Materials and Reagents

1. Fine needle (Bonn Micro Probes) (Fine Science Tools, catalog number: 10030-13 )
   
2. Cover glass (Matsunami Glass Ind, catalog number: C024241 )
   
3. Wild-type mice (Japan SLC, model: C57BL / 6JJmsSlc )
   
4. Isoflurane (e.g., Wako Pure Chemical Industries, catalog number: 099-06571 )
   
5. Lidocaine solution (80 mg/ml) (e.g., Xylocaine Pump Spray 8%) (AstraZeneca) for local anesthesia
   
6. Dental cements (Super-Bond C&B) (Sun medical) (GC's Global, UNIFAST II)
   
7. Voltage-sensitive dye (OPTICAL IMAGING LTD, catalog number: RH1691 )
   
8. NaCl
   
9. KCl
   
10. MgCl₂^.6H₂O
    
11. CaCl₂^.2H₂O
    
12. HEPES
    
13. Distilled water
    
14. RH1691
    
15. Ringer’s solution (see Recipes)
    
16. Voltage-sensitive dye (VSD) solution (see Recipes)
    

Equipment

1. Anesthesia system for isoflurane (Shinano, catalog number: SN-487-0T )
   
2. Electric clipper (e.g., Panasonic Corporation, catalog number: ER803P ) for cutting mouse hair
   
3. Feedback-controlled heating pad (Bio Research Center, catalog number: BWT-100A )
   Note: The pad monitors the mouse body temperature and maintains it at the desired temperature.
   
4. Head holder (stereotaxic apparatus) (NARISHIGE Group, model: SG-4N )
   
5. Fine scissors (Fine Science Tools, catalog number: 91460-11 )
   
6. Student Dumont #7 Forceps (Fine Science Tools, catalog number: 91197-00 )
   
7. Dumont #5SF Forceps (Fine Science Tools, catalog number: 11252-00 )
   
8. Cotton swab for absorbing the ringer’s solution
   
9. Vacuum pump (e.g., AGC TECHNO GLASS CO., model: VPUMP-140 ) for removing the ringer's solution
   
10. Surgical blade (e.g., Kai industries, catalog number: 310-A )
    
11. Head-fixation plate (handmade, Figure 1A)
    
12. Plate holder (custom-made, Figure 1B)
    
13. Stereo Microscope (OLYMPUS, model: SZX7 )
    
14. Green LED light (REVOX Inc., model: SLG-50S-G )
    
15. Dental drill (SHOFU Inc., model: Tas-35LX )
    
16. Dental round bur (SHOFU Inc., model: ELA Steel Bur HP-1 )
    
17. Lens blower (e.g., HAKUBA, model: KMC-45 )
    
18. Ultra-Fast CMOS Imaging System (Brainvision, model: MiCAM ULtima )
    Note: For reference imaging (see procedure step 13), the cortex is illuminated with a blue LED light (center wavelength: 465 nm) through a 506-nm dichroic mirror; green fluorescence is corrected through a 536/40-nm filter.
    For voltage-sensitive dye imaging, VSD fluorescence is excited with a red LED light (center wavelength: 625 nm). The excitation light is filtered with a 632/22-nm band-pass filter, reflected using a 655-nm dichroic mirror, and focused 375 μm below the cortical surface. Fluorescence is filtered with a 665-nm long pass filter.
    
19. Blue LED light (Brainvision, model: LEX2-B )
    
20. Red LED light (REVOX Inc., model: SLG-50S-R )
    
21. Stimulator (e.g., NIHON KOHDEN CORPORATION, model: SEN-5201 )
    
22. Metal electrodes for hindpaw stimulation (handmade, Figure 1C)
    
23. Vortex mixer (e.g., Scientific Industries Inc., model: VORTEX-GENIE 2 )
    
    
    Figure 1. Head-fixation equipment. A. Handmade head-fixation plate; B. Custom-made plate holder; C. Handmade metal electrodes for hindpaw stimulation.
    

Software

1. Acquisition software (Brainvision, model: UL-Acq)
   
2. Analysis software (Brainvision, model: BV_Ana)
   

Procedure