The project was supported by Award AAES 461Hatch/Multistate Grants ALA08-045 and ALA015-1-10026 to N.L.

1. Preparation of Instruments, Stimuli Solutions, and Bed Bugs
<ol>
	<li>Prepare a 50% KNO2 solution (w/v) in a 20 ml bottle.</li>
	<li>Sharpen two tungsten microelectrodes in KNO2 solution at 5 V by repeatedly dipping the tungsten electrodes in and out of the solution.
	<ol>
		<li>Roughly sharpen the tungsten wire by dipping about 10 mm of the tungsten wire in and out of the KNO2 solution at the speed of 2 dips/sec&#160;for about 5 min, which can greatly consume the front end of the tungsten wire.</li>
		<li>Delicately sharpen the electrode by dipping about 1 mm of the wire tip in and out of the solution at the speed of 2 dips/sec&#160;for at least 1 min&#160;so as to make a fine and sharp point of the electrode. Check the electrode tip diameter under the microscope frequently until it reaches 0.2-0.5 &#181;m, which should be fine enough to puncture the cuticle of bed bug olfactory sensillum. 
		Note: While manually sharpening the electrode, the dipping speed of the tungsten wire into the KNO2 solution is not constant all the time. With more practice, it is much easier to keep a relatively constant speed in sharpening the electrode. The sharpening time is also uncertain depending on how fine the electrode should be. Here, an electrode tip with the diameter of ~0.2 &#181;m is sufficient enough to puncture through the olfactory sensillum.</li>
	</ol>
	</li>
	<li>Dilute each of the chemical stimuli in dimethyl sulfoxide (DMSO) from the neat compound to an initial concentration of 1:10 v/v as a stock solution. Create a series of decadic dilutions depending on how many doses required in the experiment, again with DMSO, from each of the stock solutions for each chemical. Here, use 10% (+)-&#946;-pinene and eucalyptol.</li>
	<li>Place the unfed or seven days post-feeding adult bed bugs (either male or female) from the Ft. Dix colony (a gift from Dr. Haynes in University of Kentucky) to be used in the experiment in a petri dish. 
	Note: There is no exact number for bed bugs placed in the Petri dish. It can be a few or a lot.</li>
</ol>
2. Bed Bug Antennae Preparation
<ol>
	<li>Anaesthetize the bed bugs on ice (2-3 min).</li>
	<li>Fix both the antennae and insect body on a microscope coverslip with double-sided tape and remove the legs with fine scissors.</li>
	<li>Use a small pin to gently touch the antennae so as to stick them on the tape steadily.</li>
	<li>Rest the coverslip against a small ball (~1 cm diameter) of dental wax to facilitate manipulation and adjust it to an appropriate angle (~ 90&#176;) for the recording electrode (Figure 1).</li>
	<li>Once secured, place the bed bug under a stereo microscope, turn on the cold light source and adjust the intensity of illumination until the antenna is clearly presented, and focus the microscope on the second flagellum of the bed bug antenna at high magnification (720X). 
	Note: The intensity of illumination used in the experiment is not quantitated, which really depends on how the experimenter&#39;s eyes feel the intensity of illumination.</li>
</ol>
3. Single Sensillum Recording
<ol>
	<li>Connect the preamplifier (10X) with the signal acquisition controller, which is connected with the computer for signal recording and visualization. Turn on the computer and launch the software, e.g., AutoSpike32 and click the &#34;Record&#34; mode from the menu bar. Then choose the &#34;wave&#34; so as to start recording the wave signals. 
	Note: A flat line running from the left to the right of the monitor repeatedly should now be visible. Here, the recording window lasts 40 sec. Max wave recording is 10 sec. Selected sampling rate is 96000 and digital sampling rate is 240. There is 0% offset and no filtering, no rectification for the recording signals. All these parameter settings in the software can be modified as needed.</li>
	<li>Turn on the speaker connected to the preamplifier, which is used to present the toning mode for the neuronal responses from antennal sensillum.</li>
	<li>Insert the reference electrode into the abdomen of the stabilized bed bug. 
	Note: The reference electrode was held by a metal stand magnetically attached to the air-table.</li>
	<li>After the reference electrode has been connected to the bed bug&#39;s abdomen, move the recording electrode, which is connected to the preamplifier and manipulated by a micromanipulator, towards the posterior end of the bed bug&#39;s antenna.</li>
	<li>When the recording electrode is in contact with the right tip of the antenna, switch on the microscope and locate the electrode at low magnification.</li>
	<li>Adjust the recording electrode while gradually increasing the magnification until both the electrode and the antennal sensillum are in the same plane and clearly visible under the microscope. 
	Note: By this time, the microscope is usually at the highest magnification.</li>
	<li>Insert the recording electrode into the shaft of the sensillum using the micromanipulator and go a little deeper if the background noise is high compared to the action potential.</li>
	<li>Once clear action potentials are observed from the recorded sensillum, fill a micropipette with 10% (+)-&#946;-pinene. Use the micropipette to deposit 10 &#181;l aliquot of 10% (+)-&#946;-pinene onto a filter paper strip (~3 x 15 mm) placed inside a glass Pasteur pipette.
	<ol>
		<li>Connect the loaded pipette to the outlet of the pulse flow tube of the stimulus controller and place the tip of the pipette into the small hole in the tube oriented towards the antenna.</li>
	</ol>
	</li>
	<li>When all these connections have stabilized, depress the footswitch of the stimulus controller to deliver a 0.5 sec puff of stimulus (0.5 L/min) into the continuous humidified air stream. The recording of action potentials will be initiated simultaneously when the footswitch is depressed. The recording process will be last for 10 sec starting 1 sec before the stimulation.</li>
	<li>Count the action potentials off-line for two 500 msec periods, one before and one after the stimulation. Subtract any change in the spike rate during the 500 msec post-stimulation from the spontaneous activity recorded during the preceding 500 msec and convert the counts into the conventional scale of spikes/s by multiplying them by 2.</li>
</ol>
4. Stimulus Replacement in the SSR
<ol>
	<li>Once the footswitch has been triggered, deliver the 10% (+)-&#946;-pinene in the pipette onto the bed bug antennae and record the response to this specific odorant for 10 sec, after which the pipette is removed.</li>
	<li>Label another new pipette with the 0.001% eucalyptol to be tested. Place a small piece of filter paper onto which 10 &#181;l of the stimulus has been applied, into the new pipette.</li>
	<li>Wait for 2-5 min until the stimulus is completely vaporized in the glass pipette. Attach the pipette onto the outlet of the pulse flow tube.</li>
	<li>Insert the pipette tip into the small hole of the tube oriented towards the antenna. Depress the footswitch and start the 10 sec recording.</li>
	<li>Disconnect the pipette and prepare another pipette with 0.01% eucalyptol.</li>
	<li>Test all the rest of the doses of eucalyptol (from 0.001% to 10%) on the antennal sensilla to observe the dose-dependent responses. Test from the most dilute to the least dilute doses.</li>
</ol>

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L., Leffler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bed+bug+infestations+in+the+news:+a+picture+of+an+emerging+public+health+problem+in+the+United+States.">Bed bug infestations in the news: a picture of an emerging public health problem in the United States.</a> J Environ Health. 70 (9), 24-27 (2008).</li><li>Boase, C., Robinnson, W., Bajomi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bed+bugs+(Hemiptera:+Cimicidae):+an+evidence-based+analysis+of+the+current+situation.">Bed bugs (Hemiptera: Cimicidae): an evidence-based analysis of the current situation.</a> Sixth international conference on urban pests. OOK-Press Kft. , (2008).</li><li>Doggett, S. L., Geary, M. J., Russell, R. 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The authors have nothing to disclose.

The Single Sensillum Recording technique has been extensively used in testing the neural responses of insects such as fruit flies, mosquitoes and bed bugs to different chemical stimuli in the environment. These chemical stimuli are often dissolved and diluted in a common solvent in order to prepare different doses of treatments. However, different solvents can produce quite different release rates for the stimuli. Previous studies on some extensively studied insects such as Drosophila melanogaster, Anopheles gambiae, Culex quinquefasciatus and Aedes aegypti have usually used paraffin oil as the solvent to dissolve the stimuli, as these insects are relatively insensitive to paraffin oil17-20. Paraffin oil has also been used in earlier single sensillum recording studies of bed bugs, for the same reason14. However, the most commonly used solvent may not be the best for each insect species. In the case of bed bugs, both paraffin oil and DMSO, to which bed bugs also exhibit insensitivity, have been used to dissolve the stimuli in different studies14,15, but the same doses of stimuli diluted in DMSO appear to elicit much stronger neural responses on the sensilla of bed bugs. For instance, DMSO-dissolved R-(+)-limonene and S-(-)-limonene generated neural responses of &#8805;70 spikes/sec from D&#947; sensilla on bed bug antennae, while the paraffin oil-dissolved limonene elicited neural responses of only &#8804;25 spikes/sec from D&#947; sensilla. This decrease in the neural responses is quite common in stimuli that have been diluted with paraffin oil, probably due to the slower release rate of paraffin oil compared with DMSO. This slower release rate reduces the amount of stimulus delivered onto the surface of the sensillum and may result in a misleading conclusion regarding the sensitivity of insects to certain semiochemicals.
Two critical steps for conducting the single sensillum recording are 1) sample preparation and 2) signal recording. For the sample preparation, since the bed bugs have very strong legs and actively moving antennae, it is very important to remove all legs and stick the antennae tightly onto the double-sided tape. In the signal recording process, sometimes, it is positionally impossible for the electrode to point into the sensillum shaft. If this is the case, the electrode may puncture the posterior end of the sensillum, which always gives a very clean and clear signal with very little background noise.
As there are multiple neurons housed in the D and C type sensilla, it is often hard to distinguish between individual neurons based on the amplitudes and shapes of the action potentials produced during SSR. However, it is still possible to see differences in the bed bug&#39;s responses to different stimuli based on the combined firing frequency of all the neurons in the same sensillum. Theoretically, bed bugs are sensitive to certain stimuli with strong stimulation while insensitive to other stimuli with weak stimulation at the same dose. Further studies integrating behavior tests and information of their neural response to these stimuli would therefore provide meaningful information on ecologically related semiochemicals for bed bugs.
In this study, we also used the SSR technique to test the neural responses of olfactory sensilla to different doses of stimuli. We observed a dose-dependent pattern in the bed bug&#39;s neural responses to different chemicals. However, considering the complex environment bed bugs live in, the actual dose of volatiles encountered by bed bugs in their normal surroundings will be very low. As a result, the semiochemicals that elicit strong neural response at low doses down to 1:105 v/v and 1:104 v/v are more likely to be biologically meaningful for bed bugs than other chemicals that only function at high doses. Therefore, those semiochemicals that act at low doses probably play an important role in the chemoreception of bed bugs, helping them to locate a host or avoid adverse factors, and will thus provide useful guidance in screening for promising bed bug attractants or repellents for use in both laboratory and field assays.

The common bed bug Cimex lectularius L (Hemiptera: Cimicidae), as a temporary ectoparasite, is an obligated blood-sucking insect, which means their survival, development, and reproduction require blood sources from hosts, including both humans and animals1,2. Although virus transmission has rarely been reported due to C. lectularius, the biting nuisance generated by an infestation seriously affects hosts both physically and psychologically3. The introduction and widespread use of chemical insecticides, especially DDT, lowered the risk of infestations and by the end of the 1950s infestations were at such a low level that they were no longer a serious public concern. However, a number of possible factors have led to resurgence in bed bug populations worldwide, such as the reduced use of insecticides, a decline of public awareness, increased traveling activity, and the development of resistance to insecticides4-9.
Chemical cues in the environment are detected and recognized by insects through olfactory organs such as antennae and maxillary palps. The olfactory sensilla on the insect antennae play a crucial role in detecting these chemical cues. The chemical molecules enter the antennal cuticle through pores on the cuticle surface. Odorant binding proteins in the antennal lymph bind to these chemical molecules and transport them onto the odorant receptors10. The odorant receptors and their co-receptor from the non-selective cation ion channel on the neural membrane, which will be depolarized once these chemical molecules are recognized by the odorant receptors11.
Single sensillum recording (SSR) was developed to detect the extracellular change in the action potential caused by the application of either chemical or non-chemical stimuli. By inserting a recording electrode into the sensillum lymph and a reference electrode into some other part of the insect body (usually either the compound eyes or the abdomen), the firing rate of the neurons in response to stimuli can be recorded12. Changes in the number of spikes represent the sensitivity of the insect to specific stimuli. Chemical stimuli of different identities and concentration will elicit different neural responses, with different firing rates and temporal structures, and can thus be used to investigate the coding process of the insect to specific chemicals.
For the common bed bug, both sexual forms share the same pattern of olfactory sensilla on the antennae: nine grooved peg C sensilla, 29 hair-like E (E1 and E2) sensilla, and one pair each of D&#945;, D&#946;, D&#947; smooth peg sensilla 13,14. As multiple neurons have been identified in each type of sensillum, it is not easy to distinguish the action potentials from different neurons housed in the same sensillum, so for this experiment the total numbers of action potentials were counted off-line for a 500 msec period before and after stimulation. The number of action potentials after stimulation was then subtracted from the number of action potentials before stimulation and multiplied by two in order to quantify the changes in the firing rate in each individual sensillum in spikes per second15.

<table><tbody><tr><td>Tungsten wire</td><td>A-M SYSTEMS</td><td>#716500</td><td>Used for preparing the electrode</td></tr><tr><td>KNO2</td><td>Sigma</td><td>#310484</td><td>Used for sharpening the tungsten wire</td></tr><tr><td>AC Power Supply</td><td>BK Precision</td><td>1653A</td><td>Providing the voltage in sharpening the tungsten wire</td></tr><tr><td>Leica Z6 APO Microscope</td><td>Leica</td><td>10447424</td><td>Used for observing the sensilla on antennae</td></tr><tr><td>Simulus controller</td><td>Syntech</td><td>CS-55</td><td>Used for controlling the stimulus application</td></tr><tr><td>4-Channel USB 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><td>Light Source</td><td>SCHOTT</td><td>A20500</td><td>Providing light sources for observation</td></tr><tr><td>Micromanupulator</td><td>Leica</td><td>115378</td><td>Used for minor movement of electrode</td></tr><tr><td>Speaker</td><td>Juster</td><td>95a</td><td>Connected with Acquisition Controller IDAC-4 and providing sound for the signal</td></tr><tr><td>Magnetic stand</td><td>Narishige</td><td>GJ-1</td><td>Used to hold the reference electrode, stablized bed bug and stimulus delivery tube</td></tr><tr><td>TMC Vibration Isolation Table</td><td>TMC</td><td>63-500</td><td>Used for isolating the vibration from the equipments</td></tr><tr><td>Coverslip</td><td>Tedpella</td><td>2225-1</td><td>Used for holding the bed bug</td></tr><tr><td>Double-sided Tape</td><td>3M</td><td>XT6110</td><td>Used for stablizing the bed bug on the coverclip</td></tr><tr><td>Dental Wax</td><td>Dentakit</td><td>DK-R012</td><td>Used for supporting the coverclip where bed bug is stablized&amp;nbsp;</td></tr></tbody></table>

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.

Single sensillum recording is a powerful investigative technique used in studies of insect chemical ecology and neural physiology. Investigating the neural responses of insects to different volatile compounds, especially those thought to be ecologically related to the survival and development of the insects, not only gives us invaluable insights into the insect olfaction process, but also opens up promising new avenues that could potentially lead to the development of useful new reagents for pest control.
The common bed bug, as a notorious urban pest, has certainly attracted the attention of many researchers. Among the various study areas related to bed bugs, their olfaction mechanism is of the utmost importance for the chemical ecology of bed bugs. Previous studies have explicitly described the quantity and distribution of different types of olfactory sensillum on bed bug antennae. As shown in Figure 2A, bed bug antennae possess four segments (SC, PE, F1 and F2). The majority of the olfactory sensilla are presented on the posterior end of the 2nd flagellum (F2), but their distribution is distinctly different for each type: the D sensilla, namely D&#945;, D&#946; and D&#947;, are only located along the inner side of the antennae (Figure 2C), while the C and E (E1 and E2) sensilla are found on both sides of the antennae (Figure 2B). Therefore, in order to ensure that we record the neural response of the D sensilla, careful positioning of the antenna is essential.
Since both sexual forms of bed bug share the same pattern of sensilla types and the inner side of their antennae contains all types of sensilla, targeting this area makes it much easier to record the chemical responses of all the different types of sensilla separately on the antennae (Figure 3A). In single sensillum recording, different olfactory sensilla exhibit neural signals with distinctively different action potential types and amplitudes (Figure 3B). For example, E sensilla are known to have one or two neurons inside, while D type sensilla house more neurons than either E or C sensilla, producing more complicated action potentials than the others as a result. The amplitudes of the neural responses from C sensilla are much smaller than those of the other sensillum types.
Once the electrode connections have been set up, the neural responses from each type of sensillum to each stimulus can be recorded based on their identity and intensity. For some stimuli to which bed bugs are extremely sensitive, the neural response can be very strong and last several seconds beyond the termination of stimulation. For instance, in response to 10% (+)-&#946;-pinene, bed bugs showed a strong reaction with a huge firing rate (&#8805;200 spikes/sec) and super-sustained temporal dynamics compared to the control with solvent alone as the stimulus (Figure 4A and B). Different stimuli may elicit totally different neural responses from the same sensillum and different concentrations of the same stimulus are likely to generate quite different firing frequencies. As shown in Figure 5, increasing the concentration of eucalyptol raised the firing frequencies from 30 spikes/sec at 0.001% to 240 spikes/sec at 10% in a dose-dependent manner.
<img alt="Figure 1" src="/files/ftp_upload/53337/53337fig1.jpg" /> 
Figure 1.&#160;A schematic diagram showing the fixation procedure for bed bugs. The bed bug is stabilized on the coverslip with the antennae fixed on the tape. The assembled sample is then placed on a magnetic stage. The orientation and height of the sample can be adjusted to an appropriate angle between the bed bug antennae and the recording electrode. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53337/53337fig2.jpg" /> 
Figure 2.&#160;Distribution of olfactory sensilla on the bed bug antennae.&#160;(A) A Scanning Electrode Microscope (SEM) image of a bed bug antenna. The antenna has four segments, the Sculpus (SC), Pedecel (PE), the first flagellum (F1) and the second flagellum (F2). Most of the olfactory sensilla are located on F2, although a few olfactory sensilla have also been found on F1, which is thought to be related to their function of aggregation pheromone detection for the bed bug16. (B) A SEM image of the outer side of F2, which houses the C and E olfactory sensilla. (C) A SEM image of the inner side of F2, which was found to house all the different types of olfactory sensilla: D (D&#945;, D&#946;, D&#947;), C and E. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53337/53337fig3.jpg" /> 
Figure 3.&#160;Typical neural signals of different types of olfactory sensilla on bed bug antennae. (A) High resolution SEM pictures of each type of olfactory sensillum on the bed bug antennae. (B) Typical neural signals of the different olfactory sensilla before exposure to a stimulus. D&#945;, D&#946;, D&#947; and C sensilla, which house multiple olfactory sensory neurons (OSNs), exhibit more complicated action potentials than E1 and E2 sensilla, which contain only one or two OSNs. The amplitudes of the action potentials from C sensilla are much smaller than those in other sensillum types. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53337/53337fig4.jpg" /> 
Figure 4.&#160;Representative neural response to stimuli that bed bugs are sensitive to.&#160;(A) Signal trace showing the typical neural response of an olfactory sensillum (D&#947;) to the solvent, which is used as the control in the single sensillum recording. The signal traces are set to start 1 sec before the 0.5 sec&#160;puff of stimulus. The signal traces continue recording for 10 sec after initiating the stimulus puff. (B) Signal trace showing the extremely strong neural response of an olfactory sensillum (D&#947;) to a botanical stimulus, 10% (+)-&#946;-pinene. After the puff of (+)-&#946;-pinene is delivered to the D&#947; sensillum, OSNs housed inside this sensillum are fired with high frequency and a long-lasting temporal dynamic. The white bar above the signal trace indicates the 1 sec interval before stimulus exposure, the red bar above the trace corresponds to the delivery of the stimulus puff onto the olfactory sensillum, and the black bar above the trace indicates the signal recorded after the termination of stimulus puff. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53337/53337fig5.jpg" /> 
Figure 5.&#160;Representative dose-dependent responses of OSNs to the stimuli. Using another botanical stimulus, eucalyptol as an example, the D&#947; sensilla displayed a dose-dependent response to different concentrations of eucalyptol. As the concentrations increased from 0.001% to 10%, the firing frequencies rose from 30 spikes/sec to 240 spikes/sec. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals at JoVE.com

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

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 ...

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9.8K Views.  Auburn University. 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.

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

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.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (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 ...

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

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 ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

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.

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

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, ...

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 ...

A Novel Method for Enhanced Analysis of Insect Taste Neuron Activity

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

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

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

A rarely used method of electrophysiological recording, base recording, allows analysis of features of taste coding that cannot be examined by conventional recording methods. Base recording also allows the analysis of taste responses to hydrophobic stimuli that cannot be studied using traditional electrophysiological methods.

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

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

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Chapters in this video

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological Measurements from a Moth Olfactory System

Electrophysiology of Scorpion Peg Sensilla

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

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

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

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