Transplantation of Interneuron Precursor Cells into the Brain of a Mouse Pup

Begin with an anesthetized recipient mouse pup. Secure its head with tape, ensuring that the skull remains exposed.

Use the anatomical reference lambda to locate the target brain region.

Position a micropipette over the target region, loaded with a suspension of interneuron precursor cells capable of differentiating into mature interneurons.

These precursor cells are derived from the cortex, striatum, or hippocampus of a donor pup at the same developmental stage as the recipient.

Inject the suspension into the brain to perform either a homotopic transplant, in which precursors are delivered to their region of origin, or a heterotopic transplant, where they are introduced into a different brain region.

Allow the suspension to diffuse before retracting the micropipette to prevent backflow.

Transfer the pup onto a warming pad for recovery.

The survival and maturation of the donor precursors in the brain of the recipient pup can now be evaluated.

Load a fine-tipped micropipette with mineral oil. Attach the micropipette to a nanoliter injector, and secure the nanoliter injection apparatus to a manipulator attached to a magnetic base. While the first pup is being anesthetized on ice, pipette the cell solution several times to ensure a homogeneous cell distribution.

Eject the mineral oil and completely fill the pipette with the single cell suspension. After confirming a lack of response to toe pinch, place the pup on a Petri dish cover with the head resting on adhesive putty so that the top of the head is relatively flat.

Cover the head with a piece of lab tape with a diamond hole, pulling the tape taut until the skin is stretched so that the head is firmly secured and lambda is visible through the hole. Move the secured pup under the nanoliter injector and lower the micropipette so that the tip is directly above lambda.

Observe the x, y-coordinates on the manipulator and adjust the manipulator knobs until the micropipette is positioned at the appropriate coordinates along the mediolateral and anteroposterior axes of the head. Lower the micropipette until the tip creates a small concavity on the skin, and turn the z-axis manipulator knob firmly but gently to drive the micropipette through the skin and skull into the brain.

Retract the micropipette slightly until the tip is surrounded by a cone of skin, shaped like a tent, and view the coordinates along the z-axis. Then lower the micropipette to the appropriate experimental depth and inject the cells. Wait 15 seconds after the cells have been delivered before retracting the micropipette and repeat injection at a second location if desired. Place the pup on a heating pad and monitor until full recovery is achieved before transferring back to home cage.