Name: Electrophysiology of Scorpion Peg Sensilla (Video) | JoVE Journal
Uploaded: 2011-04-13T13:00:00.000+00:00
Duration: 7 min 50 s
Description: Learn electrophysiological tip-recording techniques to study chemosensory physiology in scorpion peg sensilla. This method isolates chemical stimulation to individual sensilla using mineral oil chambers.

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5 minutes

We thank the Life fund, University of Oklahoma Undergraduate Research Opportunities Program, and the Department of Zoology for funding this work. We also thank Dr. Mari&euml;lle Hoefnagels for editorial help.

1. Pre-recording, Live Animal Preparation
<ol>
	<li>Remove the scorpion from its home jar and place it in a pre-chilled glass container; next, place the jar containing the scorpion into a freezing environment (-5 &deg;C) for a minimum of two minutes. This time may vary according to the age, size, species, etc. of the specific scorpion under study. But generally, the freezing environment phase is complete once the animal is motionless.</li>
	<li>After two minutes, remove the immobilized scorpion from the cold glass container, and place it ventral side up on a microscope slide. Secure the stinger, tail, legs, and pedipalps with moldable clay (oil-based, Van Aken Plastalina).</li>
	<li>Make a platform on which to put the scorpion's pectines. Adjust the platform's length and width according to the size of the pecten. Typically, our platforms are constructed out of microscope cover glass and are approximately 10(L) X 18(W) mm.</li>
	<li>Adhere double-sided adhesive tape to the pecten platform, trimming the tape (if necessary) to fit.</li>
	<li>To make a pectinal chamber, put three of the platform's four sides into pre-melted wax (we use 100% beeswax chips) to a depth of 3-4 mm. We melt the wax by placing the chips within a glass petri dish, located on a hot plate. Once the wax on the edges of the pectinal chamber is cooled to room temperature, this should create walls of wax about 1 mm high.</li>
	<li>On the animal, locate the point of pectinal insertion, and place the un-waxed edge of the platform (tape side-up) just posterior to it. Afterward, stabilize the platform (so that it will not move later on) by gently pressing its right and left peripheral edges against the clay used to restrain the animal's legs.</li>
	<li>Use fine-tipped forceps to carefully place the pecten or pectines inside the chamber. Specifically, using forceps, hold the pecten along the pectinal spine, and gently bring the pecten out from underneath the cover glass and lower it onto the adhesive platform floor. Establish a pecten-to-tape bond by carefully applying pressure to the pectinal spine.</li>
	<li>Now apply melted wax (we use a pre-heated metal spatula) to the un-waxed edge of the platform. The purpose is two-fold, to secure the pecten and to fully enclose the chamber for future mineral oil introduction. Use caution when applying the wax over the animal's pecten, as it is possible to damage the pecten with wax that is too hot.</li>
	<li>The pecten chamber construction is complete. Next, establish an indifferent electrode connection with the animal's hemolymph by inserting a silver wire between two tail segments.</li>
</ol>
2. Simultaneous Extracellular Recording and Chemical Stimulation
<ol>
	<li>We prepare the animal 24 h prior to conducting extracellular, tip-recordings, which, among other reasons (i.e. animal adjustment to fixed position), allows time to custom-make stimulant pipettes of a specific (sensillum-dependent) tip-diameter. We use a micropipette puller for achieving such specifications. Ideally, the diameter of the pipettes is &asymp; 2 &mu;m larger than the sensillar pore diameter. We adjust our micropipette specifications accordingly for each animal.</li>
	<li>1 h prior to recording, we add 5 &mu;l of mineral oil to the pectinal chamber, submerging the pecten under oil. Then, we position the animal beneath a high-powered microscope, which has long working distance objectives and epi-illumination.</li>
	<li>To chemically stimulate peg sensilla, we introduce aqueous tastants (i.e. citric acid and salt) through the oil medium. First, we use a micropipette filler to inject the stimulant into the stimulant pipette. Note: all stimulant solutions should contain an electrolyte for electrical conductance.</li>
	<li>Next, after positioning the stimulant pipette on a micromanipulator (in the recording rig), insert the recording electrode into the blunt, open end of the stimulant pipette. It is important to ensure contact between the metal electrode and electrolytic stimulant solution.</li>
	<li>Once the preparation is electrically grounded (via the indifferent electrode), simply lower the pipette through the oil medium and onto a sensillar pore. The duration of chemical stimulation per pectinal sensillum can vary from as much as thirty minutes to as little as one second. Generally, we can sample multiple pegs (&gt; 30) per pipette. In the event that the stimulant pipette becomes clogged with minute debris, simply exchange the clogged pipette for a new, unused pipette.</li>
</ol>
3. Preparing an Animal for Multiple Days of Experimentation
<ol>
	<li>If the animal is to be used over an extended time period, we recommend reusing the pectinal chamber and replacing the drop of mineral oil for each recording session. After a given day's recording session is complete, remove the animal from the recording set-up and perform a mineral oil wash.</li>
	<li>Specifically, add another drop (5 &mu;l) of mineral oil to the chamber, and remove all oil. This helps minimize the presence of residual stimulant, which may have leaked out of the pipette during the recording session.</li>
	<li>Before the next session, add one drop of oil as described previously.</li>
</ol>
4. Representative Results:
Depending on the recording equipment used (e.g. the particular amplifier, digitizing hardware, etc.), a representative result is an extracellular recording with a S:N ratio of at least 3:1.
Successful construction of the mineral oil chamber is necessary for isolating chemical stimulation to individual sensilla, as it provides a medium for delivering polar (water-based) chemical tastants8. If there is too little oil or none at all, the stimulant solution will spread from the recorded sensillum to its neighboring sensilla, which is likely to cause a prolonged, uncontrolled bout of chemical stimulation8.

<ol><li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiology+of+a+Primary+Chemoreceptor+unit%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Physiology of a Primary Chemoreceptor unit.</a> Science. 12, 417-418 (1955).</li>
<li>Newland, P. L. Taste processing in the insect nervous system. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNewland%2C+PL+%22Methods+in+Insect+Sensory+Neuroscience%22">Methods in Insect Sensory Neuroscience</a>. Christensen, T. A. , CRC Press. Boca Raton. 289-318 (2005).</li>
<li>Foelix, R. F., Müller-Vorholt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+fine+structure+of+scorpion+sensory+organs+II.+Pecten+sensilla%5BTitle%5D)+AND+%22Bull.+Br.+Arachnol.+Soc%22%5BJournal%5D)">The fine structure of scorpion sensory organs II. Pecten sensilla.</a> Bull. Br. Arachnol. Soc. 6, 68-74 (1983).</li>
<li>Carthy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fine+structure+and+function+of+the+sensory+pegs+on+the+scorpion+pecten%5BTitle%5D)+AND+%22Experientia%22%5BJournal%5D)">Fine structure and function of the sensory pegs on the scorpion pecten.</a> Experientia. 22, 89-91 (1966).</li>
<li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Response+properties+of+chemosensory+peg+sensilla+on+the+pectines+of+scorpions%5BTitle%5D)+AND+%22J.+Comp.+Physiol.+A%22%5BJournal%5D)">Response properties of chemosensory peg sensilla on the pectines of scorpions.</a> J. Comp. Physiol. A. 181, 291-300 (1997).</li>
<li>Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pectine+organs+of+the+scorpion%2C+Vaejovis+spinigerus%3A+Structure+and+%28glomerular%29+central+projections%5BTitle%5D)+AND+%22Arthropod+Struct.+Dev%22%5BJournal%5D)">The pectine organs of the scorpion, Vaejovis spinigerus: Structure and (glomerular) central projections.</a> Arthropod Struct. Dev. 37, 67-80 (2008).</li>
<li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+evidence+of+synaptic+interactions+within+chemosensory+sensilla+of+scorpion+pectines%5BTitle%5D)+AND+%22J.+Comp.+Physiol.+A%22%5BJournal%5D)">Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines.</a> J. Comp. Physiol. A. 181, 301-307 (1997).</li>
<li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+approach+to+examining+scorpion+peg+sensilla%3A+the+mineral+oil+flood+technique%5BTitle%5D)+AND+%22J.+Arachnol%22%5BJournal%5D)">A new approach to examining scorpion peg sensilla: the mineral oil flood technique.</a> J. Arachnol. 37, 379-382 (2009).</li>
<li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+tip-recording+method+to+test+scorpion+pecten+chemoresponses+to+water-soluble+stimulants%5BTitle%5D)+AND+%22J.+Neurosci+Methods%22%5BJournal%5D)">A new tip-recording method to test scorpion pecten chemoresponses to water-soluble stimulants.</a> J. Neurosci Methods. , Forthcoming Forthcoming.</li>
<li>Haupt, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antennal+sucrose+perception+in+the+honey+bee+%28Apis+mellifera+L.%29%3A+behaviour+and+electrophysiology%5BTitle%5D)+AND+%22J.+Comp.+Physiol.+A%22%5BJournal%5D)">Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology.</a> J. Comp. Physiol. A. 190, 735-745 (2004).</li>
<li>Bland, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensilla+on+the+antennae%2C+mouthparts%2C+and+body+of+the+larva+of+the+alfalfa+weevil%2C+hypera+postica+%28gyllenhal%29+%28Coleoptera+%3A+Curculionidae%29%5BTitle%5D)+AND+%22Int.+J.+Insect+Morphol.+Embryol%22%5BJournal%5D)">Sensilla on the antennae, mouthparts, and body of the larva of the alfalfa weevil, hypera postica (gyllenhal) (Coleoptera : Curculionidae).</a> Int. J. Insect Morphol. Embryol. 12, 261-272 (1983).</li>
<li>Cate, H. S., Derby, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hooded+Sensilla+Homologues%3A+Structural+Variations+of+a+Widely+Distributed+Bimodal+Chemomechanosensillum%5BTitle%5D)+AND+%22J.+Comp.+Neurol%22%5BJournal%5D)">Hooded Sensilla Homologues: Structural Variations of a Widely Distributed Bimodal Chemomechanosensillum.</a> J. Comp. Neurol. 444, 345-357 (2002).</li>
</ol>

We have previously published these methods in two journals, Knowlton and Gaffin (2009) and Knowlton and Gaffin (in press).

The protocol above describes how to prepare a desert grassland scorpion (Paruroctonus utahensis) for electrophysiological study. Specifically, we show how to build a chamber for controlled extracellular tip-recordings of pectinal chemosensory neurons under oil. Because oil and water do not mix, it is possible to isolate chemical stimulation (with water-based stimulants) to single sensilla. It should be stressed that P. utahensis, is a relatively small animal (&asymp; 2.5-5 cm) and applying this protocol to larger animals may require many size adjustments, such as the size of the animal's platform, chamber, and amount of oil applied. We recommend conducting pilot studies to test the minimum amount of oil necessary for controlled delivery of water-based chemical stimulants.
Furthermore, we found no effect of oil on the baseline neural activity within pectinal sensilla8. This should be confirmed for other model systems as well, prior to assessing chemoresponsiveness.
This method may be used in conjunction with another method to test for chemoresponse interaction effects among sensilla in the sensory field. For example, it is possible to tip-record (under oil) from one sensillum as we base-record (via a tungsten electrode) from a neighboring sensillum9. Such a recording configuration may be used to assess whether or not chemically stimulating one sensillum affects the neural activity of the base-recorded sensillum. To date, no one has tested this for scorpion pectinal sensilla, and it remains an open question for other chemosensory systems as well.
To summarize, we think the tip-recording method under oil advances the scope of electrophysiological investigations into the neural basis of arthropod gustation. In this manuscript, we provide a protocol for preparing animals for this method, and we hope it provides a firm foundation for further study of peripheral sensory nervous systems.

<table><tbody><tr><td>Lift-N-Press tab</td><td>Ted Pella, Inc.</td><td>16082</td><td>Doubled-sided adhesive</td></tr><tr><td>MicroFil</td><td>World Precision Instruments, Inc.</td><td>MF34G</td><td>Micropipette filler</td></tr><tr><td>Standard Glass Capillaries</td><td>World Precision Instruments, Inc.</td><td>1B100F-6</td><td>Glass pipettes</td></tr><tr><td>Micr&amp;#8211;lectrode Holder</td><td>World Precision Instruments, Inc.</td><td>MEH3SBW10</td><td></td></tr></tbody></table>

electrophysiology of scorpion peg sensilla

Electrophysiology of Scorpion Peg Sensilla

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the mid-ventral surface of all scorpions are two appendages called pectines, which have dense fields of mechano- and chemosensory peg sensilla5,6. One method for assessing chemoresponsiveness of these sensilla uses a tungsten electrode for extracellularly recording neural activity within a sensillum as a volatile odorant is introduced to the sensory field5,7. The limitations of this method include slow data collection and uncontrolled stimulant introduction to, and removal from, the peg field. To overcome these limitations, we developed a new tip-recording technique that uses nonpolar mineral oil as a medium through which to deliver water-based tastants to individual peg sensilla8,9. We have successfully applied this method to obtain sensillar chemoresponses to citric acid, ethanol, and salt. Here we describe the experimental protocol for such a study9. We think this new method may be useful for studying the response properties of other arthropod chemosensory systems, including those of insects10, 11 and crustaceans12.

This article describes an electrophysiological method for isolating chemical stimulation to individual sensilla via extracellular, tip-recordings under mineral oil.

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

The overall goal of this procedure is to study the chemo sensory physiology of individual peg illa on a scorpion pectin. This is accomplished by first immobilizing, an anesthetized scorpion ventral side up on a glass slide. Using modeling clay to set up requires submerging the pectins in a custom made oil-filled chamber and making electrodes filled with a chemical stimulant.Chemos sensory responses are recorded at individual illa on the pectin, which ultimately produce similar response profiles to aqueous chemo stimulants. We Used to record from the basis of illa while blowing volatile stimulants across the pores. The main advantage of the tip recording technique is that it allows the stimulation to be isolated to one pig centum without affecting the activity of its neighbors.This method can help us answer key questions in the field of chemo sensory biology, for example, why do animals use so many repeated silla in chemical detection and perception? Scorpions might not seem like the safest animals to study, however, not all species are dangerous or difficult to deal with. For example, the desert grassland scorpions are only four to five centimeters long are easy to maintain in the lab and rarely sting if handled properly.If stung, the venom artificially triggers your sensory neurons, some of which transmit pain information. The sensation usually subsides in about 15 minutes. Scorpions are hardy subjects and can endure multiple days of experimentation, often being returned to their habitations.When the experiment is complete to immobilize the scorpion, place it in a pre chilled glass container. Then place the jar containing the scorpion into a freezing environment for a minimum of two minutes and remove it from the freezer when the animal is motionless. Now place the immobilized scorpion ventral side up on a microscope slide.Secure the stinger tail legs and pedi pals with moldable clay. The scorpion's pectins are paired comb like structures near the sex organs and are unique to these animals. A chamber must be made to contain a single pectin without removing it from the animal.First, make a platform for the pectin to fit onto a glass cover slip cut to approximately five millimeters by 18 millimeters. Usually serves well. Second adhere double-sided adhesive tape to the platform and trim the tape to fit if necessary.Third, melt some 100%beeswax chips in a glass dish using a hot plate. Now dip three of the platform's four edges into melted beeswax to a depth of three to four millimeters. The cooled wax serves as a wall to make a chamber.Now, place the unwed edge of the platform just posterior to the point of pectal Insertion on the animal. Stabilize the platform by gently pressing its right and left peripheral edges against the clay used to restrain the animal's legs using fine tipped forceps. Carefully hold one of the pectins along the pectal spine and gently bring it out from underneath the cover glass and lower it onto the adhesive platform floor.Stick the pectin down by carefully applying pressure to the pectal spine. Complete the chamber by using a preheated metal spatula to apply melted wax to the unwed edge of the platform. The animal is now almost prepared for doing electrophysiology recordings.Now is an easy time to make an indifferent electrode connection. Insert a silver wire between two tail segments into the scorpion's hemolymph. Press some of the wire that exits the animal into the clay to secure it and leave enough length to reach the amplifier.Now set the preparation aside for at least an hour before making recordings to allow the preparation to settle for recordings. Use a micro pipette puller to make pipettes with an inner diameter of about two microns larger than the sensor pore diameter, which varies between animals. One hour before recording, fill the pectin chamber with five microliters of mineral oil.Then position the animal beneath the high powered microscope with long working distance objectives and epi illumination to chemically stimulate peg illa. Load the stimulant pipette with aqueous tastes mixed in an electrolytic solution. Using a micro pipette filler, then attach the loaded stimulant pipette to a micro manipulator.Now insert the recording electrode into the open end of the stimulant pipette. It is important to ensure contact between the metal electrode and electrolytic stimulant solution. Connect leads of the recording and indifferent electrodes to a differential amplifier.The amplifier is connected to an oscilloscope to visualize electrical activity and to a speaker to audibly detect electrical change. The setup is now ready for taking recordings. Using the micro manipulator carefully lower the pipette onto a sensor pore.A change to higher frequencies will be heard when electrical contact is made between the pipette electrolyte and the fluid within the peg sens. The distinct popping of neural action potentials can be heard at this time as well. The duration of chemical stimulation per perpetual sens can vary from as much as 30 minutes to as little as one second.Generally, one pipette can sample more than 30 pegs. Replace clogged pipettes with clean pipettes. Pipettes are easy to prepare and clogging is not uncommon, so always keep extra pipettes at the ready.It is possible to record from a single animal for several hours in a single recording session before having to change the mineral oil. A single animal can be kept in place in the recording rig for multiple recording sessions over several successive days. For subsequent sessions, reuse the pectal chamber, but replace the mineral oil.Just remove all the old oil with a pipette and add a fresh drop. This helps minimize the presence of residual stimulant, which may have leaked out of the stimulant pipette during the recording session. Here are representative tip recordings to different stimulants.This trace shows a relatively consistent response to 0.01 molar potassium chloride. This peg sends response to 0.1 molar citric acid begins much more intensely then tapers down in intensity within a fraction of a second, followed by some diminishing in frequency. In a typical response to 30%ethanol, the trace shows bursts with multiple spikes between short gaps of inactivity when comparing tip recordings of different sensor responses to the same stimulant, like 0.1 molar citric acid.The responses are very similar. While attempting this procedure, it's important to remember to fill the pipette with a water-based stimulant, which doesn't mix with the oil medium and allows you to chemically stimulate one lum at a time. After watching this video, you should have a good understanding of how to controllably stimulate individual CIN on a scorpion pectin using mineral oil as a medium.

electrophysiology-of-scorpion-peg-sensilla

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Learn electrophysiological tip-recording techniques to study chemosensory physiology in scorpion peg sensilla. This method isolates chemical stimulation to individual sensilla using mineral oil chambers.

Electrophysiology of Scorpion Peg Sensilla

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