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

The overall goal of this procedure is to measure changes in membrane properties of olfactory sensory neurons in response to odor stimulants. This is accomplished by first dissecting the olfactory epithelium and transferring it into a perfused chamber. The second step is to locate and choose an OSN of interest under the microscope.

The dendritic knob of the chosen OSN is then patch clamped and analyzed while being stimulated with odorants. Ultimately, perforated patch clamping of OSN dendritic knobs characterizes OSN functional properties at the membrane level. This method can help address key questions in the olfaction fields, such as recording olfactory, sensory neurons, expressing defined odor art receptors, And also patch clamp recordings.

Provide information about the membrane properties of these neurons. Prepare the stimulating pipettes from one millimeter glass or purchase them pre pulled using a flame. Bend six pipettes about one centimeter from the tip to a 45 degree angle.

Then arrange the six bent tip pipettes around a seventh straight pipette, using an eyelet as a guide and wrap them in heat shrink tubing. Heat the shrink wrap at the other end of the pipettes. Apply more shrink wrap.

This makes a prepo seven barrel stimulating pipette. Now using a multi barrel polar pull the seven barrel stimulating pipette. Once pulled, strengthen the assembly with some white glue around the eyelet.

Then allow the glue to dry overnight of the required solutions. The Nystatin solution must be made fresh just before it is used. When needed, weigh out three milligrams of Nystatin and add it to 50 microliters of DMSO.

Then vortex mixture for 20 seconds. Next, sonicate the mixture for two to three minutes to ensure a complete dilution. Now add 20 microliters of the mix to five milliliters of intracellular stock solution.

Vortex's mix for 20 seconds, followed by two to three minutes of sonication. Then chill the working solution on ice and keep it shielded from light prepared. This way the working solution is good for a few hours.

Load a microfilm equipped syringe with working solution. It will be good for an hour of use until it is used. However, preserve the loaded syringe by keeping it on ice.

Now pull some recording electrodes with long necks and two micron openings. The goal is to get a resistance of 15 to 20 mega ohms once loaded with Nystatin solution. Finally, load the stimulating pipette with odorant diluted in ringer.Solution.

For this protocol, have mice available between two and four weeks old. In this presentation, mice expressing or IRES tau GFP are used By using or IS tau GFP mice. All neurons expressing the or of interest will be labeled with GFP.

This method can be used for or expressed in all zones. However, dissections and recordings will be easier for ORs expressed in the dorsal zone. Begin by anesthetizing and then decapitating a mouse.

The outcome of this protocol depends on the quality of the dissection. These dissection steps must be as short as possible and be precise. To begin the surgery, use dissecting scissors to make a longitudinal cut along the dorsum.

Pull the skin apart to remove it. Then cut along the lower jaw and make a coronal cut parallel to the teeth. Thus, remove the upper incisor teeth.

Now make a coronal cut around the head, behind the eyes, discard the posterior head and transfer the anterior portion to ice cold ringers. Now under a dissection microscope, isolate the septum within five to 10 minutes. Start with a longitudinal cut along the ventral side.

Then cut the dorsal bones longitudinally along the dorsal lateral side of the nasal cavity. Next, remove most of the bones and the palate. Transfer the isolated septum and attach epithelium to a container of oxygenated ringers at room temperature.

Within five minutes, make the final preparations before recording. The solution must not be too warm, or preparation will look damaged. To access the neuro epithelium for recording.

First, peel the epithelium from the underlying septum with forceps. Then using micro Vanna scissors, cut the anterior end of the septum to release the epithelium. Now carefully remove the vomer nasal organ.

Cut it off where it is attached dorsally. The isolated epithelium can now be moved to the recording chamber mucus side up. Once in place, use a harp to flatten the tissue.

Then transfer the chamber to an upright microscope. Visualize the neuro epithelium under a 40 x water immersion objective while perfusing it continually with fresh ringers at room temperature. To begin search for the cells of interest, which in this case express EGFP, so 480 nanometers light is used to scan for cells that emit light in the 530 to 550 nanometer range.

The targeted dendritic knobs and osn when reviewed at 80 to 160 x under bright field should be clearly distinguishable from the supporting cells. Now load the electrode with an S statin solution. Tap out any bubbles that form and attach the electrode to the holder.

Now apply some positive pressure. The expected resistance is between 15 and 20 mega ohms. Then bring the electrode to the cell while maintaining the positive pressure.

When the resistance increases to about 40 mega ohms, release the pressure and apply a slight negative pressure. If a giga seal is made, the membrane potential should be clamped at about negative 75 millivolts. Now that the cell is open, proceed with the experiment.

Apply stimulation, protocols, and perfuse with pharmacological treatments as needed to test with a single odorant. Record 200 to 500 milliseconds of spontaneous activity. Apply the odorant for 500 milliseconds and then measure responses for up to 10 seconds Using the described protocol.

MOR 23, expressing neurons tagged with EGFP, were patched and stimulated with different concentrations of al. Under current clamp mode, lyal is a ligand for MOR 23. Alternatively, the cells were simulated under the voltage clamp mode.

In voltage clamp mode recordings, different characteristics were monitored. To quantify the response as performed classically in electrophysiology using maximum amplitude response, the dose response was plotted using the hill equation. These results provide information about the encoding properties of each OSN, which includes its detection threshold, temporal, dynamic, dynamic range, and saturation level.

Once mastered, the dissection part of this technique can be done within 10 to 15 minutes. After watching this video, you should have a good understanding on how to explore functional properties of olfactory sensory neurons expressing defined odorant receptors in mice.