6-OHDA lesion in medial forebrain bundle

Weigh mice.

Administer intraperitoneally a mixture of ketamine (Butler Animal Health Supply) and xylazine (Lloyd Laboratories) at concentrations of 110 and 10 mg/kg of body weight, respectively.

Administer desipramine 25mg/kg intraperitoneally 30 minutes prior to the intracerebral injection of 6-OHDA.

Prepare 6-OHDA hydrobromide solution:

6-OHDA hydrobromide (Sigma-Aldrich) in PBS with 0.1% ascorbate at a concentration of 3.0 mg/mL

Each mouse will be administered injected with a total volume of 0.6 μL.

*Note that this solution is light-sensitive and loses efficacy after 2 hours. Stock solution can be made and stored in -80°C and used for 2 hours before discarding.

Place mouse into stereotactic frame (David Kopf Instruments) and place eye lubricant.

Remove fur from skull area using either electric shaver or depilatory cream.

Sterilize skull area and then cut open with scalpel.

Adjust brain so that it is level. Locate bregma, and zero X and Y axes on stereotaxic frame.

Move to injection coordinates and drill small hole into the skull.

Coordinates: AP -1.1 mm, ML -1.1 mm, DV -5.0 mm relative to bregma and the dural surface

Zero the Z axis on the stereotaxic frame.

Using a 10 μL stereotactic syringe with a 30 G needle attached to a micro-infusion pump (World Precision Instruments), withdraw the 6-OHDA solution.

With the loaded syringe, navigate the needle to the desired Z coordinate.

Inject 0.6uL of 6-OHDA at a flow rate of 0.1 μL/min.

To prevent reflux, leave the injection needle in place for 5 min, withdraw a short distance (0.3 - 0.5 mm), and then leave in the new position for an additional 2 min before complete removal of the needle.

Close skin with non-absorbable sutures.

Give meloxicam 2mg/kg subcutaneously immediately after surgery and 24 hours post-surgery.

Give 1-2mL of dextrose 0.833% S.C. This is made by diluting 5% stock dextrose 1:6.

*Note that mice will weaken and lose body weight. Extra care must be given until body weight stabilizes to ensure survival.

• Perform health checks twice a day for 15 days post-surgery or until the mice fully recover.
• Check for any sign of distress, infection, and inflammation.
• Give 1-2mL of dextrose 0.83% S.C. every day for the first 15 days post-injection.
• Give diet supplements (e.g. condensed milk solution, moistened pellets).
• Remove any bacterial plaques that form on genitals.

Remove sutures 15 days post-injections.

Mice were allowed 4 to 6 weeks to recover before being subjected to the apomorphine-induced a behavioral test to estimate the extent of dopamine depletion in the substantia nigra. See protocol on protocols.io for more details.