TITLE: Ex vivo Brain Slice Preparation for electrophysiology and 2PLSM recordings
AUTHORS: taylor.panczyk

[DESCRIPTION]
In this protocol we detail the steps to obtain brain slices containing the pedunculopontine nucleus (PPN) for ex vivo electrophysiology and 2PLSM recordings. We perform parasagital slices with a 20º angle to maximize the PPN area in each slice. We make both electrophysiological and imaging recording of PPN cholinergic neurons to understand the mechanisms that rely in their vulnerability in Parkinson's disease.


[STEPS]
SECTION: Prepare Bubbling Solution
1. On the experiment day prepare the working aCSF and slicing solution in a 1:10 dilution from stock solutions
SECTION: Prepare Bubbling Solution
2. Place slicing solution in -20 °C freezer for 30-40 minutes.

SECTION: Prepare Bubbling Solution
2.1. Then place the slicing solution in a large Styrofoam container of ice to remain cold.
SECTION: Prepare Bubbling Solution
3. Place two 95mm diameter glass petri dishes on top of ice bucket and fill with cold slicing solution. Bubble the slicing solution in the bottle and the two petri dishes.
SECTION: Prepare Bubbling Solution
4. Turn on water bath and set to 34 °C°.

SECTION: Prepare Bubbling Solution
5. Fill a 250 mL beaker with 150 mL of slicing solution.


SECTION: Prepare Bubbling Solution
6. Place a second container that has a sieve bottom into the beaker containing the slicing solution.
SECTION: Prepare Bubbling Solution
7. Place the beaker with the slicing solution and second container in water bath and begin bubbling it.
SECTION: Prepare Bubbling Solution
8. Fill a 95mm diameter glass petri dish with slicing solution and set aside.
SECTION: Prepare Vibratome
9. Cut a 1 cm piece of the 20° agarose wedge and super glue it to the specimen plate.
SECTION: Prepare Vibratome
10. Insert razor blade into blade holder.
SECTION: Prepare Vibratome
11. Set parameters (speed, amplitude, and section thickness) on the Vibratome control panel.
SECTION: Prepare Vibratome
12. Place ice in the chamber below the specimen plate and prepare a glass with water next to the vibrotome.
SECTION: Preparing Apparatus and Anesthesia:
13. Fill one 10 mL syringe with cold slicing solution, connect with tubing, and place 27G needle at end of tubing.

SECTION: Preparing Apparatus and Anesthesia:
14. Fill a styrofoam tray with ice.
SECTION: Preparing Apparatus and Anesthesia:
15. Anesthetize the mice with a mixture of ketamine (50 mg/kg) and xylazine (4.5mg/kg).
SECTION: Preparing Apparatus and Anesthesia:
16. Use toe pinch-response method to determine depth of anesthesia.
SECTION: Preparing Apparatus and Anesthesia:
17. Place the animals on ice in tray lying on the back with face upward.
SECTION: Perfusion Surgery
18. Make an incision through the abdominal skin.
SECTION: Perfusion Surgery
19. Make two additional skin incisions from the xiphoid process along the base of the ventral ribcage laterally.
SECTION: Perfusion Surgery
20. Gently reflect the two flaps of skin to expose thoracic field completely.

SECTION: Perfusion Surgery
21. Grasp the cartilage of the xiphoid process with blunt forceps and raise it slightly to insert pointed scissors. Cut through the thoracic musculature and ribcage between the breastbone and medial rib insertion points and extend the incision rostrally to the level of the clavicles.

SECTION: Perfusion Surgery
22. Separate the diaphragm from the chest wall on both sides with scissor cuts.

SECTION: Perfusion Surgery
23. Clamp the reflected ribcage laterally with a hemostat to expose the heart.
SECTION: Perfusion Surgery
24. Secure the beating heart with fingers or blunt forceps, and immediately insert a blunt 27G syringe needle.
SECTION: Perfusion Surgery
25. Cut the right atrium with scissors, and at the first sign of blood flow, begin the infusion of 1x slicing solution at 2-4 ml/min.
SECTION: Perfusion Surgery
26. Continue perfusion with slicing solution until the 10 mL syringe is empty.
SECTION: Dissection
27. Decapitate the mouse with large surgical scissors.
SECTION: Dissection
28. Place the decapitated head in one of the petri dishes containing cold, bubbling slicing solution. Removal of brain must be done in cold, bubbling slicing solution.
SECTION: Dissection
29. Cut down the midline to expose the skull.
SECTION: Dissection
30. Make two lateral and one dorsal cut using sharp surgical scissors on the base of the skull.
SECTION: Dissection
31. Cut the olfactory bulbs/optic nerve at rostral end of skull.
SECTION: Dissection
32. Gently peel off the skull using blunt forceps.
SECTION: Dissection
33. Once brain is fully exposed, remove it from the skull and place it in a petri dish with clean, cold and bubbled slicing solution.
SECTION: Dissection
34. Cut the brain in two halves along the midline.
SECTION: Dissection
35. Super glue the flat section (midline) onto the agarose wedge glued to the specimen plate so that the vibratome cuts along parasagittal plane with a 20° angle. Place the posterior part of the brain in the top of the agarose wedge. If bilateral sections are needed, super glue both hemisferes to the agarose wedge.
SECTION: Slicing
36. Place specimen plate in buffer tray and fill buffer tray with slicing solution.
SECTION: Slicing
37. Place O2/CO2 tubing into buffer tray to bubble slicing solution.
SECTION: Slicing
38. Play start to the vibrotome and cut the desired slices.
SECTION: Slicing
39. With the glass transfer pipette, transfer the slices to the beaker containing the sieve bottom container at 34 °C.

SECTION: Slicing
40. When finishing slicing, turn of the water bath and let it cool to room temperature.
SECTION: Slicing
41. Leave brain slices in beaker for 60 min to recover.