TITLE: Electrophysiological recording from Brain Slices Protocol
AUTHORS: HaiDun

[DESCRIPTION]
This protocol details the steps for performing slice electrophysiological recordings with DART.

[STEPS]
SECTION: Brain slice preparation
1. Take 150 mL-200 mLhigh sucrose solution cutting solution, store at-80 °C for 15 min-20 min to make chilled half ice solution.

SECTION: Brain slice preparation
4. Deeply anesthetize the mouse with isoflurane, and then decapitate.
SECTION: Brain slice preparation
5. Take brain and place the brain into pre-chilled high sucrose cutting half ice solution. For VTA sections trim the brain by cutting off the cerebellum, which provides a flat surface to mount the brain with, and a small part of the prefrontal cortex.
SECTION: Brain slice preparation
6. Mount the brain onto the Vibratome specimen disc using superglue, orienting the sample such that the cortex faces the razor blade. Add a supporting piece of agar (3-4%) behind the brain, away from the side of the Vibratome, to provide structural support during the slicing.
SECTION: Brain slice preparation
7. Use the Up-rocker button to move the buffer tray and brain to a position where the exposed surface is just below the razor blade edge and press start to begin the brain slicing  (300 1534 VTA, coronal section). Using a transfer pipette, transfer each individual brain slice containing the region of interest to a holding chamber pre-filled and with aCSF (saturated with carbogen). 
SECTION: Brain slice preparation
8. Incubate the slices for 60 min at 33 °C for 50 min-60 min in the carbogen bubbled aCSF solution and then allowed to cool to Room temperature (22 °C-24 °C) until the recordings initiate.

SECTION: Brain slice preparation
2. Saturate with carbogen (95% O2/5% CO2) for >15 min.
SECTION: Brain slice preparation
3. Set up Vibratome (Leica VT 1200s) chamber and adjust the cutting speed 0.3, amplitude 0.7.
SECTION: Electrophysiology recording
9. Fill up a bottle with aCSF solution bubble with carbogen and adjust the flow rate of aCSF solution to approximately 2 mL/min. 
SECTION: Electrophysiology recording
10. Place brain slice into recording chamber using small brush or transfer pipette.
SECTION: Electrophysiology recording
11. Fill the glass recording pipette with intracellular solution using Microfil and filter again with syringe  filter (0.2 µm), making sure the solution is all the way down at the tip of the pipette (no air).
SECTION: Electrophysiology recording
12. Attach the pipettes to the electrode holders of the patch-clamp amplifier headstages and turn into position.
SECTION: Electrophysiology recording
13. Using fine control micromanipulators, descend the recording pipettes to the region of interest within the brain slice.
SECTION: Electrophysiology recording
14. If required, use a coarse-manipulator to position an appropriate stimulating electrode again to the appropriate region of the brain slice to stimulate inputs to recorded neurons (from soma 60 1622-100 1622 distance).

SECTION: Electrophysiology recording
15. Once pipette is in contact with a neuron within the brain slice, apply negative pressure to pipette via 1 ml or 2 ml syringe. Monitor resistance of seal formation on oscilloscope or computer.
SECTION: Electrophysiology recording
16. Once seal resistance has exceeded 1 GΩ, using amplifier and computer software to compensate transients and apply further negative pressure to rupture cell membrane gaining whole-cell access to neuron.
SECTION: Electrophysiology recording
17. Perform current-voltage relationship using computer-controlled software to access neuronal health and to assess for presence of active membrane conductance.
SECTION: Electrophysiology recording
18. Once happy with quality of recording, perform set experiment applying test compounds (DART, DNQX 20 µM, AP-V 50 µM, etc) via connected in-tube line with the aCSF flow.