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

The overall goal of this procedure is to outline the surgical steps involved in the implantation of microelectrode arrays for chronic electrophysiologic recording in freely behaving marmosets. Marmosets are small primates that are increasingly being used in neuroscience research. These animals share many of the practical and logistical advantages of rodents.

Their propensity for twinning and high fecundity at young ages means ready access to large and genetically similar cohorts. In addition, their small body size makes them easy and relatively cheap to care for. As primates, the marmoset has a central nervous system that is much more similar to a human than that of a rodent.

This includes a highly developed prefrontal cortex and visual system. An advantage that marmosets have over many other species of primates however, is the progress made in genetically modifying them. With a deeply sequenced genome and success generating founder knock-in and knock-out animals, it is clear that the marmoset is poised to become a powerful model for studying brain function in health and disease.

A common method for study brain function involves implanting electrodes in anatomically specific locations. This allows one to chronically record the neural activity in different target areas in awake and freely behaving animals. Prior to the surgery determine the intraoral zero coordinate for all implants and guides.

To do so, attach each electrode array that will be implanted to an electrode holder and set one micro wire to the intraoral zero. This is where the ear bars meet. Repeat this for the additional electrode arrays and holders, if necessary.

In addition, repeat this with a 24 gauge needle similarly attached to a stereotaxic probe holder. For electrophysiologic arrays, a ground wire should be soldered to every other screw that will be used. Finally, organize and sterilize all instruments, equipment, and disposables in preparation for the surgical procedure.

On the day of the surgery insure the animal has not eaten for six hours prior to beginning anesthesia. Atropine and ketamine will be used to induce anesthesia by sequential, intramuscular injections. Next, shave the animals head using barber clippers.

It is important to intubate the animal during surgery. Our preferred method involves attaching an elastic band to the surgical table which we insert behind the animals canines while it is in a supine position. We next use a cotton tipped probe to swab dry the marmoset's tongue and grasp it in one hand.

This allows a technician to open the mouth and airway with only one hand. After inserting the endotracheal tube, attach it to the anesthesia assembly with the artificial ventilator set to 40 breaths per minute. Secure the tube and assembly with tape, as needed.

With the marmoset now in prone position fix the animal's head into the stereotaxic frame. To do so, first, insert the tip of the right ear bar into the animal's right auditory canal. Followed by the left ear bar into the left auditory canal.

Next, the head must be leveled such that the horizontal plane is parallel with the frame, while the anterior/posterior plane is centered. This is achieved by first centering the animal's head using the ear bar measurements secured at equal distances. Following this, the mouth piece is inserted into the animal's mouth, and adjusted until it touches the animal's palette, while the orbital bars are just touching the lower margin of the orbital bone.

As the horizontal plane is defined as the plane passing through the lower margin of the orbital bone and the center of external auditory medius, one should make sure that the lower surface of the orbital bone is now horizontally aligned with the center of the ear bars before proceeding. During the procedure, it is important to maintain all vital signs. Thus one should connect a portable pulse oximeter to the marmoset's hand to ensure that heart rate and oxygen saturation are within the acceptable ranges.

In addition a rectal temperature probe connected to a homeothermic heating pad set for 37 degrees centigrade, and taped to the tail, will ensure the marmoset's body temperature is not adversely affected by anesthesia. The final pre-operative step is to clean and disinfect the animal's shaved head with chlorhexidine and iodine, before covering the animal with a sterilized surgical field. Perform an incision in the midline of the scalp after applying a local analgesic at this site.

To prepare the surface of the skull, first, carefully detach the temporal muscle from the cranium. This is done by cutting the fossa at it's insertion into the skull with a scalpel. After which, the temporal muscle should be gently separated from the cranium, using a periosteal raspatory.

Continuing with the periosteal raspatory remove the periosteum from all exposed cranium by scraping. Next, mark the location of the craniotomy by creating shallow burr holes into the bone surface and drilling out the perimeter of the craniotomy. Drill six to eight screw holes, as needed, into the cranium and implant the screws.

At this time, wind the ground wires soldered to a given screw around the adjacent unsoldered screw. Remove the bone at the center of the craniotomy using McPherson forceps. While the brain and dura mater are uncovered keep them hydrated with sterile saline.

To remove the dura mater, a 25 or 26 gauge needle should be bent at approximately 90 degrees to facilitate puncturing and lifting the surface of the dura mater away from the brain's surface. At this point, micro scissors are used to excise the dura mater. Attach the electrodes to the stereotaxic micromanipulator and position it, such that the electrode is at the desired anterior/posterior and medial/lateral coordinates.

Now, lower the tip of the electrode until it touches the surface of the brain. Slowly insert the array into the brain, until it reaches the desired dorsal/ventral coordinates. Add a drop of silver paint between the ground wire and each screw to ensure an electrical connection.

Secure the electrode to the skull by applying dental acrylic to the exposed skull, one screw, and the electrode array. Repeat this implantation procedure with any additional arrays. Finally, using dental acrylic, make a sturdy head cap around the lateral extent of the arrays and completely in case the ground wires and any exposed skull or screws.

If necessary, one can insert a support bar into the head cap and seal it into place with dental acrylic. Following creation of the head cap suture the skin if muscle or bone tissue is exposed. An antiseptic solution should also be applied to the wound at this time.

With the surgery completed it is time to begin recovery. First, turn off the isoflurane supply, but not the oxygen, and remove the animal from the stereotaxic frame. Place the animal onto the heating page, with the oxygen maintained through the endotracheal tube.

When the first sign of neurogenic reflexes such as laryngeal spasms begin, remove the endotracheal tube. Oxygen should now be supplied with a mask until the animal presents clear signs of anesthetic recovery, such as protective reflexes, postural tone, and attempts to ambulate. The marmoset should be housed individually, and left to recover in a clean cage and room for 24 to 48 hours, before returning to it's home cage.

Implanted animals should be housed in separate cages. It is important to observe the animal for at least one hour following the surgery to watch for signs of distress and specifically, uncoordinated movements. Antibiotics and analgesics and antiinflammatory drugs should be administered in the days following surgery to control pain and prevent infection.

In successful surgeries, the animal should be completely recovered within three to five days. We recommend electrophysiologic recording sessions begin at least one week after the surgery. At the beginning of each recording session lightly anesthetize the animal using isoflurane and connect the electrode arrays connector to a neural recording system.

Place the animal inside the experimental chamber and wait for full anesthetic recovery before beginning the recordings. After a successful surgery, it is possible to record spikes and local field potentials from the implanted electrodes over several weeks. Ultimately a final confirmation that the electrode arrays were successfully implanted into the target structures should be preformed post-mortem.

Nissl stained sections containing electro tracks can be used to determine the position of each implanted micro wire. Chronic electrophysiologic recordings in awake behaving animals are powerful techniques used in many lines of neuroscience research. As the common marmoset offers many advantages over historically popular models, the purpose of this video, is to help other neuroscientists adopt the common marmoset for their research.

In addition to the implantation of chronic recording electrodes, as detailed in this video, the same stereotaxic surgery protocol can be adapted for many other experimental ends, including implantation of stimulating electrodes, guide cannulas, and microinjections. When adapting this protocol for one of the fore mentioned techniques, the surgeon will principally modify the drilling procedure to conform of the size of the desired implant. Additionally, the ground procedure is not necessary for many of these techniques and may thus be omitted.

Regardless of the experimental goal, after watching this video you should have an understanding on how to successfully perform stereotaxic neurosurgery in the common marmoset.