マウス鋤鼻器官の急性組織スライスで定義された細胞集団の中で、深さ生理学的解析

The overall goal of this procedure is to perform single cell electro-physiological analysis of a defined population of sensory neurons in acute tissue slices from the mouse vomeronasal organ. This method can help elucidate key questions in the field of chemosensation, such as the mechanisms of see-no chemical detection. The main advantage of this technique is that by recording from sensory neurons in acute tissue slices, these cells can be investigated in their native environment.

To begin this procedure, after sacrificing the animal, remove its lower jaw, by inserting a pair of large surgical scissors through the mouth cavity, and cut the mandible bones and muscle of each side separately. Next, remove the skin of the upper jaw and around the tip of the nose with medium forceps, to gain better access to the incisors. Then use the bone scissors to cut away the largest part of the incisors at a 45 degree angle in the rostral direction to ease the VNO capsule removal from the nasal cavity.

Do not cut the root of the tooth to prevent damage to the tip of the VNO capsule. After that, grab the rigid upper palate at its rostral part with medium forceps, and carefully peel back in one piece at a flat angle. Repeatedly rinse the naval cavity with ice cold solution S2.Then use micro spring scissors to cut the bone effusion between the tip of the vomer bone and the jaw.

Insert the scissor tips with the curved part pointing away from the VNO. And carefully cut the bone in small steps on both the left and right side, lateral to the VNO capsule. To remove the VNO capsule, cut through the vomer bone at the caudal part, and carefully lift the vomer bone out of the nasal cavity using medium forceps.

Immediately transfer the VNO to a small petri dish under a stereo microscope on an ice gel pack, where the remaining steps of the dissection should be performed. Next, rinse the VNO in a small amount of ice cold S2, to prevent the tissue from drying out. Then, separate the cartilaginous capsules that contain the VNO soft tissues by grabbing the back of the vomer bone with medium forceps.

For that, position the capsule for a dorsal view so that a split between both VNOs becomes visible. Use the tip of fine forceps to separate both cartilage VNO capsules from the central bone, while keeping the vomer bone pinned down at the rear part. Make sure the medial cartilage wall that was previously attached to the vomer bone is removed.

When removing cartilage around the VNO use forceps only at the rim of the cartilage capsule. And be very careful not to damage the delicate sensory epithelium. To remove remaining cartilage, turn the VNO with its curved lateral side to the bottom of the dish and securely pin down the cartilage on one side using forceps.

Then carefully move the second fine forceps from the back side at a very flat angle between cartilage and VNO to loosen the connection between tissue and cartilage. To avoid damaging the sensory epithelium slowly peel the VNO away from the cartilage by holding it at its caudal tip. Once the VNO is levered from the capsule, remove all the remaining small cartilage parts as any remaining pieces of cartilage will detach the tissue from the surrounding agarose during the slicing process.

Next, place a small drop of ice cold S2 on the first dissected VNO to prevent tissue damage. To embed the VNOs, fill both small petri dishes to the rim with melted S3.After that, immerse each VNO in agarose and move it horizontally back and forth several times to remove the film of extracellular solution as well as any air bubbles from its surface. Position the VNO vertically, with the rostral tip facing the bottom of the dish.

Subsequently, place both the dishes on a gel ice pack, and wait until agarose has solidified. Do not change VNO orientation once the agarose has started solidifying, as this will detach the tissue from the surrounding agarose. In this procedure, use a small spatula to remove the agarose block from the small dish into the lid of a large petri dish, and flip the agarose upside down, leaving the rostral tip of the VNO facing upwards.

Next, cut the block into a pyramidal shape using a surgical scalpel, and take care not to damage the embedded tissue. Then, use super glue to fix the pyramid shaped block to the center of the vibratome specimen plate. And wait about one minute for the glue to dry completely.

Slice the specimen into 150 to 200 micrometer thick sections. Briefly inspect the slice morphology under the dissecting microscope. Then transfer all the slices to the oxygenating chamber until use.

In this step, transfer a VNO slice to the imaging chamber and fix the slice position using stainless steel anchor wired with point one milometer thick synthetic fibers. Do not cover the tissue slice with any of the synthetic fiber threads. Next, transfer the imaging chamber to the recording setup and continuously superfuse the slice with oxygenated S2 at room temperature.

Adjust the suction capillary to the surface of the solution to create a constant exchange of bath solution. Then adjust the eight in one multi barrel profusion pencil above and close to the non sensory part of the VNO slice that contains the blood vessel. Afterward connect the reference electrode and bath solution using the L shaped agar bridge filled with 150 millimolar potassium chloride.

Subsequently fill the patch pipette with pipette solution S4.Mount the pipette over the silver chloride coated electrode connected to the head stage without scraping off the coating and attach firmly. Then apply slight positive pressure to the patch pipette before entering the bath. Monitor the pipette and make sure that it is between four megaohms and 10 megaohms.

Visualize the VNO slice with a CCD camera using infrared optimized DIC, and identify FPR-rs3 i-Venus expressing cells or similarly labeled neurons using florescence illumination and an appropriate filter cube. Approach the cell body using hand wheels for maximum sensitivity. Due to the positive pressure a small dent on the cell soma membrane becomes visible once the pipette tip is close proximity.

Then release the positive pressure and apply slight negative pressure to suck in the cell membrane in order to gain a high resistance seal of one to 20 gigaohms. Subsequently apply short and gentle suction to disrupt the cell membrane and establish the whole cell configuration. In this figure, a confocal image of a coronal VNO tissue slice shows the distribution of fluorescent FPR-rs3 Tau Venus positive neurons in the vomeronasal sensory epithelium.

The FPR-rs3 Tau Venus positive neurons exhibit a single apical dendrite ending in a knob like structure at the luminal border. And shown here are the whole cell patch clamp recordings performed from the VSN soma. Here are the representative traces from wholesale patch clamp recordings of a TTX sensitive fast activating sodium current, and FPR-rs3 positive VSNs.

Voltage step recordings under control conditions reveal a voltage dependent fast and transient inward current. TTX treatment at one micromolar strongly diminishes the current. And the digitally subtracted trace shown here reveals the TTX sensitive voltage gated sodium current.

The representative current clamp traces show depolarization and hyper-polarization and trains of action potentials generated upon step-wise positive and negative current injection. Once mastered, the whole dissection and tissue slicing process can be done in approximately one hour for both VNOs. Following this procedure, other methods such as extra-cellular loose patch recordings or calcium imaging can be performed in order to increase throughput when analyzing larger populations of neurons.

After watching this video, you should have a good understanding of how to make acute tissue slices from the mouth vomeronasal organ, and how to perform single cell electro-physiological recordings from vomeronasal sensory neurons.