Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice
Transcrição
For this protocol, have mice available between 2 and 4 weeks old. In this presentation, mice expressing OR/IRES/tauGFP are used.
By using OR/IRES/tauGFP 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 OR 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 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 Ringer's.
Now, under a dissection microscope, isolate the septum within 5 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 attached epithelium to a container of oxygenated Ringer's at room temperature. Within five minutes, make the final preparations before recording.
The solution must not be too warm, or the preparation will look damaged. To access the neuroepithelium for recording, first, peel the epithelium from the underlying septum with forceps. Then, using micro-Vannas scissors, cut the anterior end of the septum to release the epithelium.
Now, carefully remove the vomeronasal 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 neuroepithelium under a 40x water immersion objective while perfusing it continually with fresh Ringer's 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 OSNs, when reviewed at 80 to 160x under bright field, should be clearly distinguishable from the supporting cells.
Now, load the electrode with the nystatin 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 megaohms. Then, bring the electrode to the cell while maintaining the positive pressure.
When the resistance increases to about 40 megaohms, release the pressure and apply a slight negative pressure. If a gigaseal 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.
