TITLE: 6-OHDA lesion in medial forebrain bundle
AUTHORS: Santiago Unda, Mihaela Stavarache, Michael G. Kaplitt, Roberta Marongiu

[DESCRIPTION]
Lesion of the medial forebrain bundle using 6-OHDA to induce parkinsonian-like deficits in mice.

[GUIDELINES]
Note that sterile technique must be followed during this procedure to avoid health complications.

[STEPS]
SECTION: Anesthesia
1. Weigh mice.
SECTION: Post-surgery Procedure
15. Close skin with non-absorbable sutures.
SECTION: Post-surgery Procedure
16. Give meloxicam 2mg/kg subcutaneously immediately after surgery and 24 hours post-surgery.
SECTION: Anesthesia
2. 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.
SECTION: Anesthesia
3. Administer desipramine 25mg/kg intraperitoneally 30 minutes prior to the intracerebral injection of 6-OHDA.
SECTION: Post-surgery Procedure
19. Remove sutures 15 days post-injections.
SECTION: Post-surgery Procedure
18. *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.
SECTION: Post-surgery Procedure
17. Give 1-2mL of dextrose 0.833% S.C. This is made by diluting 5% stock dextrose 1:6.
SECTION: Apomorphine-induced Behavioral Test
20. 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.
SECTION: Surgical Procedure
5. Place mouse into stereotactic frame (David Kopf Instruments) and place eye lubricant.
SECTION: Surgical Procedure
9. 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
SECTION: Surgical Procedure
7. Sterilize skull area and then cut open with scalpel.
SECTION: Surgical Procedure
6. Remove fur from skull area using either electric shaver or depilatory cream.
SECTION: 6-OHDA Preparation
4. 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.

SECTION: Surgical Procedure
8. Adjust brain so that it is level. Locate bregma, and zero X and Y axes on stereotaxic frame.
SECTION: Surgical Procedure
10. Zero the Z axis on the stereotaxic frame.
SECTION: Surgical Procedure
11. 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.  
SECTION: Surgical Procedure
12. With the loaded syringe, navigate the needle to the desired Z coordinate.
SECTION: Surgical Procedure
13. Inject 0.6uL of 6-OHDA at a flow rate of 0.1 μL/min. 
SECTION: Surgical Procedure
14. 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.