2.3. Grafting

The animals were first anesthetized with a 50/50 mix of O₂ and N₂O supplemented with 2% Isoflurane (Wako) and then attached to a stereotaxic injector (Narishige). In the case of neonates (5-days old), because the ear bar could not be used, the animals were taped into position on a heating plate set to 37 °C. In addition, all distances for targeting the striatum were halved, since the size of the neonate brain is approximately half that of the adult brain.

The front skin of the animal was then cut to expose the skull, and a hole was drilled 3 mm to the right and 1 mm anterior of the Bregma. The skull was drilled until the bone was so thin it could easily be pierced by the tip of sharp tweezers and without burning the bone through friction. A pre-hole was formed by lowering a sharp-tipped syringe (Hamilton 80,330) 5.5 mm into the brain (0 being the surface of the brain, not the skull).

Twenty DA neurospheres (each being composed of 20.000 cells) were placed on the inner side of a piece of parafilm. The cell culture medium was carefully pipetted away in order to leave only the spheres and a minimal amount of solution. The spheres were then loaded into a blunt-end, zero dead volume Hamilton syringe (Hamilton 88,000) that had been previously hydrated with cell media. The total volume loaded into the syringe was preferably kept under 1 μL.

The loaded syringe was then set into the stereotaxic injector and lowered slowly 5 mm into the brain. The cell mix was injected at a speed of 0.5 μL/min. The syringe was left in place for 3–5 min in order to let the pressure dissipate and was then removed slowly.