TITLE: Fast-scan cyclic voltammetry to assess dopamine release in ex vivo mouse brain slices while optogenetically activating astrocytes
AUTHORS: Shinil Raina, Bradley M Roberts, Stephanie J Cragg

[DESCRIPTION]
This protocol is to assess whether optogenetically activating astrocytes affects the dopamine concentration released following electrical stimulation. 

[BEFORE_START]
Stereotaxic intracranial injections were performed to deliver AAV5/hgfaABC1D-ChR2(H134R)_EYFP in the mouse striatum. After injection, mice were kept for 3 weeks to ensure appropriate construct expression. These animals were then used in the subsequent steps.

Prior to starting the experiment, you first need to determine a stable baseline. The variance permitted in the baseline will depend upon the effect size of your manipulation. 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 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 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 cutting solution, chill and oxygenate (see Materials).
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 (Schedule 1 method), confirm death by exsanguination. 
SECTION: Preparation of ex vivo mouse brain slices
4. Decapitate mouse and remove brain on to iced plate. 
SECTION: Preparation of ex vivo mouse brain slices
5. Block brain with razor blade to remove olfactory bulb and posterior part of brain.
SECTION: Preparation of ex vivo mouse brain slices
6. Add small amount of cyanoacrylate glue to magnetic chuck, smooth with pipette tip.
SECTION: Preparation of ex vivo mouse brain slices
7. Transfer brain containing striatum to glue, mounted on the posterior surface, using filter paper to pick up block, and dampening filter paper to remove paper.
SECTION: Preparation of ex vivo mouse brain slices
8. 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
9. Section mounted striatum to 300 μm-thick coronal slices.
SECTION: Preparation of ex vivo mouse brain slices
10. Move slices to slice saver chamber at room temperature for >1 hr.
 

SECTION: Setting-up rig
12. Prepare artificial cerebrospinal fluid (aCSF) solution and oxygenate (see Materials).
SECTION: Setting-up rig
13. Pump in dH2O through inflow pipes and recording chamber to ensure flushed clean before placing slice.
SECTION: Setting-up rig
14. Transfer silver weight pins from isopropanol storage vial to recording chamber to rinse.
SECTION: Setting-up rig
15. Pump aCSF in to recording chamber, ensuring no wicking up the temperature probe or reference electrode (Ag/AgCl2 pellet).

 

SECTION: Setting-up rig
16. Camera on, heater on, light on, stimulating box on, computer on. Voltammeter*  stays off until electrode is in circuit.
SECTION: Setting-up rig
17. Place striatal section in recording chamber and place silver pins on cortex to keep slice in place. 

 

SECTION: Setting-up rig
18. Visualised using microscope, 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
19. Check waveform applied to electrode, and read detected current to ensure electrode viability and low noise. 
SECTION: Setting-up rig
20. Allow electrode to charge and stabilise for >30 mins in tissue.
SECTION: Determining evoked extracellular dopamine (DA) concentration
21. The below parameters were used to assess the effect of optogenetically activating astrocytes 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: Full signal gain: ~3-10 mV/nA, to avoid saturation of amplifier. Additional faradaic current gain: x20. 
Light stimulation: 470 nm wavelength light, 4-5 mW/mm2, 500 ms.
Drugs: Control experiments conducted in drug-free aCSF, dihydro-β-erythroidine hydrobromide (DHβE, 1 μM), (+)-bicuculline (10 μM), CGP 55845 hydrochloride (CGP, 4 μM), 8-cyclopentyl-1,3-dimethylxanthine (CPT, 10 μM), oxotremorine (10 µM).


 

SECTION: Determining evoked extracellular dopamine (DA) concentration
22. Check settings on axoscope and set file directory and filenames. 
SECTION: Determining evoked extracellular dopamine (DA) concentration
23. Make up drugs, if using.
SECTION: Determining evoked extracellular dopamine (DA) concentration
24. Visualised through a microscope (and/or camera), place stimulation electrode (bipolar concentric electrode) on surface of tissue. Surface location minimises damage.
SECTION: Determining evoked extracellular dopamine (DA) concentration
25. Place recording electrode ~100 µm from stimulating electrode, 100 µm vertical depth into the tissue.
SECTION: Determining evoked extracellular dopamine (DA) concentration
27. Wait 2.5 mins.
SECTION: Determining evoked extracellular dopamine (DA) concentration
28. Repeat steps 26 and 27  until around 6 approximately stable recordings in control condition.
 

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

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


SECTION: Determining evoked extracellular dopamine (DA) concentration
35.3. Connect electrode to voltammeter and turn on voltammeter to allow the electrode to settle for >10 mins.
SECTION: Determining evoked extracellular dopamine (DA) concentration
35.4. Switch trigger settings on axoscope to “immediate” to record without triggering stim electrode.
SECTION: Determining evoked extracellular dopamine (DA) concentration
35.5. 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
35.6. Watch bubbles to know when DA is washing on. 
SECTION: Determining evoked extracellular dopamine (DA) concentration
35.7. Start recording axoscope.
SECTION: Determining evoked extracellular dopamine (DA) concentration
35.8. Refresh voltammeter immediately before DA washes on.
SECTION: Determining evoked extracellular dopamine (DA) concentration
35.9. Watch oxidation peak rise with DA wash-on, for ~30 s.
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
35.10. Switch tube to aCSF, and repeat as for wash-on to record negative oxidation potential for wash-off.
SECTION: Determining evoked extracellular dopamine (DA) concentration
35.11. Repeat as desired to gain a reproducible estimate of electrode sensitivity. 
SECTION: Determining evoked extracellular dopamine (DA) concentration
35.12. Plot peak oxidation potential over time for an example DA wash-on.

 
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
35.13. 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
35.14. Keep electrode until data has been analysed. 

 

SECTION: Determining evoked extracellular dopamine (DA) concentration
36. At end of the day, wash recording chamber through with dH20 and then empty. Use cotton bud with HCl to clean bath and dissolve any salt build-up around temperature probe or ref electrode.
SECTION: Determining evoked extracellular dopamine (DA) concentration
37. Rinse aCSF bubblers and aerate them until dry to ensure they do not remain full with liquid and become contaminated or grow mould.
SECTION: Determining evoked extracellular dopamine (DA) concentration
38. Release pressure points from the peristaltic pump on inflow and outflow tubes to prolong the life of the tubing.  
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
29. When stable recordings are obtained with 1p stimulation, follow the stimulation protocol outlined for a baseline set of recordings.

1p

Wait 2.5 min

5p at 50Hz

Wait 2.5 min

1p
SECTION: Determining evoked extracellular dopamine (DA) concentration
30. To explore impact of prior light activation of ChR2-expressing astrocytes, repeat step 29 pairing each electrical stimulation with a 470 nm light stimulation starting at 400 ms preceding the electrical stimulation.  
 

SECTION: Determining evoked extracellular dopamine (DA) concentration
31. Repeat alternatively step 29 and  step 30 for example x3 to gain dataset in triplicate.
SECTION: Determining evoked extracellular dopamine (DA) concentration
29.1. Repeat step 29 only for the baseline recordings.
SECTION: Determining evoked extracellular dopamine (DA) concentration
28.1. If applying a drug, continue stimulations at same interval during drug equilibration period. This period will vary significantly depending on drug characteristics (e.g. molecular weight, diffusion profile in brain tissue, absorption, uptake/breakdown, hydrophobicity/hydrophilicity).
SECTION: Preparation of ex vivo mouse brain slices
11. Optional step: Take ear-clip for post-hoc genotyping where required (put in 2.5 ml Eppendorf).
SECTION: Determining evoked extracellular dopamine (DA) concentration
26. Activate data capture on computer recording system, stimulate and record evoked signals until return to baseline.