In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive is the most ventral part of the medulla, and thus is extremely difficult to reach in a living animal. Here, we introduce a protocol to expose the adult mouse brain stem from the ventral side, and use GRIN lenses to record neuronal activity in the inferior olive neurons expressing calcium sensors. This method avoids damage to life-critical brain stem, and enables investigations on spatial-temporal activity patterns and input integration in the inferior olive.

Since the operation is performed in the complex throat region with many vital structures, it is essential that it is conducted by a researcher with high-level surgical skills. Prepare an intubation tube by cutting a 5 to 6 millimeter long and 0.8 millimeter wide slit from the tip of a 20 gauge catheter. Prepare a blunt and curved needle by cutting the tip off, sand the fracture surface, and bend it with the plier.

This will be used to support a trachea during the tracheotomy. Dilute 15 milligram per milliliter ketamine with saline to 15%Assemble surgery tools and consumables. Set heating pad to 38 degrees Celsius.

Turn the nosecone 180 degrees horizontally in stereotaxic frame. Weigh the mouse whose inferior olive was previously transfected by virus-carrying GCaMP6s, and calculate the amount of diluted ketamine for injection. Pre-fill induction box with 5%isoflurane, and anesthetize the mouse.

Mount the mouse in the stereotaxic frame, ventral side up. Shave mouse throat and thigh area. Remove the residual hair with hair removal cream.

Topically apply xylocaine jelly on throat skin. Monitor the mouse temperature with a rectal thermal sensor. Inject one milliliter pre-warmed saline intraperitoneally.

Assess the depth of anesthesia by strong pinch on hindlimb toes. No detectable response should be evoked. Make a vertical incision in throat skin along the midline.

Separate neck skin from viscera under it by using blunt dissection method and cut the skin off. Free salivary glands from connective tissue, and flip them laterally to expose the sternothyroid muscle-covered trachea. Intraperitoneally inject the first dose of diluted ketamine at five milliliter per kilogram of animal weight.

Carefully split sternothyroid muscle along the midline with the tip of a fine forceps to expose trachea. Detach trachea from blood vessels and esophagus with forceps using blunt dissection method. Inject a second dose of diluted ketamine at 2.5 milliliter per kilogram of animal weight.

Crosswise insert a blunt needle under trachea. Lift trachea with the blunt needle. Make the suture thread go around the third trachea ring, caudal the thyroid gland, with a half circle needle.

Make four instrument ties on this ring. Pierce the chest skin with the same half-circle needle and lead the thread through the skin. Gently lift the trachea with the thread, and cut the trachea caudal to the thyroid gland.

Pull the trachea towards the chest skin. Raise the opening of the trachea by adding a small piece of surgical sponge under it. Remove any remaining liquid inside the opening tip of the trachea with a thin strip of cleaning tissue.

Switch the isoflurane flow from stereotaxic nosecone to the intubation tube. Insert the intubation tube into the trachea. Make sure part of the slit in the tube remains outside of the trachea to allow breathing.

Fix the trachea to mouse chest skin by making three to four instrument ties. Tie the trachea with the suture thread to secure the intubation tube. Slit the sternothyroid muscle along the muscle fibers with the fine forceps.

Cut the isolated part off with the spring scissors. Carefully free leftover trachea and larynx from muscle to minimize the damage on blood vessels in the muscle. Remove leftover trachea and larynx.

Free esophagus from attached tissue with forceps and cut it off with spring scissors. Remove the longitudinal muscle covering the brainstem and the ventral arch of the atlas. Remove the muscle covering the atlas ventral arch and the anterior tubercle.

Cut the ventral arches of atlas with the rongeur. Remove the anterior tubercle of atlas. Remove the blood and fluid to view the foramen magnum and the brain stem.

Expand the foramen magnum by removing the occipital bone with the rongeur. Remove the cartilage, and carefully peel the periosteal layer of the dura mater with the fine forceps to have a clear view of the ventral brainstem. Clamp the SpO2 sensor on the thigh of the mouse to monitor vital signs such as heart rate, oxygen saturation, and breath rate.

Mount the GRIN lens on the implantation rod. Clean the lens carefully with 70%ethanol-soaked cleaning tissue. Fix the implantation rod on stereotaxic frame and mount the miniature microscope on the implantation rod.

Add several drops of saline in the brainstem area for immersion of GRIN lens. Approach the brainstem with the GRIN lens. GCaMP6s expressing inferior olive neurons in the superficial area can be found in a rectangle shaped region, about 0.5 to 1.7 millimeter rostral to the remaining atlas, and about 2.6 to 1.1 millimeter lateral to the midline.

Turn on the excitation blue LED in miniature microscope to locate GCaMP6s transfected inferior olive neurons. The neurons show different baseline fluorescence intensity due to various GCaMP6s expression level. The change of calcium level of circled inferior olive neuron somata in the video on the left will shown us delta F divided by F traces on the right.

Although the mouse is kept warm and hydrated, it will inevitably be weakened by prolonged anesthesia and surgery. It's essential to keep the duration of surgery short so the mouse physical condition will be good for recording. A skilled researcher can finish the surgery in 70 minutes.

This method, with modifications, can be used to study other adjacent regions of the ventral brainstem.