TITLE: Voltage sensor imaging in a population of dopaminergic axons in the mouse striatum
AUTHORS: Lucille Duquenoy, Bethan O'Connor, Stephanie J Cragg

[DESCRIPTION]
This protocol describes how to perform voltage sensor imaging from a population of striatal dopaminergic axons using high frame rates (600 Hz minimum) on coronal mouse brain slices. In particular, this protocol has been optimised for the use of ASAP5f voltage sensor (AAV9-EF1α-DIO-ASAP5-WPRE) from the Lin lab and changes in fluorescence are resulting from electrical stimulation.  

[BEFORE_START]
Intracranial injections:  
Between 3 to 4 weeks prior to the experiments, we injected heterozygous DAT-Cre mice with the ASAP5f voltage sensor (AAV9-EF1α-DIO-ASAP5-WPRE). We injected the virus diluted to 1.92x1012 vg/mL bilaterally in the midbrain (1 μL per site).  
 
To target mostly VTA (to record from NAcC) we used the following coordinates from Bregma: A/P: -3, M/L: +/- 1, D/V: -4.25, raising the needle to -4.2 by the end of the injection.
To target mostly SNc (to record from DLS), we used the following coordinates from Bregma: A/P: -3, M/L: +/- 1.25, D/V: -4.25, raising the needle to -4.2 by the end of the injection.
 
We found that using the same coordinates bilaterally to target VTA dopaminergic neurons and ultimately record from NAcC were giving the best results rather than targeting VTA in one hemisphere and SNc in the second one. 
 

[STEPS]
SECTION: Brain slice preparation
1. Sacrifice mice using cervical dislocation, exsanguination and decapitation. Quickly open the skull and collect the brain in an ice-cold slicing solution saturated with 95% O2/ 5% CO2, containing (in mM): 194 sucrose, 30 NaCl, 4.5 KCl, 1 MgCl2-6H2O, 26 NaHCO3, 1.2 NaH2PO4, 10 glucose.
SECTION: Brain slice preparation
2. Cut 300 μm thick coronal slices using a vibratome in ice-cold slicing solution saturated with 95% O2/ 5% CO2.
SECTION: Brain slice preparation
3. Immediately transfer each slice to a beaker in a water bath at 32°C, the beaker filled with aCSF saturated with 95% O2/ 5% CO2, containing (in mM): 130 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 10 glucose, 2 MgCl2-6H2O, 2.5* CaCl2. Leave the beaker in the water bath at 32°C for 15 minutes. Then move the beaker to room temperature (20°C-22°C) for a minimum of 45 minutes before using the slices.  This protocol also works well with aCSF containing 2 mM CaCl2.

 

SECTION: Data acquisition
18. Go back to the Acquisition Setup menu and in the Triggering drop-down menu, change the trigger to External Start in order to have time-locked stimulations. 
SECTION: Brain slice preparation
4. While the slices are equilibrating, turn on the rig perfusion system, the heater, and run through dH2O. The temperature in the recording chamber should be 32°C ± 1°C and the perfusion rate should be ~ 2 mL/min.
SECTION: Choosing acquisition settings and placing stimulating electrode
6. Open the Andor Solis software and click on the Real Time icon to open the camera shutter and achieve visualisation of the slice. 

 

SECTION: Choosing acquisition settings and placing stimulating electrode
7. Use a 10x microscope objective to locate the region of the brain slice you are interested in. Keep the wide field light intensity to a minimum to avoid bleaching of your sample.  
SECTION: Brain slice preparation
5. Run aCSF through the perfusion system before putting your slices (or hemisected slices) in the recording chamber.
SECTION: Choosing acquisition settings and placing stimulating electrode
8. Briefly, turn on the blue LED light (λ = 470 nm, 10 mW) to confirm good viral expression.  
SECTION: Choosing acquisition settings and placing stimulating electrode
9. Using the wide field light, position your stimulating electrode on the surface of the brain slice and centre it in the field of view. 
SECTION: Choosing acquisition settings and placing stimulating electrode
10. Switch to a 60x objective, and locate the tip of the stimulating electrode. 
SECTION: Choosing acquisition settings and placing stimulating electrode
11. Navigate to around ~ 150 μm (using the conversion between pixel size to actual distance that you can find in the specification documents of the camera) away from the stimulating electrode and place that region of interest in the bottom left corner of the sensor. 

 

SECTION: Choosing acquisition settings and placing stimulating electrode
12. Click on the Acquisition Setup icon to open the acquisition settings. In the Camera Setup tab, choose Kinetic as the acquisition mode. Check the boxes for Frame Transfer and Crop Mode. Set the Electron Multiplier (EM) Gain Level to 200. 
SECTION: Choosing acquisition settings and placing stimulating electrode
13. In the Crop Mode ROI tab, choose the size of your ROI (148 pixels high x 496 pixels long) and choose a pixel binning of 4x4. 
 

SECTION: Choosing acquisition settings and placing stimulating electrode
15. Go back to the Camera Setup tab and change the exposure time to the minimum (0.0015870 seconds) to reach a frame rate of around 600 Hz (599.52 Hz). Close Acquisition Setup.
SECTION: Choosing acquisition settings and placing stimulating electrode
16. Click the Real Time icon and turn on the wide field light to position the blades of the OptoMask, ensuring that the edges of all four blades are just visible inside the ROI. 
 

SECTION: Choosing acquisition settings and placing stimulating electrode
17. Briefly turn on the blue LED light to make sure that the surface of the brain slice is in focus.
SECTION: Data acquisition
19. Calculate the right number of frames to be recorded (frame rate * seconds) and enter this value in the Kinetic Series Length box. 
 

SECTION: Data acquisition
20. Navigate to the Auto Save tab and select the folder in which your want your files to be saved. Here, you can personalise the file naming structure.


SECTION: Data acquisition
21. When you are ready to begin, click on the Take Signal icon, and start your stimulation protocol in your preferred software/hardware (we use a Multi Channel Systems stimulus generator and the accompanying MC_Stimulus II software). We usually deliver the electrical stimulation at 1.4 s into the recording, so that the initial “exponential” 'bleaching' decay has passed and the stimulation occurs when the 'bleaching' appears more linear.  
 

SECTION: Data acquisition
22. Wait for at least 1 minute before the next stimulation. 
SECTION: Choosing acquisition settings and placing stimulating electrode
14. In the Auto-Save tab, choose the folder you wish to save the data to, and any file naming options you desire.