by mixing 900 mL of distilled water with 6.312 g of NaCl, 0.373 g of KCl, 0.337 g of NaHCO3, 0.1120 g of NaH2PO4, 1.892 g of Trehalose &#1468;2H2O, 3.423 g of sucrose, 1.192 g of HEPES, and 8.2 mL of 1M MgCl2. Using distilled water, bring the total volume up to 1 L. Bring the pH to 7.4 using 1 N NaOH and sterilize the solution with a vacuum-driven filter system. For long-term storage, keep the AHL aliquots at 4 &#176;C. 
	NOTE: Consistent deliveries of palmitoleic acid are contingent upon uniformity between cartridges. It is critical that each cartridge is assembled in a reproducible manner.</li>
	<li>Using a razor blade, remove 0.9 cm from the tip of a 200 &#181;L pipette tip to create the first cartridge section, measuring 4.1 cm; refer to the dimensions detailed in Figure 1A. Use another 200 &#181;L pipette tip and remove 1.7 cm and 1.5 cm from the tip and the base, respectively, to create the second cartridge section, measuring 1.8 cm (Figure 1A). Use a ruler to ensure reproducibility.</li>
	<li>Use a &#8539;&#34; hole puncher to cut discs from filter paper.</li>
	<li>Use forceps to place a filter paper disc at the tip of the second cartridge section. Visually confirm that there is an opening in the cartridge tip through which air can pass.</li>
	<li>Attach the first and second cartridge sections together, as shown in Figure 1A. Angle the second cartridge section downward to facilitate square aiming at the prep (Figure 1B).</li>
	<li>Connect the cartridge with the odor-delivery tube, which is mounted on a micromanipulator. 
	NOTE: This design allows the cartridge to be swiveled outward to facilitate exchange (Figure 1C).</li>
	<li>Set the constant humidified airflow to 2 L/min in one mass controller and the odorant flow to 500 mL/min in another mass controller.</li>
	<li>Using the software (see the Table of Materials), program the procedure to administer a 500 ms odor puff.</li>
</ol>
2. Preparation of Palmitoleic Acid Odorant Solutions for Delivery
NOTE: Or47b ORNs respond to both cis- and trans-palmitoleic acid. As palmitoleic acid is unstable at RT, stocks are stored at -20 &#176;C and used within a month upon opening. Ethanol is the solvent of choice for palmitoleic acid.
<ol>
	<li>Use a vortex mixer to thoroughly mix 10 &#181;L of cis- or trans-palmitoleic acid stocks or dilutions with 90 &#181;L of 100% ethanol for ten-fold serial dilutions in 1.7 mL microtubes. Prepare fresh palmitoleic acid dilutions daily prior to experiments and use within a day. 
	NOTE: For odorants that are not soluble in ethanol, a glass vial is recommended for preparing odor dilutions with other types of organic solvents.</li>
	<li>Using a P10 micropipette, apply 5 &#181;L of cis-palmitoleic acid solutions of the desired dilutions to the filter paper in each corresponding cartridge. 
	NOTE: The highest dosage (10-1) contains 450 &#181;g of the compound. For trans-palmitoleic acid solutions, apply 4.5 &#181;L instead so that the highest dosage (10-1) also contains 450 &#181;g of the compound.</li>
	<li>To completely evaporate the solvent, place the palmitoleic acid cartridges in a vacuum desiccator for 1 h at RT and 7.59 mmHg of pressure. 
	NOTE: The cartridges can be used for up to 4 h at RT.</li>
</ol>
3. Preparation of Drosophila for Ready Access to the at4 Sensilla for In Vivo Electrophysiological Recordings
NOTE: WT flies (Berlin) are reared in standard cornmeal medium at 25&#176;C in a 12:12 light-dark cycle. Upon eclosion, flies are separated by sex into groups of ten, whereby they are group-housed until 7 d&#160;of age. Or47b ORNs in both male and female flies respond to palmitoleic acid. For simplicity, only male flies are examined in the current study.
<ol>
	<li>Assemble a fly-prep slide: on a glass slide, place a glass coverslip (18 x 18 mm2) on a small amount of modeling clay, forming an ~3&#176; angle with the glass slide. Place double-sided tape on the inner edge of the coverslip and on the area of the slide immediately below. Replace with fresh tape for every day of recording (Figure 2A).</li>
	<li>Use a fly aspirator15 to collect the fly of interest in the tubing and then fit a 200-&#181;L pipette tip over the end of the tubing. Simultaneously flick the tube forward while blowing air into the tube to push the fly to the end of the pipette tip. Use a razor blade to cut just below the body of the fly and 2 heads&#39; lengths above the fly.</li>
	<li>Tamp the bottom of the pipette tip with modeling clay, pushing the fly upward until both the antennae and the clypeus are exposed (Figure 2B). To avoid killing the fly, add only enough clay to expose the antennae and aristae, as this prevents the fly&#39;s abdomen from being crushed. Furthermore, add clay slowly and gently to prevent any sudden constriction. Confirm that the fly is alive by checking for antennal or proboscis movement.</li>
	<li>Use forceps to maneuver the pipette tip that houses the fly. Orient the head so that the clypeus is facing to the right of the observer. Adjust the prep along the coverslip using fine forceps until the lateral side of the antenna lies against the taped coverslip surface (Figure 2B).</li>
	<li>Place a holding rod on the arista to secure the antenna to the double-sided tape to prevent movement (Figure 2B). 
	NOTE: The holding rod is pulled from a borosilicate glass capillary with a pipette puller and held in position with modeling clay (Figure 2A).</li>
	<li>Place the prep on the stage of the rig (Figure 2C). Using the microscope, confirm that the trichoids are visible along the distal-lateral edge of the third segment of the antenna. 
	NOTE: Ideally, the sensilla should be clearly silhouetted against the background, which simplifies their identification and facilitates recording (Figure 3). In this preparation, the majority of the accessible trichoid sensilla are of the at4 type.</li>
	<li>Keep the prep under constant humidified airflow (2 L/min) delivered via a separate air delivery tube from a distance of about 2 cm from the prep (Figure 4), as described previously2,15.</li>
</ol>
4. Recording of at4 Sensillum Activity from Or47b ORNs in the at4 Trichoids in Response to Palmitoleic Acid
<ol>
	<li>Insert the reference electrode into the clypeus (Figure 3A). To avoid tissue damage, ensure that the electrode is inserted just below the surface, where it can contact the hemolymph under the cuticle, with a swift and smooth motion.</li>
	<li>Lower the recording electrode slowlyuntil it enters the same plane of view as the target sensillum (Figure 3B). Record under a 50X objective lens. 
	NOTE: The tough trichoidal cuticle necessitates inserting the recording electrode into the sensillar base, whose wider area provides a larger target that reduces the likelihood that the electrode is deflected away (Figure 3B, inset).</li>
	<li>Before applying odor stimuli to a sensillum, observe the following selection criteria; any trichoid failing to meet these standards should be rejected and another sensillum chosen instead.
	<ol>
		<li>Observe a high signal-to-noise ratio (see Figure 3C for an example).</li>
		<li>Observe identifiable spikes from at4A and at4C neurons (Figure 3C). 
		NOTE: Of note, the at4B spike amplitude appears very similar to at4A10 and cannot be identified easily without odor stimulation.</li>
		<li>Observe that the basal firing rate of the at4A neurons is around or under 20 Hz. 
		NOTE: This criterion is specific for at4A because the basal firing rate for the neuron is higher than that of the basiconic ORNs2. A much higher basal firing indicates that the neurons may have been damaged during electrode insertion.</li>
	</ol>
	</li>
	<li>Connect the cartridge to the odorant-delivery tube. Start with the solvent control and then the odorants, from low to high concentrations. Use the micromanipulator to maneuver the cartridge towards the prep while aiming the cartridge squarely at the head of the prep. Visually confirm that the cartridge is pointed directly at the antenna (Figure 4) from a few millimeters away. 
	NOTE: The goal is to orient the opening of the cartridge directly at the antenna and position it in close proximity to the target tissue.</li>
	<li>Ensure that the odorant cartridge is separated from the recording electrode on its right by 1 - 2 mm and from the fly prep slide below by approximately 1 mm. 
	NOTE: In the setup described here, the odorant cartridge is closely bordered by the recording electrode, the reference electrode, and the fly-prep slide (Figure 4). 
	NOTE: Pay attention to the distance between the cartridge and the recording/reference electrodes. A distance of around 4 mm is recommended1. Unintentional contact may terminate the signal and break the tip of the recording electrode, damaging the current neuron and complicating further recordings. 
	NOTE: Consider the distance separating the cartridge and the fly-prep slide. Touching the coverslip may also dislodge the recording electrode to disrupt the recording.</li>
	<li>Press &#34;Record&#34; in the data acquisition software to begin the recording. 
	NOTE: For each 10 s recording, a single 500 ms odor pulse is delivered directly to the antenna, as described in step 1.9.</li>
	<li>After odorant application, carefully retract the cartridge before replacing it with a cartridge of the next-highest concentration. Continue until the entire dosage range is obtained. 
	NOTE: It is recommended that only one Or47b ORN is recorded from each fly to avoid any possible effects of adaptation.</li>
	<li>Thoroughly rinse the recording electrode with distilled water after finishing recording for the day.</li>
	<li>Analyze and plot the data using commercially available offline analysis software.</li>
</ol>

<ol><li>Lin, H. -H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hormonal+modulation+of+pheromone+detection+enhances+male+courtship+success%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Hormonal modulation of pheromone detection enhances male courtship success.</a> Neuron. 90 (6), 1272-1285 (2016).</li>
<li>Hallem, E. A., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coding+of+odors+by+a+receptor+repertoire%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Coding of odors by a receptor repertoire.</a> Cell. 125 (1), 143-160 (2006).</li>
<li>Silbering, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complementary+function+and+integrated+wiring+of+the+evolutionarily+distinct+Drosophila+olfactory+subsystems%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems.</a> J Neurosci. 31 (38), 13357-13375 (2011).</li>
<li>Min, S., Ai, M., Shin, S. A., Suh, G. S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dedicated+olfactory+neurons+mediating+attraction+behavior+to+ammonia+and+amines+in+Drosophila%5BTitle%5D)+AND+%22Proc+Nat+Acad+Sci+USA%22%5BJournal%5D)">Dedicated olfactory neurons mediating attraction behavior to ammonia and amines in Drosophila.</a> Proc Nat Acad Sci USA. 110, 1321-1329 (2013).</li>
<li>Kurtovic, A., Widmer, A., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+class+of+olfactory+neurons+mediates+behavioural+responses+to+a+Drosophila+sex+pheromone%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone.</a> Nature. 446 (7135), 542-546 (2007).</li>
<li>Van der Goes van Naters, W., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Receptors+and+neurons+for+fly+odors+in+Drosophila%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Receptors and neurons for fly odors in Drosophila.</a> Curr Biol. 17, 606-612 (2007).</li>
<li>Schlief, M. L., Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Olfactory+processing+and+behavior+downstream+from+highly+selective+receptor+neurons%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Olfactory processing and behavior downstream from highly selective receptor neurons.</a> Nat Neurosci. 10 (5), 623-630 (2007).</li>
<li>Laughlin, J. D., Ha, T. S., Jones, D. N. M., Smith, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+Pheromone-sensitive+neurons+is+mediated+by+conformational+activation+of+pheromone-binding+protein%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Activation of Pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein.</a> Cell. 133 (7), 1255-1265 (2008).</li>
<li>Gomez-Diaz, C., Reina, J. H., Cambillau, C., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ligands+for+pheromone-sensing+neurons+are+not+conformationally+activated+odorant+binding+proteins%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">Ligands for pheromone-sensing neurons are not conformationally activated odorant binding proteins.</a> PLoS Biol. 11 (4), e1001546(2013).</li>
<li>Dweck, H. K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pheromones+mediating+copulation+and+attraction+in+Drosophila%5BTitle%5D)+AND+%22Proc+Nat+Acad+USA%22%5BJournal%5D)">Pheromones mediating copulation and attraction in Drosophila.</a> Proc Nat Acad USA. 112, 2829-2835 (2015).</li>
<li>Cappa, C. D., Lovejoy, E. R., Ravishankara, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaporation+rates+and+vapor+pressures+of+the+even-numbered+C8-C18monocarboxylic+acids%5BTitle%5D)+AND+%22J+Phys+Chem+A%22%5BJournal%5D)">Evaporation rates and vapor pressures of the even-numbered C8-C18monocarboxylic acids.</a> J Phys Chem A. 112 (17), 3959-3964 (2008).</li>
<li>Kimura, K. -I., Sato, C., Yamamoto, K., Yamamoto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+the+back+or+front%3A+the+courtship+position+is+a+matter+of+smell+and+sight+in+Drosophila+melanogaster+males%5BTitle%5D)+AND+%22J+Neurogenet%22%5BJournal%5D)">From the back or front: the courtship position is a matter of smell and sight in Drosophila melanogaster males.</a> J Neurogenet. 29 (1), 18-22 (2015).</li>
<li>Grosjean, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+olfactory+receptor+for+food-derived+odours+promotes+male+courtship+in+Drosophila%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">An olfactory receptor for food-derived odours promotes male courtship in Drosophila.</a> Nature. 478 (7368), 236-240 (2011).</li>
<li>Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-photon+calcium+imaging+reveals+an+odor-evoked+map+of+activity+in+the+fly+brain%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.</a> Cell. 112 (2), 271-282 (2003).</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. (36), e1-e5 (2010).</li>
</ol>

The authors have nothing to disclose.

Here, we described a procedure by which the responses of Or47b ORNs to palmitoleic acid can be robustly induced and recorded. We modified a conventional long-distance odor delivery method2,7,10 to troubleshoot the problem of insufficient pheromone odorant delivery. We addressed the issue of low odorant volatility by delivering the compound via odorant cartridges, the opening of which are positioned within millimeters of the prep...

Drosophila ORNs respond to a vast number of odorants, with widely ranging carbon chain lengths and a variety of functional groups, including esters, alcohols, ketones, lactones, aldehydes, terpenes, organic acids, amines, sulfur compounds, heterocyclics, and aromatics2,3. Odorants varied in their physicochemical features can have markedly different volatilities, indicated by the vapor pressure of the compound. Notably, biologically relevant odorants for Drosophila melanogaster differ tremendously in their volatility. For example, Ir92a ORNs respond to ammonia4, which is highly volatile, with a vapor pressure of 6,432 mmHg at 20 &#176;C. In contrast, Or67d ORNs respond to a male pheromone, cis-vaccenyl acetate (cVA)5,6, the vapor pressure of which is 43 mmHg at 20 &#176;C.
Studying the olfactory response to odorants of low volatility is particularly challenging with conventional odor-delivery methods, in which odorants are delivered via a carrier air stream over a relatively long distance (i.e.&#160;several centimeters). As such, the reported olfactory responses to a given low-volatility odorant can vary greatly, depending on the design of the odor-delivery system. For example, the reported response of Or67d ORNs to a high dose of cVA ranges from ~407&#160;- &#62;200 spikes/s6. Moreover, the ineffective delivery of cVA with conventional delivery methods is likely attributed to false-negative results, leading to the interpretation that cVA by itself is not sufficient to activate Or67d ORNs8. This interpretation was later challenged by another study using a close-range odor-delivery method9. It is therefore imperative to develop a robust odor-delivery system for the effective presentation of odorants of low volatility.
Recently, we identified several long-chain cuticular fatty acids as ligands for Or47b ORNs. They are housed in the type 4 Antennal Trichoid Sensillum (at4). Among the long-chain fatty acid odorants, we found that palmitoleic acid functions as an aphrodisiac pheromone that promotes male courtship by activating Or47b ORNs1. However, in another study using a conventional odor-delivery method, methyl laurate was shown to elicit responses from Or47b ORNs, while palmitoleic acid evoked no response when presented from the same distance10. Compared to cVA, long-chain fatty acids are even less volatile, with vapor pressures less than 0.001 mmHg at 25 &#176;C11. The inherently low volatility of long-chain fatty acid odorants, which precludes efficient presentation to the antenna via conventional odor-delivery systems, likely accounted for the false-negative results10. This inconsistency highlights the inadequacy of conventional odor-delivery systems in presenting low-volatility odorants. It was previously shown that the effective delivery of fly cuticular odors requires close proximity between the odor source and the target tissue6. Thus, to fully characterize the effects of biologically active pheromones while mimicking the distance from which they are likely encountered by fruit flies in nature12,13, we agreed that minimal distance must be accorded high priority in our procedure.
Our method holds further advantages, including compatibility with standard electrophysiology rigs and techniques. Pre-existing rig setups require minimal modification to accommodate this protocol, and most SSR steps require only minor adjustments. This renders our technique readily accessible to researchers experienced in SSR. Furthermore, our technique allows for the presentation of low-volatility odorants with sharp onset and offset, correlating stimulus delivery with neuronal response. Finally, the hardware layout facilitates rapid exchanges between odorant cartridges, expediting data collection over a desired dosage range.
We begin by reviewing the preparation of reference and recording electrodes, Adult Hemolymph-Like (AHL) solution, odorant delivery cartridges, and the corresponding olfactometer. We then discuss the preparation of the palmitoleic acid odorant solutions, followed by the preparation of the fly for recording. We proceed to consider the criteria for selecting a trichoid sensillum to record and more closely examine the positioning of the odorant cartridge before presenting representative data acquired using this method. Finally, we conclude by exploring useful applications of this technique, some encountered issues, and their solutions.

<table><tbody><tr><td>&lt;strong&gt;Prep Setup &amp;amp; Miscellaneous Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pipette Puller Instrument&amp;nbsp;</td><td>Sutter Instruments&lt;br/&gt; Novato CA USA</td><td>P97</td><td>Pipette Puller</td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instruments&lt;br/&gt; Sarasota FL USA</td><td>1B100F-4</td><td>to make holding rods</td></tr><tr><td>Aluminosilicate Glass Capillaries&amp;nbsp;</td><td>Sutter Instruments&lt;br/&gt; Novato CA USA</td><td>AF100-64-10</td><td>to make electrodes</td></tr><tr><td>Superfrost Microscope Slides</td><td>Fisher Scientific&lt;br/&gt; Pittsburgh PA USA</td><td>12-550-143</td><td>for fly-prep station</td></tr><tr><td>Permanent Double Sided Tape</td><td>Scotch&lt;br/&gt; St. Paul MN USA</td><td>NA</td><td>for fly-prep station</td></tr><tr><td>Upright microscope</td><td>Olympus&lt;br/&gt; Shinjuku Tokyo Japan</td><td>BX51</td><td>for recording rig</td></tr><tr><td>Plastalina modeling clay</td><td>Van Aken&lt;br/&gt; North Charleston SC USA</td><td>B0019QZMQQ</td><td>for prep station and to stablize the holding rod</td></tr><tr><td>Rapid-Flow Sterile Disposable Filter Unit with SFCA Membrane, 0.45 mm</td><td>Nalgene&lt;br/&gt; Rochester NY USA</td><td>#156-4045</td><td>to sterilize AHL solution</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cartridge Materials&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>200 &amp;micro;L pipette tip&amp;nbsp;</td><td>VWR&lt;br/&gt; Radnor PA USA</td><td>53508-810</td><td>to make odor cartridges and fly prep</td></tr><tr><td>Filter Paper</td><td>Whatman&lt;br/&gt; Maidstone Kent UK</td><td>740-E</td><td>to make odor cartridges&amp;nbsp;</td></tr><tr><td>Vacuum Desiccator&amp;nbsp;</td><td>Cole-Parmer&lt;br/&gt; Vernon Hills IL USA</td><td>VX-06514-30</td><td>to vaporize ethanol solvent</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Odorant Materials&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>cis-palmitoleic acid</td><td>Cayman Chemical&lt;br/&gt; Ann Arbor MI USA</td><td>#10009871 (CAS # 373-49-9)</td><td>Or47b odorant</td></tr><tr><td>trans-palmitoleic acid</td><td>Cayman Chemical&lt;br/&gt; Ann Arbor MI USA</td><td>#9001798 (CAS # 10030-73-6)</td><td>Or47b odorant</td></tr><tr><td>Ethanol</td><td>Spectrum Chemical MFG.&amp;nbsp;&lt;br/&gt; New Brunswick NJ USA</td><td>E1028-500MLGL</td><td>to dilute palmitoleic acid&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Rig Setup Materials&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>Odorant Cartridge Micromanipulator</td><td>Siskiyou&lt;br/&gt; Grants Pass OR USA</td><td>MX130R</td><td>to position the olfactometer</td></tr><tr><td>Flow Vision software&amp;nbsp;</td><td>Alicat&lt;br/&gt; Tuscon AZ USA</td><td>FLOWVISIONSC</td><td>software to control flow rate</td></tr><tr><td>Mass Controller</td><td>Alicat&lt;br/&gt; Tuscon AZ USA</td><td>MC-2SLPM-D</td><td>to control the flow rate for humidified air</td></tr><tr><td>Mass Controller</td><td>Alicat&lt;br/&gt; Tuscon AZ USA</td><td>MC-500SCCM-D</td><td>to control the flow rate for odor stimulation</td></tr><tr><td>Clampex</td><td>Molecular Devices&lt;br/&gt; Sunnyvale CA USA</td><td>Ver. 10.4</td><td>Data acquisition software</td></tr><tr><td>Air delivery tube</td><td>Ace Glass&lt;br/&gt; Vineland NJ USA</td><td>8802-936&amp;nbsp;</td><td>to deliver humidified air</td></tr><tr><td>50X objective lens&amp;nbsp;</td><td>Olympus&lt;br/&gt; Shinjuku Tokyo Japan</td><td>LMPLFL50X</td><td>recording rig</td></tr><tr><td>Clampfit 10</td><td>Molecular Devices&lt;br/&gt; Sunnyvale CA USA</td><td>Ver. 10.4</td><td>software for spike analysis&amp;nbsp;</td></tr><tr><td>Igor Pro 6</td><td>WaveMetrics&lt;br/&gt; Lake Oswego OR USA</td><td>Ver. 6.37</td><td>software for data analysis&amp;nbsp;</td></tr><tr><td>Audio Monitor</td><td>ALA Scientific Instruments&lt;br/&gt; Farmingdale NY USA</td><td>NPIEXB-AUDIS-08B</td><td>Aurally reports individual spikes</td></tr><tr><td>Extracellular Amplifier</td><td>ALA Scientific Instruments&lt;br/&gt; Farmingdale NY USA</td><td>NPIEXT-02F</td><td>to increase the amplitude of electrical signals</td></tr><tr><td>Valve Controller</td><td>Warner Instruments&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>VC-8</td><td>to control the opening of the valve for odor stimulation</td></tr><tr><td>Recording Electrode Micromanipulator</td><td>Sutter Instruments&lt;br/&gt; Novato CA USA</td><td>MP-285</td><td>to position recording electrode</td></tr><tr><td>Headstage Amplifier</td><td>ALA Scientific Instruments&lt;br/&gt; Farmingdale NY USA</td><td>EQ-16.0008</td><td>to increase the amplitude of electrical signals</td></tr><tr><td>Oscilloscope</td><td>Tektronix&lt;br/&gt; Beaverton OR USA</td><td>TDS2000C</td><td>Visual report of individual spikes</td></tr></tbody></table>

electrophysiological recording from drosophila trichoid sensilla in response to odorants of low volatility

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Our technique was successfully applied to determine the relative efficacy of the trans (Figure 5A) versus cis (Figure 5B) isomers of palmitoleic acid. Our representative data demonstrates that trans-palmitoleic acid is a more effective ligand for Or47b ORNs when compared to the cis isoform (Figure 5C). A single neuron was recorded from each fly, with twelve flies r...

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.

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

The overall goal of this technique is to present odorants of low volatility effectively to olfactory receptor neurons and drosophila for electrophysiological recording. Those neurons are housed in the trichoid sensilla and respond to long chain cuticular pheromones. This method can help answer key questions in the field of insect pheromone communication by providing an improved odor delivery technique.The main advantage of this technique is that it allows odorants which were previously inaccessible due to their low volatility to be effectively presented during in vivo recording. Specifically, pheromones are presented from close distances that simulate the range between courting males and target females. The preparation of the odor delivery cartridge component for AT4 recordings begins with removing 0.9 centimeters from the narrow end of a 200-microliter pipette tip.Then, from a second pipette tip, remove 1.7 centimeters from the narrow end, and 1.5 centimeters from the wide end to make a 1.8 centimeter length. Next, use forceps to place a 1/8 inch diameter disk of filter paper at the tip of the 1.8 centimeter section. Leave a small opening through which air can pass.Then secure the pipette sections together with the smaller section angled downward to aim at the preparation. Now use a P10 to apply the odorant at the desired dilution to the filter paper. Then, to evaporate the solvent, let the loaded cartridges sit in a vacuum desiccator for an hour.First, assemble a fly preparation slide. On a glass slide, attach a glass cover slip with modeling clay, to create an angle of about three degrees between the slide and cover slip. Then attach double-sided tape at two places, the inner edge of the cover slip and the slide immediately below the cover slip.Always use fresh tape. Now collect the fly of interest in an aspirator tube. Then fit a 200-microliter pipette tip over the end of the tube, and simultaneously flick the tube while blowing, to push the fly into the end of the pipette tip.Then use a razor blade to cut the tip just below the body of the fly. Now carefully load molding clay into the wide end of the tip to force the fly's head into the smaller opening. Once both antennae are exposed, stop packing in clay.Now use forceps to position the prep onto the slide. Position the clipeus to face horizontally with the lateral side of the antenna against the taped cover slip surface. Then place the holding rod on the arista to secure the antenna to the tape.Next, place the prep onto the stage of the rig. Confirm that the trichoids are visible along the distal lateral edge of the third segment of the antenna. The sensilla should be clearly silhouetted against the background.Keep the prep under a constant humidified air flow delivered via separate tube at a distance of about two centimeters. To take recordings, insert the reference electrode into the clipeus using a swift and smooth motion. Place it just below the cuticle where it will be in contact with the hemolymph.Next, slowly lower the recording electrode until it enters the same plane of view as the target sensillum. Then insert the recording electrode into the sensillar base. Before proceeding, ensure that there is a high signal-to-noise ratio, with identifiable spikes from AT4-A and AT4-C neurons.The AT4-A neurons should fire no faster than 20 hertz. Now connect the cartridge to the odorant delivery tube. Begin with the solvent control and progress towards the highest odorant concentration.In this case, a palmitoleic acid cartridge is attached. Next, use the micromanipulator to maneuver the cartridge towards the prep while aiming it to the head. Position the cartridge tip pointing directly at the antenna from a distance of about four millimeters.The cartridge must also be about one millimeter above the slide. Precise positioning of the cartridge with respect to the prep is the single most critical step of this procedure. Consistent neuronal responses are contingent on the reproducible position of the cartridge across trials.The cartridge might be closely bordered by the electrodes and the slide. It should ideally be at least four millimeters from either electrode. For stimulation, set the odorant flow controller to 500 millisecond bursts, and use a 500 milliliter per minute flow rate.For each stimulus burst, record the electrical activity for 10 seconds. After an odorant application, carefully retract the cartridge before replacing it with a cartridge containing the next highest concentration. After taking all the recordings for the day, thoroughly rinse the recording electrode with distilled water.Using the described methods on seven-day-old male Berlin wild-type flies, the relative efficacy of the trans and cis palmitoleic acid was compared. Trans-palmitoleic was a stronger stimulant of Or47b olfactory receptor neurons. From each fly, only a single neuron was recorded.The distance between the opening of the odor cartridge to the head of the fly had a significant influence on neuronal responses. At four millimeters there were many more spikes fired in response to palmitoleic acid. At a distance of 11 millimeters, there were hardly any observable responses by the O47b neurons.Close-range presentation of palmitoleic acid was quite important for studying its effects as an odorant. We hope that after watching this video you will have a clear understanding of how to present palmitoleic acid or other low-volatility odorants into your drosophila olfactory receptor neurons during in vivo single sensillum recording. Once this technique is mastered, recording an entire dosage curve from a single fly can be achieved in around 20 minutes.After its development, this technique paved the way for researchers in the field of insect olfaction to explore the neurophysiology and signal transduction mechanisms for pheromone detection.

electrophysiological-recording-from-drosophila-trichoid-sensilla

Research

JoVE Journal

Neuroscience

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Learn single-sensillum recording in Drosophila for studying low-volatility pheromone detection using odor delivery cartridges. This method enables precise electrophysiological analysis of olfactory receptor neuron responses.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

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