TITLE: In vivo voltammetry and fiber photometry in the mouse striatum
AUTHORS: Yan-Feng Zhang, Stephanie J Cragg

[DESCRIPTION]
This protocol describes how to measure extracellular dopamine concentrations and calcium dynamics in striatal dopaminergic axons in response to electrical stimulations using in vivo voltammetry and fiber photometry. 

[BEFORE_START]
The in vivo voltammetry was performed in wild-type mice (C57BL/6J, 21-40 days).

The in vivo fiber photometry was performed in two different animal backgrounds:

(A) For measuring calcium dynamics, we use heterozygous DAT-Cre:Ai95D (4-7 weeks) mice that were bred from homozygous DAT-Cre mice (B6.SJL-Slc6a3tm1.1(cre)Bkmn/J, JAX stock number 006660) crossed with homozygous Ai95D mice (B6;129S-Gt(ROSA)26Sortm95.1(CAG-GCaMP6f)Hze/J, JAX stock number 028865). These mice express the genetically encoded calcium indicator GCaMP6f in striatal dopaminergic axons. 

(B) For measuring evoked extracellular dopamine concentrations, we followed the steps described in the Protocol: Intracranial injections of viral vectors in mouse midbrain and striatum to inject the AAV serotype 5 vector containing GRAB-DA2m (AAV5-hSyn-GRABDA2m) in the dorsal striatum of wild-type mice. The titer dilution was 1E+13 vg/mL. 


[STEPS]
SECTION: Preparing the mouse for surgery
1. Anesthetize the mouse in an induction chamber with urethane (1.4–1.9 g/kg, i.p.; Biolab), supplemented with additional urethane (0.2 g/kg) every 1-2 hr as required. 
SECTION: Preparing the mouse for surgery
2. Infiltrate all wounds and pressure points with bupivacaine (0.5%). 
SECTION: Preparing the mouse for surgery
3. Upon reaching surgical anaesthesia, fix the head in a stereotaxic frame. 
SECTION: Preparing the mouse for surgery
4. Monitor core temperature using a rectal probe and maintain it at 35-36°C using a homeothermic blanket. 
SECTION: Preparing the mouse for surgery
5. Administer mecamylamine (2 mg/kg) by intraperitoneal injection to block nAChRs in the striatum.
SECTION: Measuring evoked extracellular dopamine (DA) concentration using fast-scan cyclic voltammetry (FCV)
6.1. Remove a round piece of skull overlying the left hemisphere to target the dorsolateral striatum (DLS) (AP +1.0 mm, ML 1.6 mm, DV 2.2 mm to bregma).
SECTION: Measuring evoked extracellular dopamine (DA) concentration using fast-scan cyclic voltammetry (FCV)
6.2. Position a stimulating and recording array consisting of a 7 µm-diameter carbon fiber microelectrodes (CFMs; tip length 50-100 µm) and a bipolar stimulating electrode (MS303/3-A/SPC, P1 Technology) in the DLS. 
SECTION: Measuring evoked extracellular dopamine (DA) concentration using fast-scan cyclic voltammetry (FCV)
6.3. Implant the Ag/AgCl reference electrode in another part of the forebrain. 
SECTION: Measuring evoked extracellular dopamine (DA) concentration using fast-scan cyclic voltammetry (FCV)
8. Use fast-scan cycling voltammetry (FCV) with carbon fiber microelectrodes and a Tarheel system (University of Washington, Seattle, US) to measure evoked extracellular dopamine concentration.
SECTION: Measuring evoked extracellular dopamine (DA) concentration using fast-scan cyclic voltammetry (FCV)
9. Apply voltage as a triangular waveform (-0.4 to +1.3 V range versus Ag/AgCl) at a scan rate of 400 V/s.
SECTION: Measuring evoked extracellular dopamine (DA) concentration using fast-scan cyclic voltammetry (FCV)
11. Apply electrical stimulus (0.65 mA current, 200 µs) using a bipolar stimulating electrode (0.005 inch, MS303/3-A/SPC, P1 Technologies).
 
 
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
12.1. Remove a round piece of skull overlying the left hemisphere to target the dorsolateral striatum (DLS) (AP +1.0 mm, ML 1.6 mm, DV -2.2 mm to bregma) and susbtantia nigra (SNc) (AP -3.1mm, ML 0.8mm, DV -4.3mm to bregma). 
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
12.2. Position the injection and recording array, consisting of a glass pipette and a 200 µm diameter fibre, in the DLS. 
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
13.3. Apply electrical stimulus (0.5 mA current, 500 µs) using a bipolar stimulating electrode (0.005 inch, MS303/3-A/SPC, P1 Technologies) at 0.1 Hz. 
 

SECTION: Verification of carbon-fibre locations in dorsal striatum
15. Sacrifice anesthetised mice and quickly remove brains.
SECTION: Verification of carbon-fibre locations in dorsal striatum
16. Fix whole brains overnight in 4% PFA.
SECTION: Verification of carbon-fibre locations in dorsal striatum
17. Section fixed brains into 50 μm slices using a vibratome.
SECTION: Verification of carbon-fibre locations in dorsal striatum
18. Mount the individual slices on glass slides and image under microscope to identify the location of recording sites. 
SECTION: Measuring evoked extracellular dopamine (DA) concentration using fast-scan cyclic voltammetry (FCV)
10. Sample data at 10 Hz. 
SECTION: Measuring evoked extracellular dopamine (DA) concentration using fast-scan cyclic voltammetry (FCV)
6. Surgical Procedure:
SECTION: Measuring evoked extracellular dopamine (DA) concentration using fast-scan cyclic voltammetry (FCV)
7. Determining evoked extracellular dopamine (DA) concentration using electrical stimulations:
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
12. Surgical Procedure:
These steps were performed in heterozygous DAT-Cre:Ai95D (4-7 weeks) mice expressing the genetically encoded calcium indicator GCaMP6f in striatal dopaminergic axons, or in wild-type mice expressing GRABDA2m in the dorsal striatum. 
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
13. Determining axonal calcium dynamics in striatal dopaminergic (DA) axons using electrical stimulations:
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
13.1. Activate GCaMP6f expression in DA axons with 480 nm light (76 µW).
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
13.2. Sample the intensity of GCaMP6f at 40 Hz with Neurophotometrics (FP3001). 
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
14. Determining evoked extracellular dopamine (DA) concentration using electrical stimulations:
These steps were performed in wild-type mice expressing GRABDA2m in the dorsal striatum. 
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
14.1. Activate GRAB-DA2m with 480 nm light (76 µW).
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
14.2. Sample the intensity of GRAB-DA2m at 40 Hz with Neurophotometrics (FP3001). 
SECTION: Measuring axonal calcium dynamics and evoked extracellular dopamine (DA) concentration using fiber photometry
14.3. Apply electrical stimulus (0.5 mA current, 500 µs) using a bipolar stimulating electrode (0.005 inch, MS303/3-A/SPC, P1 Technologies) at 0.05 Hz.