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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Deep brain stimulation (DBS) Surgery
- Inject the Ketamine/Xylazine mix intraperitoneally into the animal and confirm that it has reached a surgical plane of anesthesia (by checking for the toe-pinch reflex, respiratory rate depth, and regularity of breathing). Remove hair from the region of the scalp that will be incised and disinfect the scalp with three alternating scrubs, each of betadine and either alcohol or sterile saline. Secure and position the animal on the stereotactic frame.
- Use circulating warm water pads to maintain the animal’s body temperature at an optimal level. Apply eye lubricant to protect the animal’s eyes from overdrying.
- Use the following stereotactic coordinates for targeting the ATN (Anterior thalamic nucleus): anterioposterior -1.6 mm, mediolateral 1.5 mm, and dorsoventral 5.2 mm.Make an incision in the scalp sagittally to reveal the skull. Using a pair of retractors, secure the incised scalp to expose the skull. Use sterile saline to flush the incision. If the incision is too wet, use sterile cotton swabs to dry it. Locate the bregma and mark it with a black marker. To guide the position of the burr holes, make two more marks at approximately 1.5 mm mediolaterally on both sides from the sagittal suture and 1.6 mm posterior to the coronal suture.
- Use a hand-held drill to make the burr holes. Make sure the tip of the burr hole is sterile by sterilizing it with ethanol. Hold the drill at about a 45° angle to the skull surface when drilling. Frequently switch between the two burr holes to avoid excessive heat at the location of any burr hole.
- Continue drilling until the dura is exposed. Using a needle with its tip bent resembling the shape of an ‘L’, remove any broken pieces of bone that would obstruct the insertion of the electrode. Take care to avoid damaging the underlying dura and/or brain tissue while removing bone fragments using the bent needle.
NOTE: Using a blunted needle or fine blunt forceps is also an option. - Fix the dual-electrode assembly to the rotating handle of the stereotactic frame and fix the handle at a 90° angle. Using the adjustments in the stereotactic frame, position the left electrode exactly above the bregma.
- Using the stereotactic adjustments for mediolateral positioning, precisely move the left electrode 1.5 mm to the left side of the bregma so that there are now two electrodes perfectly aligned along the coronal suture but spaced apart 1.5 mm mediolaterally from the bregma.
- Using the anteroposterior stereotactic adjustments, move the electrodes 1.6 mm posterior to the coronal suture.
- Use the dorsoventral adjustments to lower the electrodes to first check if the burr holes have been made at the right location such that the electrodes can be inserted with ease, without touching the rough edges of the burr holes. If so, insert the electrodes to a depth of 5.2 mm from the surface of the skull.
- Connect the electrodes via leads to a stimulator set at 130 Hz, 2.5 V, and 90 µsec pulse-width.
- Deliver high-frequency stimulation for an hour (or for a desired period of time as per experimental setup). During the course of the simulation, remember to ensure proper anesthetic depth regularly by checking for the absence of foot withdrawal in response to a toe pinch. Supplement anesthesia with approximately half the initial dose used to induce anesthesia. Avoid contamination of your sterile gloves when checking anesthetic depth, and change them if necessary. Perform unilateral or bilateral stimulation based on one’s experimental needs. Include controls such as low-frequency stimulation (e.g., 10 Hz) and unstimulated animals (inserting electrodes with no subsequent stimulation).