When the DV coordinate at the point situated on the vertical axis of the target has been surveyed and identified as described in the previous paragraph, the skull bone is drilled to access the dura. For this, a dental drill with drill bit and handpiece that have been previously sterilized are usually used. After the motor is turned on, the drill bit is applied to the skull surface applying just enough force to pierce the bone. This gesture should be carried out as quickly as possible to avoid generating heat caused by friction of the rotating bit, which can be further reduced by simultaneous irrigation with sterile saline. Particular care should be taken regarding the amount of force applied while drilling.

If too much force is applied and the bone is penetrated too deeply, there is a risk of damaging the dura and the surface of the cortex. After trepanation, the dura is delicately hooked using the tip of a small sterile hypodermic needle and then incised in a circular motion. The correct execution of this step is evident by the appearance of cerebrospinal fluid seeping out to the surface of the skull.

To facilitate the introduction of the cannula into the brain, the dura mater is perforated, or a small surface of it resected, using a sterile beveled syringe needle (Fig. 6.3b).

In order not to contaminate the cannula, it should be installed on the object carrier and inserted at the last moment, immediately after the dura mater has been perforated.

After having installed a sterile cannula support or holder, which fixes the guide cannula to the object carrier (Fig. 6.4), it is imperative to check the verticality of its axis relative to the coronal and median planes before it is lowered (Fig. 6.5). If this is not done, the cannula could end up being inserted at an angle, thus missing its intended target.

When verticality has been verified, the object carrier is fixed to the vertical stereotaxic manipulator arm. The tracking of the arm should be carefully checked with respect to the graduations marked on its base. For convenience, it is preferable to align the arm with the principle graduation mark as reference. Any rotation of the arm necessary for this alignment does not affect its verticality. The validity of this reference point is indispensable if it becomes necessary to set an angle for inserting the cannula (Fig. 6.6). The guide cannula that will remain permanently implanted in the brain is then mounted onto the cannula support for insertion (Fig. 6.7).

Next, the guide cannula is carefully lowered according to the predetermined dorsoventral coordinates (in this example, DV = −3.5 mm for the rat, or DV = −2.4 mm for the mouse), checking that its displacement is smooth and even along the dorsoventral axis (Fig. 6.8). This step is very important: if the orifice of the guide cannula is not centered correctly with respect to the target coordinates, the cannula risks touching the bone at the edge of the insertion hole, deviating its verticality and compromising an accurate trajectory towards the target.

The rest of the surgical operation may have different experimental goals requiring specific procedures. Some of these are described in the examples that follow in this chapter.

The guide cannula and anchoring screws must be fixed solidly to the skull of the animal using dental acrylic cement (e.g., Paladur® powder and solvent, Heraeus Kulzer & Co. GmbH, or Unifast Trad® powder and solvent, GC America Inc.) that can flow easily and finely to coat the guide cannula and anchor screws (Fig. 6.9). The cement should have a fluid consistency so that it acquires a maximum hardness after it sets. It is recommended to use aliquots of resin powder and solvent in vessels that should be kept sterile on the instrument tray so as not to break the chain of asepsis during preparation of the cement.

Before applying the dental cement, it is recommended to first apply a self-curing adhesive resin such as (Super-Bond C&B^®, Sun Medical Co., Japan), especially if the skull is to be subjected to high mechanical stress (Gervasoni et al. 1998, 2000) or if the implant is required to hold for several months. This type of adhesive exists in the form of a monomer powder and a liquid polymer that are mixed together with a few drops of catalyst just prior to use. For optimal adhesion, it is necessary to etch the bone surface by using a cotton swab to apply a solution, such as mixture B in the Super-Bond^® kit, containing 10 % citric acid. Because of its corrosive property, care should be taken not to prolong the contact of this solution with the bone for more than half a minute and to carefully dry the bone surface after its preparation. The Super-Bond® resin is then applied using two brush techniques. In the case of a large area to cover, the technique of bulk mixing can be used, whereby the monomer and polymer are premixed together in a single vessel and the cement picked up and applied with the brush. For a quicker setting option, the brush-dip technique is used; this consists of first dipping the brush into the polymer solution, draining it of the excess and using it to pick up and apply the monomer powder. In both cases, the bone surface must first be well dried and free of any signs of hemorrhaging.

When the cement is completely dried, the object carrier is raised slowly so that the guide cannula remains in place and visible on the skull (Fig. 6.10a). An obturator (“dummy” cannula) (Fig. 6.10b) is then inserted into the guide so that the latter does not become plugged during the postoperative recovery period (Fig. 6.10c).

It is useful to protect the guide cannula-obturator assembly with a plastic ring of the appropriate dimensions, which encloses the guide cannula but leaves enough space for the operator to easily manipulate the obturator. This protective collar is slipped around the guide cannula and encircled with a second layer of cement to fix it in place (Figs. 6.10c and 6.11).