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 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.
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.
Part 3: Preparing odorant solutions
The odorants amyl acetate and heptaldehyde evoke large responses and are thus good choices as EOG stimulants.
Part 4: Recording the EOG and analyzing data
Representative Results
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 (τ, 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.
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.
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.
The authors have nothing to disclose.
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.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Air delivery tube | equipment | Custom fabricated | The barrel of a 1-mL syringe with a T-fitting can be used as a substitute | |
Air table | equipment | Newport | LW3030B-OPT | |
Amplifier | equipment | Warner | DP-301 | |
Computer and Data Acquisition Software | equipment | Axograph 4.9.2 on Apple Macintosh | Updated versions of Axograph for Mac OS X and Windows are available from http://axographx.com/. | |
Butane torch | equipment | A crème brûlèe torch works well | ||
Digitizer | equipment | Axon Instruments | Digidata 1322A | |
Dissecting Scope | equipment | Scienscope | SSZ | |
Electrode holder | equipment | Harvard Apparatus | 64-1021 | |
Magnetic Holding Devices (12 mm) | equipment | World Precision Instruments | M10 | |
Micromanipulators | equipment | World Precision Instruments | M3301R M3301L |
|
Micropipette Puller | equipment | Sutter Instrument Co. | P2000 | |
Oscilloscope | equipment | Tektronix | 5110 | |
Picospritzer III | equipment | Parker Instrumentation | ||
Silicone tubing | equipment | Nalge Nunc | ||
Specimen stage | equipment | Custom fabricated | Any small solid object can be used to elevate the mounting dish. Immobilize the dish with modeling clay. | |
18 gage needles | material | Becton Dickinson | 305195 | |
2 oz. glass bottles | material | VWR International | 16152-201 | |
Glass capillaries | material | World Precision Instruments | TW150F-6 | |
Silicone stoppers size 16D | material | Chemware | D1069809 | |
Silver wire | material | World Precision Instruments | AGW1010 | |
SylGuard 184 | material | Dow Corning | SYLG184 | From World Precision Instruments |
Agarose | reagent | Invitrogen | 15510-027 | |
Amyl acetate | reagent | Aldrich | W504009 | |
Calcium chloride (CaCl2) | reagent | Sigma | C-1016 | |
Dimethyl sulfoxide (DMSO) | reagent | Sigma | D5879 | |
HEPES | reagent | Fisher | BP310 | |
Heptaldehyde | reagent | Aldrich | H2120 | |
Magnesium chloride hexahydrate (MgCl2+6H2O) | reagent | Sigma | M9272 | |
Sodium chloride (NaCl) | reagent | JT Baker | 3624-05 | |
flowmeter | equipment | Gilmont | GF-2260 |