This work is supported by a grant from the National Natural Science Foundation of China (No.31472037). Any mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply a recommendation.

1. Preparation of Instruments and Insect
<ol>
	<li>Preparing tungsten electrodes and stimuli solutions
	<ol>
		<li>Fix a new tungsten wire (diameter of 0.125 mm, length of 75 mm) into a micromanipulator and sharpen it in a 10% (w/v) sodium nitrite (NaNO2) solution in a syringe at 10 V provided by a power supply for about 1 min under a stereomicroscope (40X magnification).</li>
		<li>Dip the sharpened tungsten wire repeatedly into the 10% NaNO2 solution, about 4 mm at 5 V in &#60; ...

<ol>
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R., 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=Cuticular+hydrocarbon+pheromones+for+social+behavior+and+their+coding+in+the+ant+antenna.">Cuticular hydrocarbon pheromones for social behavior and their coding in the ant antenna.</a> Cell Reports. 12 (8), 1261-1271 (2015).</li><li>de Bruyne, M., Clyne, P. J., 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=Odor+coding+in+a+model+olfactory+organ:+the+Drosophila+maxillary+palp.">Odor coding in a model olfactory organ: the Drosophila maxillary palp.</a> Journal of Neuroscience. 19 (11), 4520-4532 (1999).</li><li>Syed, Z., Leal, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maxillary+palps+are+broad+spectrum+odorant+detectors+in+Culex+quinquefasciatus.">Maxillary palps are broad spectrum odorant detectors in Culex quinquefasciatus.</a> Chemical Senses. 32 (8), 727-738 (2007).</li><li>Lu, T., 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=Odor+coding+in+the+maxillary+palp+of+the+malaria+vector+mosquito+Anopheles+gambiae.">Odor coding in the maxillary palp of the malaria vector mosquito Anopheles gambiae.</a> Current Biology. 17 (18), 1533-1544 (2007).</li><li>Pellegrino, M., Nakagawa, T., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+sensillum+recordings+in+the+insects+Drosophila+melanogaster+and+Anopheles+gambiae.">Single sensillum recordings in the insects Drosophila melanogaster and Anopheles gambiae.</a> Journal of Visualized Experiments. 36, e1725 (2010).</li><li>Grant, A. J., Wigton, B. E., Aghajanian, J. G., O'Connell, R. J. <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+receptor+neurons+in+mosquito+maxillary+palp+sensilla+to+carbon+dioxide.">Electrophysiological responses of receptor neurons in mosquito maxillary palp sensilla to carbon dioxide.</a> Journal of Comparative Physiology A. 177 (4), 389-396 (1995).</li><li>Blaney, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ultrastructure+of+an+olfactory+sensillum+on+the+maxillary+palps+of+Locusta+migratoria+(L.).">The ultrastructure of an olfactory sensillum on the maxillary palps of Locusta migratoria (L.).</a> Cell and Tissue Research. 184 (3), 397-409 (1977).</li><li>Hassanali, A., Njagi, P. G. N., Bashir, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+ecology+of+locusts+and+related+acridids.">Chemical ecology of locusts and related acridids.</a> Annual Review of Entomology. 50, 223-245 (2005).</li><li>Chapman, 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=Contact+chemoreception+in+feeding+by+phytophagous+insects.">Contact chemoreception in feeding by phytophagous insects.</a> Annual Review of Entomology. 48, 455-484 (2003).</li><li>Chapman, R. F., Sword, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+palpation+in+food+selection+by+a+polyphagous+grasshopper+(Orthoptera:+Acrididae).">The importance of palpation in food selection by a polyphagous grasshopper (Orthoptera: Acrididae).</a> Journal of Insect Behavior. 6, 79-91 (1993).</li><li>Winstanley, C., Blaney, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+mechanisms+of+locusts+in+relation+to+feeding.">Chemosensory mechanisms of locusts in relation to feeding.</a> Entomologia Experimentalis et Applicata. 24, 750-758 (1978).</li><li>Blaney, W., Duckett, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+palpation+by+the+maxillary+palps+of+Locusta+migratoria+(L.):+an+electrophysiological+and+behavioural+study.">The significance of palpation by the maxillary palps of Locusta migratoria (L.): an electrophysiological and behavioural study.</a> Journal of Experimental Biology. 63, 701-712 (1975).</li><li>Blaney, W. <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+the+terminal+sensilla+on+the+maxillary+palps+of+Locusta+migratoria+(L.)+to+some+electrolytes+and+non-electrolytes.">Electrophysiological responses of the terminal sensilla on the maxillary palps of Locusta migratoria (L.) to some electrolytes and non-electrolytes.</a> Journal of Experimental Biology. 60, 275-293 (1974).</li><li>Jin, X., Zhang, S., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+odorant-binding+and+chemosensory+proteins+and+spatial+map+of+chemosensilla+on+labial+palps+of+Locusta+migratoria+(Orthoptera:+Acrididae).">Expression of odorant-binding and chemosensory proteins and spatial map of chemosensilla on labial palps of Locusta migratoria (Orthoptera: Acrididae).</a> Anthropod Structure &amp; Development. 35 (1), 47-56 (2006).</li><li>Zhang, L., Li, H., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+olfactory+pathways+to+detect+aldehydes+on+locust+mouthpart.">Two olfactory pathways to detect aldehydes on locust mouthpart.</a> International Journal of Biological Sciences. 13 (6), 759-771 (2017).</li><li>Dweck, H. K. M., 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=Olfactory+channels+associated+with+the+Drosophila+maxillary+palp+mediate+short-+and+long-range+attraction.">Olfactory channels associated with the Drosophila maxillary palp mediate short- and long-range attraction.</a> eLife. 5, e14925 (2016).</li><li>Bohbot, J. D., Sparks, J. T., Dickens, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maxillary+palp+of+Aedes+aegypti,+a+model+of+multisensory+Integration.">The maxillary palp of Aedes aegypti, a model of multisensory Integration.</a> Insect Biochemistry and Molecular Biology. 48, 29-39 (2014).</li><li>Delventhal, R., Kiely, A., 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=Electrophysiological+recording+from+Drosophila+labellar+taste+sensilla.">Electrophysiological recording from Drosophila labellar taste sensilla.</a> Journal of Visualized Experiments. 84, e51355 (2014).</li><li>Ng, R., Lin, H. H., Wang, J. W., Su, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.</a> Journal of Visualized Experiments. 125, e56147 (2017).</li><li>Syed, Z., Leal, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+measurements+from+a+moth+olfactory+system.">Electrophysiological measurements from a moth olfactory system.</a> Journal of Visualized Experiments. 49, e2489 (2011).</li><li>Saha, D., Leong, K., Katta, N., Raman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-unit+recording+methods+to+characterize+neural+activity+in+the+locust+(Schistocerca+Americana)+olfactory+circuits.">Multi-unit recording methods to characterize neural activity in the locust (Schistocerca Americana) olfactory circuits.</a> Journal of Visualized Experiments. 71, e50139 (2013).</li><li>Liu, F., Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single+sensillum+recording+to+detect+olfactory+neuron+responses+of+bed+bugs+to+semiochemicals.">Using single sensillum recording to detect olfactory neuron responses of bed bugs to semiochemicals.</a> Journal of Visualized Experiments. 107, e53337 (2016).</li></ol>

Insects rely on palps to detect food odors, and their palps are believed to play an important role in speciation13,27. The palps are simple olfactory organs and are receiving increasing attention as an attractive model for the exploration of the neuromolecular networks underlying chemosensation28.
Insect labellar and palp SSRs have been successfully performed on Drosophila melanogaster, Anopheles ga...

Animals have evolved a range of chemosensory organs that sense exogenous chemical cues. In insects, the most important chemosensory organs are the antennae and the palps. On these organs, several types of chemosensory hairs, called chemosensory sensilla, are innervated by chemosensory neurons (CSNs) within the hairs. CSNs in chemosensory sensilla recognize specific chemical cues through signal transduction from chemical stimuli to electrical potentials that are subsequently transferred up to the central nervous systems1,2,3.
CSNs express various chemosensory receptors [e.g., odorant receptors (ORs)], ionotropic receptors (IRs), and gustatory receptors (GRs) on their membranes, which encode exogenous chemical cues associated with different types of chemosensation4,5,6. The characterization of CSNs is key to the elucidation of cellular and molecular mechanisms of insect chemoreception. Now single sensillum recording (SSR) is a widely-used technique for the characterization of insect CSNs in the antennal sensilla of many insects, including flies7, moths8, beetles9, aphids10, locusts11, and ants12. However, few studies have applied an SSR to insect palps13,14,15,16,17, because the particular structures of their sensilla make an electrophysiological recording difficult18.
Swarms of locusts (Orthoptera) often cause serious crop damage and economic loss19. The palps are believed to play an important role in the food selection of locusts20,21,22,23,24. Two types of chemosensory sensilla are investigated by a scanning electron microscope (SEM). Usually, 350 sensilla chaetica and 7 - 8 sensilla basiconica are observed on each dome of the locust palps18. Sensilla chaetica are gustatory sensilla that sense non-volatile chemical cues, whereas sensilla basiconica have an olfactory function, sensing volatile chemical cues.
On locust palps, the diameters of the hair sockets of the sensilla basiconica (ca. 12 &#181;m), are much greater than those of sensilla chaetica (ca. 8 &#181;m)18,25. The cuticular wall of the sensilla basiconica on the palps is much thicker than that of antennal sensilla18. In addition, the dome of the palp has fluid contents within a highly flexible cuticle. These characteristics mean that a penetration with a microelectrode and an acquisition of good electrophysiological signals is more difficult than for antennal sensilla. In this paper, a detailed and highly effective SSR protocol for locust palp sensilla basiconica is presented with a video.

<table border="0" cellpadding="0" cellspacing="0" style="width:1145px;" width="1145">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">Tungsten wire</td>
			<td style="width:140px;">ADVENT</td>
			<td style="width:217px;">W559504</td>
			<td style="width:485px;">Used for making the electrode and fixing the palp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaNO2</td>
			<td>Sigma-aldrich</td>
			<td>563218-25G</td>
			<td>Used for sharpening the tungsten wire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AC Power Supply</td>
			<td>Syntech</td>
			<td>A2-70</td>
			<td>Providing the voltage in sharpening the tungsten wire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stereoscope</td>
			<td>Motic</td>
			<td>SMZ-163</td>
			<td>Used for observing the sharpening of tungsten wire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope</td>
			<td>Olympus</td>
			<td>W-51</td>
			<td>Used for observing the sensilla on locust maxillary palp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Intelligent Data Acquisition Controller</td>
			<td>Syntech</td>
			<td>IDAC-4</td>
			<td>Real-time on screen display of all signals before and during recording</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stimulus controller</td>
			<td>Syntech</td>
			<td>CS-55</td>
			<td>Used for controlling the stimulus application</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electronic micromanipulator</td>
			<td>C.M.D.T</td>
			<td>CFT-8301D</td>
			<td>Used for minor movement of the recording electrode</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-151</td>
			<td>Used for minor movement of the reference electrode</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Speaker</td>
			<td>EDIFIER</td>
			<td>R101T06</td>
			<td>Connected with IDAC-4 and providing sound for the signal</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnetic base</td>
			<td>PDOK</td>
			<td>PD-101</td>
			<td>Used to hold the electrode, and stimulus delivery tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vibration Isolation Table</td>
			<td>TianHe</td>
			<td>HAP-100-1208</td>
			<td>Used for isolating the vibration from the equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass slide</td>
			<td>CITOGLAS</td>
			<td>ZBP-407</td>
			<td>Used for making the base for the MPH</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blu-tack</td>
			<td>Bostik</td>
			<td>Blu-tack-45g</td>
			<td>Fixing the tungsten wire</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Pasteur tube</td>
			<td>YARE</td>
			<td>WITEG</td>
			<td>Placing the filter paper containing stimuli solutions</td>
		</tr>
	</tbody>
</table>

single sensillum recordings for locust palp sensilla basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially for the detection of non-volatile chemical cues through sensilla chaetica (previously named terminal sensilla or crested sensilla). There is now increasing evidence that these palps also have an olfactory function. An odorant receptor (LmigOR2) and an odorant-binding protein (LmigOBP1) have been localized in the neurons and accessory cells, respectively, in the sensilla basiconica of the palps. Single sensillum recording (SSR) is used for recording the responses of odorant receptor neurons, which is an effective method for screening active ligands on specific odorant receptors. SSR is used in functional studies of odorant receptors in palp sensilla. The structure of the sensilla basiconica located on the dome of the palps differs somewhat from the structure of those on the antennae. Therefore, when performing an SSR elicited by odorants, some specific advice may be helpful for obtaining optimum results. In this paper, a detailed and highly effective protocol for an SSR from insect palp sensilla basiconica is introduced.

Two sensilla subtypes (pb1 and pb2) on the locust maxillary palp are identified based on different response dynamics to chemical odorants (10% 1-nonanol and 10% nonanoic acid). The neurons in pb1 produce significantly more spikes to 1-nonanol than to nonanoic acid while the neurons in pb2 are significantly less activated by 1-nonanol compared with nonanoic acid (Figure 4). Hexanal and E-2-Hexenal can evoke a locust palp opening response (POR)<sup class="xref"...

Watch this Scientific Journal Video about Single Sensillum Recordings for Locust Palp Sensilla Basiconica at JoVE.com

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially ...

The overall goal of this procedure is to introduce a detailed and highly effective protocol to make single sensillum recordings from insect palp Sensilla Basiconica. Since mastered can help answer key questions in the dissection chemoreception field. By allowing of specific odorant receptor in palp sensilla.The main advantage of this is that use a recorder Sensilla Basiconia of locust of the palps. To prepare the tungsten electrodes first attach a new tungsten wire into a micro manipulator and observe it under 40 times magnification. Now sharpen the wire by delivering 10 volts to it while using a syringe to apply 10%sodium nitrite.Then reduce the power to five volts and over one minute repeatedly dip four millimeters of the sharpened wire into sodium nitrite solution. When in use the tip must be fine enough to penetrate the cuticle of a locust palp olfactory sensillum. Next prepare the stimulus solutions.Dilute each of the chemical stimulus substances in mineral oil. Make 10%dilutions of 1-Nonanol and Nonanoic acid in mineral oil. Then make a series of tenfold serial dilutions of E-2-Hexenal and Hexanal in mineral oil.Next prepare Pasteur tubes loaded with stimuli. Insert filter paper strips into the tubes and add 10 microliters of the diluted stimulus solutions to the filter paper strips. Then plug the tubes with one milliliter pipette tips.From the recordings make a locust maxillary palp holder from a glass slide, a piece of plastic, a cover slip and double sided tape and rubber tape. Attach the plastic piece to a corner of the slide using double sided tape and then use another piece of tape to attach the cover slip to the plastic piece. Then attach a small piece of red rubber tape onto the cover glass to make it a non-slip surface.Next using the tape attach a tungsten wire 1.5 millimeters parallel to the inside edge of the platform and fix both ends of the wire onto the platform. This completes the holder. Rear the locust on fresh wheat seedlings and at 60%humidity 28 to 30 degrees Celsius and with an 18 hour light cycle.To collect the antenna select one day to three day old fifth instar Locust nymphs. Remove the antenna using fine scissors so they cannot interfere with the recording. To secure the locust use a 1.5 millimeter tube cut in 1/2 vertically.Leave the ventral region and the head of the locust exposed. Then fix the locust and tube to the glass slide with double sided adhesive tape. Next pull the right maxillary palp out and position it onto the platform.Put the tungsten wire at the fourth segment of the palp. Now surround the tungsten wire with blue Taft putty about two millimeters from the maxillary palp. It is critical to the to most properly set up the platform with a centimeter locust maxillary palp holder and to use the tungsten wire to comprise the four segment or the palp.Now view the preparation at 100%times magnification and position the palp perpendicularly to the recording electrode. Next using a micromanipulator insert a tungsten reference electrode into the locust eye. Then move the recording electrode close to the maxillary palp.Next position the odor delivery device about one centimeter from the maxillary palp. Now open the recording software and set the recording parameters. For the recording scale use 500 microvolts.For the high cut off of the filter use 300 hertz. Set the low cutoff to 200 hertz and set the pre-trigger to 10 seconds. Now switch the microscope to a high magnification and insert the recording electrode into the base of a basiconic sensillum on the maxillary palp.Then delicately adjust the recording electrode to obtain good spontaneous spikes. It is very important that the recording electrode penetrates into the base of the sensillum and that once into the sensillum are prepared. Now open the stimulus controller to deliver a continuous airstream at 20 millimeters per second.Next set the stimulation time to one second and record signals for 10 seconds starting 10 seconds before the onset of the stimulus pulse. Now collect the data. Two sensilla subtypes on the locust maxillary palp were distinguishable based on their response dynamics to the chemical stimulants 1-Nonanol and Nonanoic acid.The odorants Hexanal and E-2-Hexanal naturally occurring in food sources can evoke a locust palp opening response. In response E-2-Hexanal pb1 neurons have longer action potentials. Comparing the mean changes in spiking during the five seconds before stimulation to the five seconds after stimulation shows that the response to 1-Nonanol is significantly higher than Nonanoic acid in the pb1 neurons.And the reverse is true for pb2 neurons. The response by these neurons to E-2-Hexenal and hexanal is also dose dependent. The two neuron types have a different dose response pattern to E-2-Hexenal, but the response to hexanal is mostly the same.After watching this video you should have a good understanding of how to perform the highly effective single sensilla recording on locust palp Sensilla Basonica. Once mastered the technique can be done if it is performed properly. When attempting this procedure it's important the tube of the palp from when the recording electrode is inserted into the base of sensillum.After this development paved the way for research in the field of insect physiology to the and cellular mechanisms of incellular olfaction in the mouth part.

single-sensillum-recordings-for-locust-palp-sensilla-basiconica

Research

JoVE Journal

Neuroscience

8.1K vistas.  China Agricultural University. The overall goal of this procedure is to introduce a detailed and highly effective protocol to make single sensillum recordings from insect palp Sensilla Basiconica. Since mastered can help answer key questions in the dissection chemoreception field. By allowing of specific odorant receptor in palp sensilla.The main advantage of this is that use a recorder Sensilla Basiconia of locust of the palps. To prepare the tungsten electrodes first attach a new tungsten wire into a micro manipul...