TITLE: Fast-scan cyclic voltammetry to assess dopamine release in ex vivo mouse brain slices
AUTHORS: Katherine Brimblecombe, Stephanie J Cragg

[DESCRIPTION]
This protocol is to assess whether a drug changes the dopamine concentration released following a single pulse (1p) electrical stimulation. 

[BEFORE_START]
Prior to drug application, you first need to determine a stable baseline. The variance permitted in the baseline will depend upon the effect size of your drug of interest. We recommend you first perform a pilot experiment to determine the approximate effect size and then determine a cut-off for any change in dopamine release over time, allowing you to pre-determine an exclusion criteria based on your control (pre-drug) data. 

If your cut-off is too stringent, you might use more animal tissue than is necessary (not in accordance with the 3Rs ), but also you may be sub-selecting a populations of release-sites that may not reflect the properties of the striatum you hope to generalise to. Setting a cut-off too permissive may result in a larger decay component due to time and may either over-estimate or underestimate your drug effect. 

Consider designing your experiment with a time-matched control. Having a pre-determined exclusion criteria will prevent you from erroneously excluding data that does not match your hypothesis and ensures your findings are more reproducible.

[STEPS]
SECTION: Preparation of ex vivo mouse brain slices
1. Prepare HEPES buffer solution (see Materials), chill and oxygenate.
SECTION: Preparation of ex vivo mouse brain slices
2. Prepare vibratome settings: 300 μm slices, 0.44 mm/s speed, Δ1.45 mm vibration. Chill plate and buffer tray in freezer, rinse razor blade in acetone. 
SECTION: Preparation of ex vivo mouse brain slices
3. Kill mouse by cervical dislocation, confirm death by exsanguination. 
SECTION: Preparation of ex vivo mouse brain slices
4. Decapitate mouse and take ear-clip for post-hoc genotyping where required (put in 2.5 ml Eppendorf).
SECTION: Preparation of ex vivo mouse brain slices
5. Remove brain.
SECTION: Preparation of ex vivo mouse brain slices
6. Block brain with razor blade to remove olfactory bulb and brain posterior to ~-1.055 mm bregma.
SECTION: Preparation of ex vivo mouse brain slices
7. Add small amount of cyanoacrylate glue to magnetic chuck, smooth with pipette tip.
SECTION: Preparation of ex vivo mouse brain slices
8. Transfer brain containing striatum to glue using filter paper to pick up block, and dampening fliter paper to reomve paper.
SECTION: Preparation of ex vivo mouse brain slices
9. This step is optional.

Save posterior brain block by transferring to Eppendorf containing 4 % PFA to allow fixation of midbrain (SNc and VTA) containing dopaminergic neurons. Tissue block can be sectioned once fixed to 40 µm sections and used for IHC.
SECTION: Preparation of ex vivo mouse brain slices
10. Section striatum to 300 μm-thick coronal slices.
11. Move slices to slice saver chamber at room temperature >1 hr.
 

SECTION: Setting-up rig
16. Prepare bicarbonate-buffered artificial cerebrospinal fluid (aCSF) solution (see Materials).
SECTION: Setting-up rig
17. Circulate dH2O through inflow pipes and recording chamber.
SECTION: Setting-up rig
18. Transfer silver weight pins from isopropanol to recording chamber to rinse.
SECTION: Setting-up rig
19. Circulate aCSF, ensuring no wicking up temp probe or reference Ag/AgCl2 pellet.

 

SECTION: Setting-up rig
20. Heater on, light on, stim box on, computer on. Master8 and VOLTAMMETER* must stay off until in circuit.
SECTION: Setting-up rig
21. Place coronal section in recording chamber and place silver pins on cortex to keep slice in place. 

 

SECTION: Setting-up rig
22. Insert the recording electrode ~100 µm into the tissue at 45o angle, connect it to voltammeter headstage, and switch on voltammeter.
SECTION: Setting-up rig
23. Check waveform of electrode, and perform a quality control of the electrode. 
SECTION: Setting-up rig
24. Allow electrode to charge for >30 mins to gain capacitance to ensure stable baseline of DA detection before start of stimulation.
SECTION: Determining evoked extracellular dopamine (DA) concentration
25. The below parameters were used to assess the effect of application of a drug (DHBE) on dopamine release evoked by single electrical pulses.

Stimulation: 200 µs, 0.6 mA 
Temperature: 31-33ºC
Perfusion speed: >1.8 ml/min
Inter-stimulation interval: 2.5 min
Voltammeter sweep: -700V to 1300V to -700V, 800 V/s. Repeated at 8 Hz. Switches out of circuit between scans.
Default gain settings: faradaic gain: x20, full signal gain: ~3-10 mV/nA, to avoid saturation of amplifier.
Drugs: record in DHBE 1 µM throughout to exclude effect of co-activation of ACh release and nAChR activation. Wash on drug of interest added to this solution. 


 

SECTION: Determining evoked extracellular dopamine (DA) concentration
26. Check settings on axoscope and set file directory and filenames. 
SECTION: Determining evoked extracellular dopamine (DA) concentration
27. Make up drugs and ensure fully thawed.
SECTION: Determining evoked extracellular dopamine (DA) concentration
28. Place stimulation electrode (bipolar concentric electrode) on surface of tissue. Surface location minimises damge.
SECTION: Determining evoked extracellular dopamine (DA) concentration
29. Place recording electrode ~100 µm from recording electrode, 100 µm into the tissue.
SECTION: Determining evoked extracellular dopamine (DA) concentration
30. Record dopamine (DA) release.
SECTION: Determining evoked extracellular dopamine (DA) concentration
30.1. Before recording, “refresh” voltammeter. (Specific for acquisition and use of Millar Voltammeter)
SECTION: Determining evoked extracellular dopamine (DA) concentration
30.2. Record >1s of baseline prior to then delivering 1p electrical stimulation.
SECTION: Determining evoked extracellular dopamine (DA) concentration
30.3. Record for sufficient time to allow DA to return to baseline (in drug free condition ~3 s, in the presence of DAT inhibitors will need to record for longer). 
SECTION: Determining evoked extracellular dopamine (DA) concentration
30.4. Note approximate peak voltage at DA oxidation potential in a lab book to alllow investigator to follow the approximate outcome of experiments during progress. 
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
31. Wait 2.5 mins.
SECTION: Determining evoked extracellular dopamine (DA) concentration
32. Repeat steps 26 and 27  until >6 stable recordings in control condition.
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
33. Move inflow pipe to drug cylinder try to minimise bubbles.
SECTION: Determining evoked extracellular dopamine (DA) concentration
34. As drug washes into the recording chamber, watch the oscilloscope in real time as drug washes on for indication of electroactive drugs.
SECTION: Determining evoked extracellular dopamine (DA) concentration
35. Continue steps 26 and 27 until 12 recordings in drug condition.
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
36. If wash-off is required, move inflow pipe to control solutions and repeat data acquisition for approximately 20 stims.
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
37. After experiment has finished, remove electrodes and ensure voltammeter is switched off when out-of-circuit.
SECTION: Determining evoked extracellular dopamine (DA) concentration
38. Remove slice. 
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
39. Allow aCSF to run through rig before placing next slice for more experiments or before electrode calibration.
SECTION: Determining evoked extracellular dopamine (DA) concentration
40. Calibration
Immediately before calibration make up 2 µM DA (see Materials).
SECTION: Determining evoked extracellular dopamine (DA) concentration
40.1. Allow aCSF to circulate at high speed (~4 ml/min). 
SECTION: Determining evoked extracellular dopamine (DA) concentration
40.2. Place electrode directly in front of inflow tube.


SECTION: Determining evoked extracellular dopamine (DA) concentration
40.3. Connect electrode to voltammeter and turn on voltammeter to allow the electrode to settle for >10 mins.
SECTION: Determining evoked extracellular dopamine (DA) concentration
40.4. Switch trigger settings on axoscope to “immediate” to record without triggering stim electrode.
SECTION: Determining evoked extracellular dopamine (DA) concentration
40.5. Rapidly switch inflow tube from aCSF to 2 µM DA solution. Minimise bubbles which interfere with the electrode                                                                                                                                                                                                                                                                                                                                                                                

SECTION: Determining evoked extracellular dopamine (DA) concentration
40.6. Watch bubbles to know when DA is washing on. 
SECTION: Determining evoked extracellular dopamine (DA) concentration
40.7. Start recording axoscope.
SECTION: Determining evoked extracellular dopamine (DA) concentration
40.8. Refresh voltammeter immediately before DA washes on.
SECTION: Determining evoked extracellular dopamine (DA) concentration
40.9. Watch oxidation peak rise with DA wash-on, for ~30 s.
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
40.10. Switch tube to aCSF, and repeat as for wash-on to record negative ox potential for wash off
SECTION: Determining evoked extracellular dopamine (DA) concentration
40.11. Repeat 3x and average wash-on and wash-off for approx. electrode sensitivity. 
SECTION: Determining evoked extracellular dopamine (DA) concentration
40.12. Plot peak oxidation potential over time for each DA wash on.

 
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
40.13. Repeat calibration for each recording solution used (i.e. each drug you use) to check drugs are not affecting electrode sensitivity. This is especially important if changing divalent ion concentration.

 

SECTION: Determining evoked extracellular dopamine (DA) concentration
40.14. Keep electrode until data has been analysed. 

 

SECTION: Determining evoked extracellular dopamine (DA) concentration
41. At end of the day, wash through with dH20 and then air to ensure clean and dry. Use cotton bud with HCl to clean bath and check no salt build up around temperature probe or ref.
SECTION: Determining evoked extracellular dopamine (DA) concentration
42. Rinse bubblers and bubble dry to ensure they do not backfill with liquid and become contaminated or grow mould.
SECTION: Determining evoked extracellular dopamine (DA) concentration
43. Release pressure points from the peristaltic pump on inflow and outflow tubes to prolong the life of the tubing. Do not stretch tubing beyond where is necessary to hook on as they may break.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
44. 
SECTION: Optional: If comparing drug vs vehicle control from tissue taken from same mouse
12. Set up needle bubbler (carbogen inlet connected to needle (26G) that rests above glass vial containing 10 mls aCSF). 
 


SECTION: Optional: If comparing drug vs vehicle control from tissue taken from same mouse
13. Add required drug or vehicle to 10 mL of aCSF in the vial and clearly label vial.
SECTION: Optional: If comparing drug vs vehicle control from tissue taken from same mouse
14. Bisect slice and put each hemisphere into appropriate vial. 


 


SECTION: Optional: If comparing drug vs vehicle control from tissue taken from same mouse
15. Allow slices to be incubated in drug or vehicle for typically 30 mins.