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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+Primary+Chemoreceptor+unit.">Physiology of a Primary Chemoreceptor unit.</a> Science. 12, 417-418 (1955).</li><li>Newland, P. L., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+processing+in+the+insect+nervous+system.">Taste processing in the insect nervous system.</a> Methods in Insect Sensory Neuroscience. , 289-318 (2005).</li><li>Foelix, R. F., M&#252;ller-Vorholt, 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+fine+structure+of+scorpion+sensory+organs+II.+Pecten+sensilla.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+structure+and+function+of+the+sensory+pegs+on+the+scorpion+pecten.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+chemosensory+peg+sensilla+on+the+pectines+of+scorpions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pectine+organs+of+the+scorpion,+Vaejovis+spinigerus:+Structure+and+(glomerular)+central+projections.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+evidence+of+synaptic+interactions+within+chemosensory+sensilla+of+scorpion+pectines.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+examining+scorpion+peg+sensilla:+the+mineral+oil+flood+technique.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+tip-recording+method+to+test+scorpion+pecten+chemoresponses+to+water-soluble+stimulants.">A new tip-recording method to test scorpion pecten chemoresponses to water-soluble stimulants.</a> J. Neurosci Methods. , .</li><li>Haupt, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+sucrose+perception+in+the+honey+bee+(Apis+mellifera+L.):+behaviour+and+electrophysiology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensilla+on+the+antennae,+mouthparts,+and+body+of+the+larva+of+the+alfalfa+weevil,+hypera+postica+(gyllenhal)+(Coleoptera+:+Curculionidae).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooded+Sensilla+Homologues:+Structural+Variations+of+a+Widely+Distributed+Bimodal+Chemomechanosensillum.">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

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.

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Electrophysiology of Scorpion Peg Sensilla

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 ...

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9.0K Views.  University of Oklahoma. This article describes an electrophysiological method for isolating chemical stimulation to individual sensilla via extracellular, tip-recordings under mineral oil.

Video: Electrophysiology of Scorpion Peg Sensilla

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
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Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

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A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

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Environment

Electrophysiological Measurements from a Moth Olfactory System

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In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

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The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

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Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

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Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

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

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Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

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.

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 ...

Electrophysiology of Scorpion Peg Sensilla

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Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

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Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

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Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

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