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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Injection of Reprogramming Factors into the Brain
- Animal setup, stereotaxic placement and drillingNOTE: This protocol focuses on the use of Ascl1, Lmx1a and Nurr1 (ALN) for the reprogramming of NG2 (Neuron-Glial Antigen 2) glia into interneurons. In our experience, interneurons of a similar phenotype can be obtained using other factor combinations
- Move the M/L-D/V arm of the stereotaxic frame into place (above the animal) and secure it.
NOTE: The stereotaxic frame allows adjustment of the syringe along the Anterior/Posterior (A/P, Y axis), Medial/Lateral (M/L, X axis), and Dorsal/Ventral (D/V, Z axis) axis. - Move the syringe along the different axis of the stereotaxic frame to bring the tip of the glass capillary just above bregma (the junction point where the different skull plates meet).
- Make sure the capillary tip is perfectly straight in both A/P and M/L planes. In case of an ambiguous bregma, take the average of the lateral and midline sutures.
- When the tip of the capillary is correctly placed above the bregma, reset both the M/L and A/P values to 0.0 on the digital coordinate counter.
- To make sure the head of the animal is in a perfectly flat position, use the digital coordinate counter to measure the D/V coordinate value when the A/P arm is at +2.0 and -2.0 (M/L = 0.0), as well as when the M/L arm is at +2.0 and -2.0 (A/P = 0.0). Adjust the height of the tooth bar and ear bars accordingly.
- Move the syringe to the desired coordinates for injection of viral vectors in the striatum (A/P = +1.0; M/L = -2.0, relative to bregma).
- Raise the syringe slightly and, by looking at the injection site through the microscope, drill a hole using a dental drill at the injection coordinates. Start to drill at the site, working in circular and gentle manner.
NOTE: Do not put too much downward pressure, as the drill-bit should be sharp enough to go through the bone without additional force. Avoid long, sustained drilling as this creates heat. - At the end of the drilling, check that the dura mater remains intact and exposed for injection.
- Syringe setup preparation
- Place a piece of cotton gauze over the open incision and flush the syringe with saline solution.
- After flushing, take up an air bubble of 1–2 μL, followed by 1 μL of solution containing the viral vectors, avoiding any unintentional bubbles. Make sure that the viral solution can easily be visualized below the air bubble, whilst being injected.
- Viral injection
- Remove the cotton gauze from over the incision site, and lower the syringe using the D/V arm of the stereotaxic frame. As the surface of the skull is approached, carefully look through the microscope and measure the D/V level of dura mater — it should bulge slightly under gentle pressure.
- While touching the dura mater with the tip of the capillary, set the D/V coordinate to 0.0.
- Lower the syringe, progressing slowly to the desired depth (D/V = -2.7, relative to dura mater). Make sure that the trajectory is clear of bone fragments, so that no bending of the needle/capillary is observed.
NOTE: The presented coordinates refer to an injection of the reprogramming factors into the striatum of NG2-Cre mice. Coordinates corresponding to other brain regions may be used. Always test the coordinates for injection first using a dye (e.g., Trypan Blue), colored bead injection, or a reporter viral vector prior to injection of reprogramming factors into the brain of a new mouse strain. If injecting Trypan Blue, the animals need to be sacrificed immediately after surgery, and the brain dissected out. Fresh brains can be cut using a microtome, while frozen, and the site of injection determined by visualization of the dye position in the brain. If testing coordinates using colored beads, it is possible to determine the site of injection on perfused and cut brains a week after injection. Alternatively, an injection with a viral vector carrying a reporter gene can be used, and the site of injection determined in perfused cut brains. - Inject 1 μL of the viral solution at a rate of 0.4 μL/min. When the whole volume is injected, allow for a diffusion period of 2 min before syringe withdrawal.
- After diffusion, slowly retract the syringe until the tip of the capillary is completely out of the brain.
- Place a piece of cotton gauze over the wound and flush the syringe with saline solution.
- Move the M/L-D/V arm of the stereotaxic frame out of the working area.
- Wound closing and post-operative procedures
- Carefully suture the incision using suture thread.
NOTE: All suture threads used in injected animals should be disposed of in a cup/vial containing an antiviral detergent solution. The same solution is used to clean all surfaces surrounding the surgery area that have been in contact with surgical materials. All surgical tools are thoroughly washed and autoclaved at the end of each surgery day. - Remove the animal from the stereotaxic frame and place it in a post-operative station, which includes a clean, heated cage, access to food and water, and where the animal stays until fully awake. During this period, monitor the animal closely until consciousness is regained.
- Do not house operated animals with non-operated animals until the former have fully recovered from surgery.
- Supervise the operated animals daily. Depending on the sutures used, make sure they are removed if necessary. All animals were given Bupenorphinum (Temgesic at 1ml/kg) subcutaneously as post-operative care.
NOTE: If operating using the same syringe on two consecutive days, flush it with water, followed by ethanol 70% and water again. Leave the syringe filled with water overnight to allow dissolution of any possible residues.