Our laboratory investigates the effects of commonly used anesthetics on sleep-related neurons and their communication pathways. We aim to better understand how anesthetics impact endogenous sleep pathways and affect perioperative sleep and cognition. Recently we identified the anatomical substrates for some of the sleep changes caused by certain types of general anesthetics.
We did so by using genetically-modified mice in which there is endogenous expression of C-fos, a marker of neuronal activation. However, using this new technique, we will be able to directly measure cellular excitability in groups of neurons in the brainstem that are associated with sleep. This is a major advantage over simply quantifying C-fos expression as a surrogate marker of neuronal activation.
Our next step will be to pursue the cellular and molecular mechanisms underlying the sleep changes induced by certain types of general anesthetics. This will entail measuring cellular excitability in brainstem neurons associated with sleep using neuro pixel probes, as well as using spatial-transcriptomic approaches to quantify and map the expression of genes related to sleep in these areas. To begin, make an electrocorticography, or ECoG headset, by soldering Furfural alkoxy-coated stainless-steel wire to a three-pin connector header.
After identifying the insertion coordinates based on a mouse-stereotaxic atlas, position the anesthetized mouse in the stereotaxic frame. Place the animal's nose in the nose cone and stabilize the head with head bars. Then perform a toe pinch test to check the depth of anesthesia.
After making a five millimeter scalp incision above the parietal and occipital bones, scrape the meninges with the scalpel blade to remove them. Use the scalpel blade to cut muscle attachments and expose the parietal and occipital bones. Apply hydrogen peroxide to control bleeding and dry the skull surface.
Next, identify the bregma and lambda landmarks on the skull. Then, adjust the nose-cone position to level the anterior posterior position of the skull. To level the medial lateral position of the skull, pick two opposite points between bregma and lambda and check their level.
Now, measure the distance between bregma and lambda and compare it to the distance reported in the Franklin Paxino Stereotaxic Atlas. Use the difference between the measured and reported distances to scale the anterior posterior coordinate proportionally. Mark craniotomy coordinates on the skull with a sterilized pencil.
Using a stereotaxic micromanipulator, position the head plate directly on top of the Lambda suture. Apply dental cement to the head plate and around it to secure it to the skull and allow it to dry for 10 minutes. Then, drill burr holes for two cortical electrodes and one reference electrode.
Place the stripped ends of coated silver-wire electrodes within the burr holes and secure them using ultraviolet light-activated resin. Completely cover the coated stainless steel wires with dental cement so that no wire is exposed. Cover the underside and sides of the headset with dental cement to ensure it is firmly in place and allow the mouse to recover for seven days.
Position the anesthetized mouse in a stereotaxic frame. Identify bregma and lambda, ensuring no more than a 100-micrometer height difference between the two landmarks. Find and mark the calculated coordinates on the skull with a sterile pencil.
Around the coordinates, create an outline of the two-millimeter by two-millimeter craniotomy window. After checking the depth of anesthesia, use a high-speed drill to create a two-millimeter by two-millimeter craniotomy window. Apply 0.5 to one milliliter of normal saline to prevent the brain surface from drying.
Remove the dura using a syringe needle and fine forceps. Next, use a high-speed drill to create a separate burr hole for the silicon probe's reference electrode, generally one to two millimeters from the cranial window. Apply 0.2 milliliters of low-toxicity silicon adhesive on the skull to completely seal the craniotomy.
Using the head plate and screws, affix the head of the mouse to the electrophysiology recording rig. Then, coat the silicon probe shank with fluorescent dye so that the probe trajectory can be reconstructed after the experiment. After that, mount the probe on the manipulator and set the desired angle.
To lower the recording probe to the brain surface within the center of the cranial window, manually insert the probe to a depth of approximately 300 micrometers. Once inserted to this depth, slowly lower the probe automatically to 200 micrometers per minute to the targeted depth to minimize tissue damage. Finally, apply mineral oil to the brain surface within the craniotomy window to prevent drying.
Then, record data from the silicon probe and ECOG at 30 kilohertz using an Intan recording controller. Most recorded neurons decrease their firing during Sevoflurane anesthesia. vlPAG firing significantly decreased from baseline to during Sevoflurane.
This decrease was consistent across all vlPAG neurons.
