Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The overall goal of this procedure is to produce an ex vivo preparation of the functionally connected mouse ro nasal organ, or VNO, an accessory olfactory bulb, or a OB.This is accomplished by first performing a coarse dissection of the mouse cranium to isolate a single hemisphere of the snout and olfactory bulb. In the second step, the delicate VNO axons and a OB are exposed through a secondary dissection in a tissue perfusion chamber. Finally, a thin cannula is inserted into the VNO through which the odor and stimulus can be introduced.

Ultimately, electrophysiological recordings can be used to measure the neural activity within the A OB during the VNO stimulation. The made advantage of this technique over existing techniques like in vitro slice electrophysiology, is with this technique, the sensory organ and downstream neural circuits remain functionally connected. Visual demonstration of this method is critical, as there are many delicate dissection steps that are difficult to learn, and because errors on any one of these steps may result in a failed dissection To begin the primary dissection, first use Addin forceps to remove the parietal and frontal bones of the mouse's skull, taking care not to make contact with the olfactory bulbs.

Next, use a scalpel to make a coronal cut through the frontal lobe, two millimeters coddle to the sinuses, and then remove the brain coddle to the cut. Use straight scissors to remove the exposed coddle aspects of the ventral skull, leaving the sinuses and remaining nervous tissue. Remove the zygomatic arch and facial muscles on the anatomical right side of the skull, and then turn the snout ventral side up.

To expose the roof of the mouth, immediately insert the tip of the scalpel blade under the palette parallel to the roof of the mouth, and then move the blade towards the incisors. Grasp the freed rostral edge of the palette with the forceps and remove the palate by pulling coddly. Then on the anatomical left side of the skull, insert only the scalpel blade tip into the void near the molars, and rotate the wrist to extend the palatine foramen coddly.

Starting from the small foramen near the molars, use the tip of the scalpel blade to continue to cut from the palatine foramen through the incisors, starting rostral to the anatomical left VNO with multiple scoring cuts. Then turn the tissue so the dorsal skull is facing up and orient a straight razor blade vertically and parallel to the midline at the coddle edge of the preparation at the ventral edge of the tissue, touch the cutting edge of the razor blade immediately to the left of the midline and gently rock the blade moving along the left palatine for Raymond, keeping the blade parallel to the midline rock. The razor blade slowly with pressure towards the anterior of the tissue, stopping immediately after breaking through the resistance at the cribriform plate.

Grasp the exposed hemispheres near the coddle sides with gloved fingers and gently rotate them laterally and anteriorly to separate the tissues. Now apply 50 to 100 microliters of tissue glue to a thin plastic plank. Grasp the tissue with forceps and blot the preparation with a paper towel to remove excess moisture from its lateral side.

Use the forceps to gently place the lateral edge of the right hemisphere onto the glue. Then place a three to five millimeter diameter, two millimeter deep drop of vacuum grease into the middle of a secondary dissection chamber, and place the side of the plank opposite the tissue firmly against the grease. Immediately fill the dissection chamber with chilled dissection, artificial cerebrospinal fluid, or A CSF, and transfer the chamber to the fine dissection area.

Then begin profusion of the chamber with oxygenated standard A CSF orienting the plank so that the olfactory bulb is directly in the stream of the freshly oxygenated fluid to perform. The secondary dissection Begin by using fine forceps to remove any visible contralateral olfactory bulb or cortical tissue. From the preparation, locate the white dura mater along the dorsal medial surface of the olfactory bulb, and then using two pairs of fine forceps, tear the dura by grasping it at the whitest portion and pulling the forceps apart.

Next, slowly and carefully, peel away the frontal cortex with fine forceps without damaging the underlying olfactory bulbs. Once the frontal cortex has been separated, cut and remove the tissue ventral and posterior to the A OB taking care not to damage the glomerular layer. Then run a single point of the fine forceps between the septal cartilage and the VNO wing, a thin piece of bony tissue that runs along the dorsal edge of both VN Os.To carefully remove the contralateral VNO after removing the VNO completely use fine forceps to make a cut at the ventral coddle edge of the septal cartilage, where it narrows to a white avascular running along the ventral edge of the septal bone.

Pinch together the fine forceps to make a semi blunt point and insert the pinch forceps lateral to the cut. Then slowly lift the cartilage away from the septal tissue proceeding roly and without disturbing the underlying septal tissue. Grasp the bone with forceps and gently move the bone back and forth until it cracks near the crib reform plate.

Now, attach a polyamide cannula to a fast perfusion device and start a flow of ringer solution at 0.1 to 0.3 milliliters per minute through the polyamide cannula. Measure the flow rate with an inline small volume flow meter. Finally, orient the cannula parallel to the VNO entrance.

When the cannula is in place, observe as the outlet pressure causes the VNO to inflate leading to the evacuation of the blood vessel. Then immediately insert the cannula into the VNO, keeping the angle of the cannula constant so that it remains parallel to the VNO opening and commence the assessment of the physiological function. A large source of variability across the preparations is the density and brittleness of the bones of the skull and snout of the experimental animals.

It is not uncommon for the septal tissues to remain attached to the contralateral hemisphere, especially near the attachment points to the dorsal bones of the snout. Other common dissection errors arise during the secondary fine dissection, during which the axons of the RO nasal nerve can be damaged in the septal tissue near the VNO or near the a OB.These and similar events will result in incomplete connectivity between the RO nasal sensory neurons and the A OB rendering. The preparations unusable upon successful completion of the procedure.

The functional connectivity can be verified using a physiological assay such as single unit electrophysiological recordings of neural activity in response to VNO stimulation with odorants. While attempting this procedure, it is important to remain calm and take short breaks. If you become fatigued, small errors in performance of this technique can damage these delicate neural structures.

After watching this video, you should have a good understanding of how to perform ex vivo dissections of the early accessory factory system for electrophysiological or imaging studies.