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

1. Preparation of Instruments and Insect
<ol>
	<li>Preparing tungsten electrodes and stimuli solutions
	<ol>
		<li>Fix a new tungsten wire (diameter of 0.125 mm, length of 75 mm) into a micromanipulator and sharpen it in a 10% (w/v) sodium nitrite (NaNO2) solution in a syringe at 10 V provided by a power supply for about 1 min under a stereomicroscope (40X magnification).</li>
		<li>Dip the sharpened tungsten wire repeatedly into the 10% NaNO2 solution, about 4 mm at 5 V in &#60; 1 min (Figure 1A).</li>
		<li>Examine the diameter of the sharpened tungsten tip frequently under the stereomicroscope until it is fine enough to penetrate the cuticle of a locust palp olfactory sensillum (Figure 1B).</li>
		<li>Prepare the stimulus solutions. Dilute each of the chemical stimulus substance in mineral oil. Dilute 1-nonanol and nonanoic acid at 10% dilutions. Dilute E-2-hexenal and hexanal at 10-2, 10-3, 10-4, and 10-5.</li>
		<li>Prepare Pasteur tubes carrying the stimuli: insert filter paper strips (length of 2 cm, width of 0.5 cm) into the Pasteur tubes, add the diluted stimulus solutions (each 10 &#181;l) to the filter paper strips, and then plug the Pasteur tubes with pipette tips (1 ml).</li>
	</ol>
	</li>
	<li>Prepare the insect
	<ol>
		<li>Rear locusts (Locusta migratoria) with fresh wheat seedlings under crowded conditions at a relative humidity of 60%, a temperature of 28 - 30 &#176;C, and a photoperiod of 18:6 h (light:dark). Choose 1- to 3-day-old 5th instar locust nymphs and remove the antennae with fine scissors to avoid any interference when recording.</li>
	</ol>
	</li>
	<li>Preparing the locust maxillary palp holder
	<ol>
		<li>Use a glass slide (25 mm x 75 mm) as the base of the maxillary palp holder (MPH). Attach a plastic piece (1 mm in height, 10 mm in width, 35 mm in length) to a corner of the glass slide with double-sided adhesive tape, and finally fix a cover glass (18 mm x 18 mm) on top of the plastic piece with double-sided adhesive tape. Place a small piece of red rubber tape onto the cover glass as a non-slip layer. The plastic piece and the cover glass constitute the platform for the locust palp. The height of the platform is approximately 1.5 mm.</li>
		<li>Install a tungsten wire (diameter of 0.125 mm, length of 36 mm) at a distance of 1.5 mm parallel to the inside edge of the platform. Fix the two ends of the wire onto the platform with double-sided adhesive tape.</li>
	</ol>
	</li>
</ol>
2. Preparation of Locust Maxillary Palps
<ol>
	<li>Cut a centrifuge tube (1.5 ml) vertically in half and cut off the bottom. Place the locust into the prepared tube. Leave the ventral region and the head of the locust exposed. Fix the assembly to the glass slide with double-sided adhesive tape (Figure 2A).</li>
	<li>Pull the right maxillary palp onto the platform.</li>
	<li>Put the tungsten wire at the fourth segment of the palp. Place adhesive putty on each side of the tungsten wire, about 2 mm from the maxillary palp (Figure 2A and 2B).</li>
</ol>
3. Single Sensillum Recordings
<ol>
	<li>Place the locust maxillary palp preparation under a microscope at a low magnification (100X). Adjust the position of the preparation until the palp is perpendicular to the recording electrode (Figure 3A).</li>
	<li>Insert the reference electrode (tungsten electrode) into the locust eye using a micromanipulator. Move the recording electrode (tungsten electrode) close to the maxillary palp with the micromanipulator (Figure 3B and 3C).</li>
	<li>Adjust the odor delivery device to about 1 cm from the maxillary palp (Figure 3B).</li>
	<li>Open the recording software Auto Spike 32. Set the recording parameters as follows: the recording scale on 500 &#181;V; the high cutoff of the filter on 300 Hz, the low cutoff on 200 Hz; and the pretrigger on 10 s.</li>
	<li>Connect the recording electrode to a 10x universal AC/DC amplifier.</li>
	<li>Switch the microscope to a high magnification (500X). Insert the recording electrode into the base of a basiconic sensillum on the maxillary palp and delicately adjust the recording electrode to obtain good spontaneous spikes (Figure 3D).</li>
	<li>Open the stimulus controller to deliver a continuous air stream at 20 ml/s. Set the stimulation time to 1 s. Record signals for 10 s, starting 10 s before the onset of the stimulus pulse.</li>
	<li>Use a 10x universal AC/DC amplifier to amplify the signals. Feed the signals into the IDAC 4. Analyze the signals with the Auto Spike 32 software. AC signals are band-pass filtered between 200 to 300 Hz. Use Auto Spike 32 to distinguish peak-to-trough amplitudes from noises. Calculate the responses of the neurons as the increases in action potential frequencies (spikes per second) over the spontaneous frequencies. Perform a statistical analysis using GraphPad Prism 7.</li>
</ol>

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The authors have nothing to disclose.

Insects rely on palps to detect food odors, and their palps are believed to play an important role in speciation13,27. The palps are simple olfactory organs and are receiving increasing attention as an attractive model for the exploration of the neuromolecular networks underlying chemosensation28.
Insect labellar and palp SSRs have been successfully performed on Drosophila melanogaster, Anopheles gambiae, and Culex quinquefasciatus13,14,15,16,17 but have rarely been reported in the form of a video presentation16,29. In contrast, video data on antennal SSRs are available for Drosophila, the navel orangeworm moth (Amyeloistransitella), Schistocerca Americana, and the bed bug (Cimex lectularius)16,30,31,32,33.
Locust palp sensilla basiconica have a particular structure that differs from that of locust antennal sensilla and many other insect sensilla. Using the method described here, action potentials generated by locust palp sensilla basiconica subtypes pb1 and pb2 could be recorded and discriminated (Figure 4 and Figure 5).
The critical step is the penetration of the recording electrode. The recording electrode should be inserted into the base of the sensillum and advanced until good signals are acquired. In addition, it is important to prevent the dome of the palp from collapsing when the recording electrode is inserted into the base of the sensillum. To achieve this, we set up a platform including a special locust maxillary palp holder (MPH) and used a tungsten wire to compress the fourth segment of the palp. Many repetitions of this procedure demonstrate that this is effective. Based on the response patterns of the neurons in the sensilla to several odorants, we have, for the first time, identified two subtypes of sensilla basiconica on the locust maxillary palp, namely pb1 and pb2.
The limitation of the technique outlined in this publication is that it could be used to record big insects (e.g., moths, beetles, and locusts) while not to record small insects (e.g., flies and mosquitoes), which have their own platforms and techniques13,14,15,16,17. This technique is complementary to existing methods.
In conclusion, a highly effective protocol of an SSR from insect palp sensilla basiconica is described in detail. This protocol could provide researchers with a useful technique in the study of molecular and cellular mechanisms of insect olfaction on the mouthpart. This method linked with gas chromatography could be used to identify natural electrophysiologically-active ligands in extracts of favorable food resources.

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

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

single sensillum recordings for locust palp sensilla basiconica

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

Two sensilla subtypes (pb1 and pb2) on the locust maxillary palp are identified based on different response dynamics to chemical odorants (10% 1-nonanol and 10% nonanoic acid). The neurons in pb1 produce significantly more spikes to 1-nonanol than to nonanoic acid while the neurons in pb2 are significantly less activated by 1-nonanol compared with nonanoic acid (Figure 4). Hexanal and E-2-Hexenal can evoke a locust palp opening response (POR)26. Hexanal is an abundant host plant green leaf volatile which may contribute to a further confirmation to the food source26. The spikes elicited in the pb1 neurons last longer than those of pb2 when they are stimulated by E-2-hexenal (Figure 4). The neurons in pb1 and pb2 exhibit similarly robust responses to hexanal (Figure 4). Comparing the mean changes of all spikes between the periods 5 s before and 5 s after the stimulation indicates that the response to 1-nonanol is significantly higher than to nonanoic acid in pb1, but contrarily in pb2 (Figure 5). The neurons in these two subtypes of sensilla respond dose-dependently to E-2-hexenal and hexanal, and their response patterns to these two aldehydes differ (Figure 6A and 6B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57863/57863fig1.jpg" /> 
Figure 1. Electrode preparation. (A) This panel shows a general view of the electrode sharpening apparatus. The syringe containing 10% NaNO2 (left) is used to sharpen the electrode (right). (B) This panel shows a close view of the electrode tip (a: suitable; b: unsuitable). <a href="/files/ftp_upload/57863/57863fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57863/57863fig2.jpg" /> 
Figure 2. Locust maxillary palp holder (MPH). (A) The MPH and a locust are mounted on the glass slide before positioning it under the microscope. (B) This panel shows a close-up of the locust maxillary palp, fixed by tungsten wire on the platform. <a href="/files/ftp_upload/57863/57863fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57863/57863fig3.jpg" /> 
Figure 3. Single sensillum recordings. (A) This panel shows a view of the electrophysiology setup. (B) This panel shows a close view of the locust preparation mounted on the microscope. (C) This image shows the locust maxillary palp at 100X magnification. (D) This image shows the palp at 500X magnification. The arrow indicates a basiconic sensillum. <a href="/files/ftp_upload/57863/57863fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57863/57863fig4.jpg" /> 
Figure 4. Response traces of single sensillum recordings of the locust maxillary palp. In this panel, pb1 stands for subtype 1 of the palp sensilla basiconica; pb2 stands for subtype 2 of the palp sensilla basiconica. The bars above the traces indicate the stimulus duration (1 s). For these recordings, all odors are used at 10% dilutions except for E-2-hexenal and hexanal, which are diluted to 1%. This figure has been modified from Zhang et al.26. <a href="/files/ftp_upload/57863/57863fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57863/57863fig5v2.jpg" /> 
Figure 5. Comparison of mean numbers of spikes in neurons in pb1 and pb2 stimulated by nonanoic acid and 1-nonanol. The mean numbers of the spikes are calculated in the periods 5 s before and after stimulation. In pb1, the mean numbers of the spikes in the neurons responding to 1-nonanol increase significantly higher than those of the spikes in neurons responding to nonanoic acid (n = 11 palps; ANOVA with post hoc t-tests; p &#60; 0.0001), in contrast to pb2 (n = 10 palps; ANOVA with post hoc t-tests; p = 0.0110). The error bar represents SEM. This figure has been modified from Zhang et al.26. <a href="/files/ftp_upload/57863/57863fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57863/57863fig6v3.jpg" /> 
Figure 6. The patterns of neurons in pb1 and pb2 responding dose-dependently to E-2-hexenal and hexanal. (A) This panel shows the patterns of the neurons in pb1 (&#177; SEM; n = 12 palps). (B) This panel shows the patterns of the neurons in pb2 (&#177; SEM; n = 10 palps). This figure has been modified from Zhang et al.26. <a href="/files/ftp_upload/57863/57863fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

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

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

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.1K Views.  China Agricultural University. This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

Video: Single Sensillum Recordings for Locust Palp Sensilla Basiconica

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 ...

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

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 ...

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

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 ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

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.

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...