Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Santos Dumont Institute Ethics Committee (protocol 02/2015AAS).

1. Surgery preparation

1. Attach each electrode array to an electrode holder compatible with the stereotaxic frame to be used.
2. Connect one electrode holder to the stereotaxic micromanipulator and set one microwire to the interaural coordinates. Repeat this for the additional electrode arrays and holders, if necessary.
   NOTE: The interaural coordinate of any microwire can be used to calculate the implantation coordinates for the entire array, because the relative distance between the microwires is constant. When the array has bundles with different lengths, the interaural coordinate of the longest wire is the most convenient to use for setting the interaural zero.
3. Detach the electrode holders from the stereotaxic micromanipulator and sterilize the assemblies (electrode attached to the electrode holder) in an ultraviolet light (UV) cabinet for at least 2 h.
4. Attach a 24 G needle to a stereotaxic probe holder, connect it to the micromanipulator and set the interaural coordinates for the tip of the needle.
   NOTE: Prior to the surgery, the coordinates of all craniotomies must be predefined as a perimeter 200 µm² larger than the anteroposterior (AP) and mediolateral (ML) position of the array’s target implantation site. Use the 24 G needle-probe holder assembly to determine the position of the craniotomies in the skull based on the zero interaural coordinates.
5. Detach the probe holder from the micromanipulator and sterilize the assembly in a UV cabinet for at least 2 h.
6. Gather 6−8 titanium or stainless-steel screws. Solder a ground wire to half of them.
7. Organize and sterilize all remaining instruments, equipment, and disposables required for the surgery.

2. Preoperative procedures

NOTE: Two adult male marmosets (Callithrix jacchus) weighing 320–370 g were used in this study. Ensure that the animal has not eaten for 6 h prior to the induction of anesthesia.

1. Anesthetize the animal with an intramuscular injection of atropine (0.05 mg/kg) to reduce salivation and bronchial secretions. Check for the lack of pedal responses.
2. After 5 min apply ketamine (10−20 mg/kg) intramuscularly.
3. Shave the animal’s head using an electric barber clipper.
4. Administer tramadol (2 mg/kg) intramuscularly as a general analgesic.
5. Intubate the animal.
   1. Using a mask, expose the marmoset to isoflurane in 1−2% oxygen with a flow rate of 1−5 L/min to induce deep anesthesia. When the animal is deeply anesthetized, reduce and maintain isoflurane to 1−3 L/min.
   2. Attach an elastic band to the surgical table with tape.
   3. Position the marmoset in a supine position with the head toward the technician and place the elastic band in the marmoset’s mouth behind its canines.
      NOTE: It is best to position the head such that the dorsal surface is pointed toward the floor and its face is toward the technician.
   4. Using a cotton-tipped probe, swab dry the marmoset’s tongue, and grasp it in one hand to keep the mouth open.
   5. Spray 10% lidocaine on the tip of the endotracheal tube.
   6. Insert the uncuffed, 2.0 mm diameter endotracheal tube into the trachea until the 4.0 cm mark is at the entrance of the trachea.
   7. Attach the tube to the anesthesia assembly with the artificial ventilator set to 40 breaths/min and confirm proper expansion and contraction of the chest.
      NOTE: At this time the isoflurane and oxygen should be delivered via the endotracheal tube, not the mask.
   8. Remove the elastic band from the marmoset’s mouth so the endotracheal tube can be taped to the jaw.
6. Position the marmoset in a prone position in line with the stereotaxic frame and fix the animal’s head into the stereotaxic frame.
   1. First, insert the tip of the right ear bar into the animal’s right auditory canal.
   2. Next, insert the tip of the left ear bar into the left auditory canal.
   3. Center the animal’s head at the center of the stereotaxic frame and fix the ear bars in place.
   4. Insert the mouthpiece into the animal’s mouth and adjust its height so that it touches the animal’s palate. At the same time, position the orbital bars at the lower surface of the orbital bone.
   5. Make sure that the lower surface of the orbital bone is horizontally aligned with the center of the ear bars.
7. Connect a portable pulse oximeter to the marmoset’s hand. Ensure that the heart rate is within 154−180 beats/min (bpm) for the duration of the surgery; often a heart rate above 200 bpm implies the animal is waking up. Ensure that the oxygen saturation is above 95%. It may occasionally drop to 90% without harm.
   NOTE: Should the heart rate drop below 154 bpm, decrease the isoflurane.
8. Position the rectal temperature probe connected to a homeothermic heating pad into the anus, with the desired temperature set for 37 °C. Tape this sensor to the tail to keep it fixed in place.
9. Apply sterile ophthalmic lubricant to the eyes.
10. Clean and disinfect the animal’s head with chlorhexidine and povidone iodine before covering the animal with a surgical field.
    NOTE: Carry out all the following surgical procedures under aseptic conditions.

3. Surgery procedures

1. Apply local analgesic subcutaneously (e.g., lidocaine 20 mg/mL, 0.1 mL) at the site of the intended incision. Make an incision in the midline of the scalp.
2. Expose and prepare the surface of the skull.
   1. Carefully detach the temporal muscle from the cranium. First, use a scalpel to cut the fascia at its insertion into the skull. Then, gently separate the temporal muscle from the cranium using a periosteal raspatory.
   2. Remove the periosteum from all exposed cranium using a periosteal raspatory.
   3. Control the bleeding with a sterile cotton swab, if necessary.
   4. Clean the bone surface with hydrogen peroxide.
3. Delineate the location of the craniotomy by marking its corners with shallow burr holes into the bone surface. Then, drill out the perimeter of the craniotomy using a dental drill at maximum speed (i.e., 350,000 rpm). Add a few drops of sterile saline over the skull while drilling to prevent overheating. Measure the position of the craniotomy and the coordinates of the electrode implant with respect to the interaural coordinates.
4. Implant screws into the skull.
   1. Drill 6−8 screw holes into the cranium.
   2. Implant the screws such that each ground wire fused screw is adjacent to and in the proximity of an unaltered screw (i.e., without a ground wire attached to it).
   3. Wind each ground wire around the adjacent, unaltered screw.
   4. Add a drop of silver paint between the ground wire and each screw.
5. Remove the bone at the center of the craniotomy using forceps with a curved tip (e.g., McPherson forceps). Keep the dura mater hydrated with sterile saline.
6. Remove the dura mater. Use a sterile hypodermic needle (25 or 26 G) with the bevel bent at approximately 90˚ to puncture and lift the surface of the dura mater away from the brain surface. Then, cut the dura mater with microscissors. Keep the exposed brain hydrated with saline.
   NOTE: If significant dural bleeding is observed, use cautery or sterile absorbent gelatin sponges soaked in thrombine²⁵.
7. Implant microelectrode arrays.
   1. Attach the sterilized electrode holder and electrode array to the stereotaxic micromanipulator.
   2. Position the micromanipulator such that the electrode is at the desired anteroposterior and mediolateral coordinates.
   3. Lower the electrode array until the tip of the longest bundle touches the surface of the brain.
   4. Slowly insert the array into the brain tissue until it reaches the dorsoventral coordinates.
   5. Cover the exposed cortex with small pieces of sterile, absorbent gelatin sponges.
   6. Secure the electrode to the skull by applying dental acrylic to the exposed skull, one screw, and the electrode.
   7. Detach the electrode holder and remove it from the micromanipulator.
8. Repeat the implantation procedure from step 3.7 with the additional arrays, if necessary.
9. Wind together and weld the ground wires of the separate arrays and screws. Use silver paint to form a bridge around the weld to ensure an electrical connection has been achieved.
10. Using dental acrylic, make a sturdy headcap around the lateral extent of the arrays, and completely encase the ground wires and any exposed skull and screws.
11. If necessary, insert a support bar into the headcap. This can be a sturdy plastic cylinder like those from a cotton swab. Seal it into place with the dental acrylic.
    NOTE: This can be helpful in securing the electrophysiology cable connectors in place but may be unnecessary depending on the equipment used. In the present method, a similar support rod is affixed to the head stage such that an elastic band can robustly hold the head stages in place on the connectors.
12. Suture the skin around the headcap.

4. Postoperative recovery

1. Apply antiseptic solution (e.g., chlorhexidine) around the wound.
2. Turn off the isoflurane supply but not the oxygen and remove the animal from the stereotaxic frame.
3. Place the animal onto the heating pad with the oxygen maintained through the endotracheal tube.
4. Remove the endotracheal tube when the first signs of neurogenic reflexes, such as laryngospasms, are observed.
5. Keep supplying the oxygen with a mask until the animal presents clear signs of anesthetic recovery, such as protective reflexes, postural tone, and attempts to ambulate.
6. Place the animal inside a clean cage in a recovery room for 24−48 h before moving the animal to its home cage. House each implanted animal individually.
   NOTE: Because marmosets tend to climb the cage walls, use a cage with smooth walls or cover the cage walls with a smooth surface to prevent the animal from falling.
7. In the first hour following surgery, observe the animal to watch for signs of distress or uncoordinated head contact against the side of the cage.
8. Administer antibiotics (e.g., enrofloxacin 5 mg/kg, subcutaneously, once a day for 5−7 days), analgesics (e.g., oral tramadol 1 mg/kg, every 8 h for 3−5 days) and anti-inflammatory drugs (e.g., dexamethasone 0.5−1.5 mg/kg, subcutaneously, once a day for 1−3 days).
   NOTE: After a successful surgery, animals will be fully recovered within 3−5 days.

5. Chronic electrophysiological recordings in freely behaving marmosets

1. Start the electrophysiological recording sessions at least 1 week after the surgery.
   NOTE: Habituate the animals to the researcher and experimental environments before starting all experimental procedures for at least 1 month.
2. At the beginning of each session, lightly anesthetize the animal using isoflurane (1−5 L/min, 1% O₂).
   NOTE: Follow the relevant institution’s guidelines regarding the sedation of small primates. If recording sessions are very frequent, habituate the animals to be handled so that cables can be connected without anesthesia.
3. Connect the electrode arrays to a commercial neural recording system.
4. Place the animal inside the experimental chamber.
   NOTE: The experimental chamber used here is a cubic acrylic box (0.45 m x 0.45 m x 0.45 m) designed to evaluate the amount and pattern of spontaneous motor activity^26,27.
5. Wait for 30 min before starting the recordings to ensure the animal is fully recovered from anesthesia.
   NOTE: Isoflurane has a rapid onset and offset action which allows for rapid sedation and awakening²⁸. Once the isoflurane supply is turned off, the animal will start to wake up. The animal is awake when it stays in the upright position and can ambulate freely in the experimental chamber without falling. This takes less than 15 min. To ensure the absence of any sedative effects, begin the recordings 30 min after the isoflurane is discontinued.
6. Confirm the position of the microelectrode array implants postmortem by NISSL staining after fixing and sectioning the tissue²⁹.