空気相Electroolfactogram記録を用いたマウス嗅覚神経細胞の分析応答

A preparation of olfactory epithelium is mounted on a dish and placed into a stream of humidified air. Under a dissecting microscope, a reference electrode is electrically connected to the tissue through a buffer solution. A recording electrode is placed on the surface of the olfactory epithelium.

The olfactory epithelium is stimulated by computer controlled odorant puffs administered into a stream of humidified air, which carries the odorants to the olfactory epithelium. EOG signals are transmitted through an amplifier and digitizer to the recording computer. Hi, I am Catherine Ciar from the Laboratory of Hygiene XO at the Department of Biology at the Johns Hopkins University.

Today we'll show you a procedure for recording the electro olf facto gram from mice. We use this procedure in our laboratory to study the responses of olfactory sensory neurons to odors. So let's get started.

The recording apparatus consists of a recording electrode reference electrode air delivery tube, specimen stage, and dissecting microscope all anchored onto an air table within a Faraday cage. Microm manipulators are used for placement of the electrodes in the air delivery tube. A continuous airstream is bubbled through distilled water to add humidity before passing through the air delivery tube and over the specimen.

A 60 millimeter culture dish filled with sard to adapt of six to eight millimeters is used as a mounting surface for the specimen. A well and a channel are hollowed out of thes guard in the mounting dish. To provide a means to electrically connect the reference electrode to the specimen via modified ringer solution.

The recording and reference electrodes are connected to an amplifier. Signals from the amplifier are sent to a digitizer and then to a computer. XI graph software is used to control the stimulation protocol, record the signal, and analyze the responses.

An oscilloscope is connected after the amplifier for real-time monitoring of the electrical potential while placing the recording electrode. And during EOG recordings. Delivery of the odorant stimuli is controlled by a pico spritzer, which is connected to the same computer used for signal acquisition just prior to delivering an odorant stimulus.

The pico spritzer output is connected to an odorant bottle containing liquid odorant. The bottle is then connected to the air delivery tube. Now let's see how to prepare electrodes for recording experiments.

The recording electrode is a chloride silver wire in a pulled glass capillary filled with modified ringer solution. The reference electrode is a chloride silver wire. Begin preparation of both electrodes by installing a 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. To add the silver chloride coating position the wire in 0.1 molar sodium chloride and connect the electrode holder to the positive terminal of a 1.5 to nine volt DC power source.

The negative terminal of the power source should be electrically connected to the 0.1 molar sodium chloride solution. Allow the chloride in 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.

Next, pull a glass capillary using a micro pipette puller. The opening at the tip of the capillary should be around five to 10 microns in diameter. Use a diamond pencil to score and break off the blunt end of the capillary so it is about two centimeters longer than the silver wire.

Use a butane torch to fire polish the cut end now melt 0.5%agros in modified ringer solution. Use a transfer pipette to pull a small amount of molten agro solution into the tip of the electrode using a syringe that has been heated and pulled to have a long thin end. Fill the pulled capillary about half of the way.

With modified ringer solution, gently flick the capillary to dislodge any air bubbles. Store the filled electrode in the storage jar with a small amount of modified ringer solution in the bottom until ready to be used. Once a tissue sample is prepared and ready for recording, install a filled capillary over the recording electrode wire with the electrodes ready.

Let's look at a recording experiment to begin preparing tissue for recording. Remove the skin overlying the skull of a sacrificed mouse and ally bisect the head along the midline mount one half of the head medial side up on the mounting dish. Carefully remove the septum to expose the turbinates.

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. Now turn on the air tank to deliver humidified air at around 600 milliliters per minute to the turbinate surface.

Position the air delivery tube so that it is approximately 10 millimeters away from the recording location. Set the amplifier to DC mode as AC amplification will induce artifacts in the EOG signal. Acquire with a low pass filter of one kilohertz and gain of 100 x.

Now mount the recording and reference electrodes on the micro manipulators. Lower the reference electrode into the well on the mounting dish and cover it with modified ringer solution such that it is electrically connected to the tissue. Carefully lower the recording electrode onto the surface of turbinate two B or three.

The electrode should barely touch the surface of the olfactory epithelium. When the electrode comes in contact with the epithelium, a straight baseline will appear on the oscilloscope. Now, attach an odorant bottle to the side port on the air delivery tube on the computer.

Initiate a stimulation protocol. The sampling rate for data acquisition should be two kilohertz or higher. The software will trigger an odor pulse and begin recording.

A typical stimulation protocol may be a 100 millisecond duration, single pulse paired, 100 millisecond pulses separated by a one second interval or a ten second sustained pulse. To minimize adaptation, allow from one to five minutes between protocols after delivering high odor concentrations. Residual odor may remain in the tube, wash the air tube with 95%ethanol and dry before continuing with additional tissue samples.

Axo graft 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. Several parameters of the EOG are particularly useful for comparison of normalized responses between mice, including the response amplitude, the latency rise time, time to peak and time constant of termination.

Shown here are OGs from a mouse in response to stimulation with increasing concentrations of liquid amyl acetate. The black line at the top indicates the timing and duration of odorant stimulation. Here we see 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. An example of an EOG in response to a paired pulse stimulation is shown a single short pulse of odorant elicits adaptation lasting for several seconds. Shown here is an EOG in response to 10 seconds of sustained odorant stimulation.

The EOG shows desensitization during continuous odorant presentation. We've just shown you how to record the electrode factor gram from mice. When doing this procedure, it's important to remember to place the electrode just on the surface of the epithelium and not to penetrate the electrode below the surface.

Always record from the same side of the head and avoid modifications to the setup to minimize variation, keep the tissue humid, but without collecting liquid droplets, putting the tissue under the humidified air immediately after dissection will keep the tissue in the best state for recording. So that's it. Thanks for watching and good luck with your experiments.