We thank Dr. Yijun Song, and members of the Hattar Kuruvilla Zhao tri-lab of the Department of Biology, Johns Hopkins University for advice and help. Supported by NIH grants DC007395 and DC009946.

Part 1. The EOG recording setup
The recording apparatus consists of a recording electrode, reference electrode, air delivery tube, specimen stage, and dissecting microscope, all anchored on an air table within a Faraday cage. Micromanipulators are used for placement of the electrodes and the air delivery tube. A continuous air stream is bubbled through distilled water to add humidity before passing through the air delivery tube and over the specimen. A 60 mm culture dish filled with Sylgard to a depth of 6-8 mm is used as a mounting surface for the specimen. A well and a channel are hollowed out of the Sylgard in the mounting dish to provide a means to electrically connect the reference electrode to the specimen via modified Ringer's solution.
The recording electrode and the reference electrode are connected to an amplifier. Signals from the amplifier are sent to a digitizer and then to a computer. Software such as Axograph or pClamp can be used to control the stimulation protocol, to record the signal, and for subsequent analysis of the responses. An oscilloscope connected after the amplifier can be convenient for real time monitoring of the electrical potential while placing the recording electrode and during EOG recordings.
Delivery of odorant stimuli is controlled by a Picospritzer, which is connected to the same computer used for signal acquisition. The air pressure at the Picospritzer is set to 10 psi. A single air tank and regulator can be used to supply air to both the air table and the Picospritzer. A second air tank and regulator is used to provide air for the humidified air stream, as this requires a lower pressure and a large amount of airflow. Just prior to delivering an odorant stimulus, the Picospritzer output is connected to an odorant bottle. The odorant bottle is then connected to the air delivery tube.
Part 2: Preparing electrodes
The recording electrode is a chlorided silver wire in a pulled glass capillary filled with modified Ringer's solution (135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1.5 mM MgCl2, 10 mM HEPES, pH 7.4, filter sterilized). The reference electrode is a chlorided silver wire.
<ol>
 <li>Install silver wire into the electrode holder. For the recording electrode, one to two centimeters of wire should protrude from the end of the electrode holder. More wire can be left for the reference electrode.</li>
 <li>To add the AgCl coat to the silver wire, position the wire in 0.1 M NaCl and connect the electrode holder to the positive terminal of a 1.5-9 V DC power source. The negative terminal of the power source should be electrically connected to the 0.1 M NaCl solution. Allow the chloriding reaction to proceed for 10 minutes. To equalize any static charge between the recording and the reference electrode, briefly touch the electrodes together before installing them on the recording apparatus.</li>
 <li>Pull a glass capillary using a micropipette puller. The opening at the tip of the capillary should be around 5-10 microns in diameter. </li>
 <li>Use a diamond pencil to score and break off the blunt end of the capillary so it is ~2 cm longer than the silver wire. Fire-polish the cut end with the butane torch.</li>
 <li>Melt 0.5% agarose in modified Ringer's solution. Pull a small amount of molten agarose solution into the tip of the electrode using a transfer pipette. </li>
 <li>Fill the pulled capillary about 1/2 of the way with modified Ringer's solution (a syringe that has been heated and pulled to have a long thin end is useful for this purpose). Gently flick the capillary to dislodge any air bubbles. Store the filled electrode in the storage jar with a small amount of modified Ringer's solution in the bottom until they are ready to be used. Once a tissue sample is prepared and ready for recording, install a filled capillary over the recording electrode wire.</li>
</ol>
Part 3: Preparing odorant solutions
The odorants amyl acetate and heptaldehyde evoke large responses and are thus good choices as EOG stimulants.
<ol>
 <li>In microcentrifuge tubes, prepare a series of dilutions of odorant in dimethyl sulfoxide (DMSO). As a starting point for a dose-response curve, prepare 10-fold dilutions from 5 M to 5 x 10-6 M. Make fresh dilutions each day.</li>
 <li>Further dilute the odorants 50-fold in water by mixing 100 &mu;L diluted stock in DMSO with 4.9 mLs water in 2 ounce bottles with silicone stoppers. Let the solutions equilibrate in the bottles for at least 30 minutes. Note that the vapor concentration of odorants in each bottle is unknown, but will vary as a function of the concentration of odorants in the liquid phase. </li>
 <li>Insert two 18-gauge needles through the silicon stopper to provide input and output ports. The ports should be plugged when the bottle is not in use.</li>
</ol>
Part 4: Recording the EOG and analyzing data
<ol>
 <li>Sacrifice a mouse by CO2 euthanasia or anesthetic overdose followed by decapitation. Remove the skin overlying the skull and sagittally bisect the head along the midline. </li>
 <li>Mount one half of the head, medial side up, on the mounting dish. Carefully remove the septum to expose the turbinates.</li>
 <li>Place the dish with mounted tissue onto the recording stage. Align the stage so that the recording location on the turbinates is centered under the microscope.</li>
 <li>Turn on the air tank to deliver humidified air to the turbinate surface. Position the air delivery tube so that it is approximately 10 mm away from the recording location. The flow rate is ~600 mL/min. </li>
 <li>Set the amplifier to DC mode (AC amplification will induce artifacts in the EOG signal) with low-pass filter at 1 kHz, and gain at 100X. </li>
 <li>Mount recording and reference electrodes on the micromanipulators. </li>
 <li>Lower the reference electrode into the well on the mounting dish and cover with modified Ringer's solution such that it is electrically connected to the tissue.</li>
 <li>Carefully lower the recording electrode onto the surface of turbinate IIb or III. The electrode should barely touch the surface of the olfactory epithelium! When the electrode comes in contact with the epithelium (i.e. completes an electrical circuit) a straight baseline will appear in the oscilloscope. </li>
 <li>Attach an odorant bottle to the side port on the air delivery tube.</li>
 <li>On the computer, initiate a stimulation protocol. The sampling rate for data acquisition should be 2 kHz or higher. The software will trigger an odor pulse and begin recording. </li>
 <li>A typical stimulation protocol may be a 100-msec duration single pulse, paired 100-msec pulses separated by a 1-sec interval, or a 10-sec sustained pulse.</li>
 <li>Allow some time between protocols so the tissue is minimally adapted. One minute is sufficient for liquid concentrations of amyl acetate and heptaldehyde up to 10-3 M; at higher concentrations allow 5 minutes. After delivering high odor concentrations (such as at the end of a dose-response curve) residual odor may remain in the tube. Wash the air tube with 95% ethanol and dry before continuing with additional tissue samples.</li>
 <li>Axograph software provides tools for measuring key parameters of the EOG signal. Such parameters include the response amplitude, latency, time to peak, and time constants of termination. It may be desirable to digitally filter traces at 25 Hz before further analysis.</li>
</ol>
Representative Results
<img src="/files/ftp_upload/1850/1850fig1.jpg" alt="figure 1" /> Figure 1. Parameters for EOG analysis. Several parameters of the EOG are particularly useful for comparison of responses between mice, including the response amplitude, the latency (the time between when the stimulus is administered and the response begins), rise time (the time between the start of the response and the peak), time to peak (the time from the start of the stimulation to the peak of the response), and time constant of termination (&tau;, determined by fitting the decay phase of the response to a single exponential equation). For comparison of kinetic parameters such as latency, rise time, and time constant of termination, it is advisable to normalize the peak amplitude of the responses prior to analysis.
<img src="/files/ftp_upload/1850/1850fig2.jpg" alt="figure 2" /> Figure 2. Representative EOG signals under different stimulation protocols. (a) Examples of EOGs from a mouse in response to stimulation with increasing concentrations of amyl acetate. The black line at the top of the panel indicates the timing and duration of odorant stimulation. The concentrations in the legend are the concentrations of the liquid solution. (b) A dose-response relation averaged from five mice. Error bars are 95% confidence intervals. A decline in the peak amplitude is often observed at very high odor concentrations. (c) An example of an EOG in response to a paired-pulse stimulation. A single short pulse of odorant elicits adaptation lasting for several seconds. (d) An example of an EOG in response to a 10-sec sustained odorant stimulation. The EOG shows desensitization during continuous odorant presentation.

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H., Ngai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anosmia+caused+by+a+targeted+disruption+of+the+mouse+olfactory+cyclic+nucleotide-gated+cation+channel.">General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel.</a> Neuron. 17, 681-693 (1996).</li><li>Belluscio, L., Gold, G. H., Nemes, A., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+deficient+in+G(olf)+are+anosmic.">Mice deficient in G(olf) are anosmic.</a> Neuron. 20, 69-81 (1998).</li><li>Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+a+mammalian+odorant+receptor.">Functional expression of a mammalian odorant receptor.</a> Science. 279, 237-242 (1998).</li><li>Wong, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+type+III+adenylyl+cyclase+gene+leads+to+peripheral+and+behavioral+anosmia+in+transgenic+mice.">Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice.</a> Neuron. 27, 487-497 (2000).</li><li>Munger, S. 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D., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphodiesterase+1C+is+dispensable+for+rapid+response+termination+of+olfactory+sensory+neurons.">Phosphodiesterase 1C is dispensable for rapid response termination of olfactory sensory neurons.</a> Nat Neurosci. 12, 454-462 (2009).</li><li>Kleene, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrochemical+basis+of+odor+transduction+in+vertebrate+olfactory+cilia.">The electrochemical basis of odor transduction in vertebrate olfactory cilia.</a> Chem Senses. 33, 839-859 (2008).</li><li>Chen, S., Lane, A. 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Mice were handled and euthanized with methods approved by the Animal Care and Use Committees of The Johns Hopkins University.

With the setup described in this protocol, the odorant stimuli at the surface of the olfactory epithelium will be consistent between tissue preparations, allowing for comparison between wild type and mutant mice, even though the exact odorant concentration and dynamics are unknown. Several factors, particularly the recording location and the flow rate of humidified air, cause variations in the EOG. Care should be taken to record from similar positions on the same turbinate to minimize variation. This can easily be achieved by consistently recording from the same side of the head and keeping the footprint of the microscope, odor delivery tube, and micromanipulators on the air table unchanged between tissue samples. In addition, tissue samples should be immediately placed into the humidified air stream after dissection to prevent excessive drying of the tissue.
EOG recordings on mice can also be carried out with a liquid perfusion apparatus on prepared mouse turbinates7, 14, 15, or by leaving the head intact and inserting the electrode into a small hole drilled above the turbinates16, 17. Each variation of EOG recording has its own strengths: air-phase recordings on tissue preparations as described in this protocol require a minimal amount of setup and are the easiest to conduct; recordings using a liquid perfusion apparatus facilitate the use of pharmacological reagents, although the hydrophobic nature of many odorants complicates odor delivery; lastly, recordings in which the head is left intact can be used in 'artificial sniff' experiments, although electrode placement is more difficult than when the turbinates are fully exposed.

<table><tbody><tr><td>Air delivery tube</td><td>equipment</td><td>Custom Made</td><td></td><td>The barrel of a 1-mL syringe with a T-fitting can be used as a substitute</td></tr><tr><td>Air table</td><td>equipment</td><td>Newport Corp.</td><td>LW3030B-OPT</td><td></td></tr><tr><td>Amplifier</td><td>equipment</td><td>Warner Instruments</td><td>DP-301</td><td></td></tr><tr><td>Computer and Data Acquisition Software</td><td>equipment</td><td>Axograph 4.9.2 on Apple Macintosh</td><td></td><td>Updated versions of Axograph for Mac OS X and Windows are available from http://axographx.com/.</td></tr><tr><td>Butane torch</td><td>equipment</td><td></td><td></td><td>A cr&amp;#232;me br&amp;#251;l&amp;#232;e torch works well</td></tr><tr><td>Digitizer</td><td>equipment</td><td>Axon Instruments</td><td>Digidata 1322A</td><td></td></tr><tr><td>Dissecting Scope</td><td>equipment</td><td>Scienscope</td><td>SSZ</td><td></td></tr><tr><td>Electrode holder</td><td>equipment</td><td>Harvard Apparatus</td><td>64-1021</td><td></td></tr><tr><td>Magnetic Holding Devices (12 mm)</td><td>equipment</td><td>World Precision Instruments, Inc.</td><td>M10</td><td></td></tr><tr><td>Micromanipulators</td><td>equipment</td><td>World Precision Instruments, Inc.</td><td>M3301R M3301L</td><td></td></tr><tr><td>Micropipette Puller</td><td>equipment</td><td>Sutter Instrument Co.</td><td>P2000</td><td></td></tr><tr><td>Oscilloscope</td><td>equipment</td><td>Tektronix, Inc.</td><td>5110</td><td></td></tr><tr><td>Picospritzer III</td><td>equipment</td><td>Parker Hannifin Corporation</td><td></td><td></td></tr><tr><td>Silicone tubing</td><td>equipment</td><td>Nalge Nunc international</td><td></td><td></td></tr><tr><td>Specimen stage</td><td>equipment</td><td>Custom Made</td><td></td><td>Any small solid object can be used to elevate the mounting dish. Immobilize the dish with modeling clay.</td></tr><tr><td>18 gage needles</td><td>material</td><td>BD Biosciences</td><td>305195</td><td></td></tr><tr><td>2 oz. glass bottles</td><td>material</td><td>VWR international</td><td>16152-201</td><td></td></tr><tr><td>Glass capillaries</td><td>material</td><td>World Precision Instruments, Inc.</td><td>TW150F-6</td><td></td></tr><tr><td>Silicone stoppers size 16D</td><td>material</td><td>Chemware</td><td>D1069809</td><td></td></tr><tr><td>Silver wire</td><td>material</td><td>World Precision Instruments, Inc.</td><td>AGW1010</td><td></td></tr><tr><td>SylGuard 184</td><td>material</td><td>Dow Corning</td><td>SYLG184</td><td>From World Precision Instruments</td></tr><tr><td>Agarose</td><td>reagent</td><td>Invitrogen</td><td>15510-027</td><td></td></tr><tr><td>Amyl acetate</td><td>reagent</td><td>Aldrich</td><td>W504009</td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>reagent</td><td>Sigma-Aldrich</td><td>C-1016</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>reagent</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>HEPES</td><td>reagent</td><td>Fisher Scientific</td><td>BP310</td><td></td></tr><tr><td>Heptaldehyde</td><td>reagent</td><td>Aldrich</td><td>H2120</td><td></td></tr><tr><td>Magnesium chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;+6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>reagent</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>reagent</td><td>JT Baker</td><td>3624-05</td><td></td></tr><tr><td>flowmeter</td><td>equipment</td><td>Gilmont Instruments</td><td>GF-2260</td><td></td></tr></tbody></table>

analyzing responses of mouse olfactory sensory neurons using the air-phase electroolfactogram recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

Watch this Scientific Journal Video about Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording at JoVE.com

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

analyzing-responses-mouse-olfactory-sensory-neurons-using-air-phase

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

16.6K Views.  Johns Hopkins University. The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Video: Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

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 ...

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

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 ...

Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory sensitivity in high PCO2/low pH water. To assess the effects of ocean acidification, or olfactory sensitivity in marine fish in general, we propose that extracellular multi-unit recording from the olfactory nerve is the method of choice. Although invasive, it is sensitive, robust, reproducible and independent of external salinity (unlike the electro-olfactogram [EOG], for example). Furthermore, it records a primary sensory input into the CNS, prior to any central processing. We show that this method can show a reduction in olfactory sensitivity that is both temporary and odorant-dependent, using a range of amino acids to construct concentration-response curves and calculate the thresholds of detection.

Extracellular multi-unit recording from the olfactory nerve is a sensitive, robust and reproducible method for assessing olfactory sensitivity in marine fish. It records primary sensory input and is independent of external salinity.

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory ...

Environment

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 ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

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, ...

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

Source: Jarriault, D., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52652">Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor.</a> J. Vis. Exp. (2015).
This video demonstrates electrophysiological recording of olfactory sensory neurons (OSNs) in mice using a perforated patch-clamp technique. The olfactory epithelium, containing OSNs with fluorophore-tagged odor receptors, is isolated from a mouse and secured in a recording chamber. A recording pipette is clamped to the dendritic knob of an OSN, and nystatin within the pipette perforates the membrane to form electrical connectivity with the cell. Finally, another pipette is used to deliver an odor stimulant, and the electrophysiological activity of the patched cell is recorded.

Perforowany zapis węchowych neuronów czuciowych u myszy

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

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

Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts

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

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

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

Perforowany zapis węchowych neuronów czuciowych u myszy