Figure 1. Exposure of AHA-GnRH neurons

Materials and Reagents

1. GnRH-GFP mice (Spergel et al., 1999; Suter et al., 2000)
   
2. Pentobarbital
   
3. CaCl₂
   
4. MgCl₂
   
5. HEPES
   
6. NaHCO₃
   
7. D-glucose
   
8. Sucrose
   
9. O₂ Medical Grade
   
10. 5% CO₂ Carbogen
    
11. Artificial cerebrospinal fluid (aCSF) (see Recipes)
    

Equipment

1. Set Up
   1. Metal plate 200 x 200 x 1 mm
      
   2. Binocular microscope (Leica Microsystems, model: M80 )
      
   3. Cold light source (Leica Microsystems, model: CLS 150X )
      
   4. Upright microscope (OLYMPUS, model: BX51 )
      
   5. Long working distance objective (20x M Plan Apo NA 0.42) (Mitutuyo, catalog number: 378-804-2 )
      
   6. Blue fluorescence illumination (OLYMPUS, model: U-MGGFPHQ ) (excitation wavelength centered at 470 nm with high pass emission filtered at 517 nm)
      
   7. AC/DC bioamplifier (CWE, model: BMA-200 )
      
   8. Analogic-to-digital converter (Molecular Devices, model: Digidata 1332A )
      
   9. Subcutaneous electrodes (platinum ~20 G) hooked to the bioamplifer
      
   10. Ventilator (CWE, model: SAR-830/P) with the following parameters: 100 breaths/min, with constant pressured cycles set at 1.4 cm H₂O, 0.3 s/inspiration, O₂ 90–80 ml/min
       
   11. Body temperature controller (CWE, model: CT-1000) with heat pad and rectal probe, set at 37 °C
       
       
2. Surgery
   1. Stainless steel blocks (L x W x H: 60 x 40 x 10 mm)
      1. Home-made block, inclinable with 2 screw = “pillow” (Figure 2)
         
         
         Figure 2. Pillow
         
         
      2. Home-made block, inclinable with 2 screw and holding a stainless steel spatula narrowed down to 3 mm wide with a fine file to fit in the mouth of a mouse, bent into hook (about 0.80 cm diameter) = central separator (Figure 3)
         
         
         Figure 3. Central separator
         
         
   2. Syringe (26 G needle) for intraperitoneal and intramuscular injections
      
   3. Syringe (23 G needle) for chilled aCSF
      
   4. Forceps Dumont No 5
      
   5. Small scissors
      
   6. Spring scissors 8 mm blades (Fine Science Tools, catalog number: 15024-10)
      
   7. Bone scissors Bone Cutting Spring Scissors (Fine Science Tools, catalog number: 16144-13)
      
   8. Agricola retractor 3.5 cm spread (Fine Science Tools, catalog number: 17005-04)
      
   9. Micro-drill (GEBR Brasseler; Ideal Micro-Drill) (PW Stoelting, catalog number: 58610)
      
   10. Round burr tip (o.d. 0.5 mm)
       
   11. Bone wax (Johnson & Johnson, Ethicon, model: W810)
       
   12. Syringe body holding a 23 G needle taped into a hook = needle hook
       
   13. Dental vacuum connected with 1-ml syringe body holding a cut 23 G needle (23 G-needle dental vacuum) or 1-ml insulin syringe holding a cut 30 G needle (30 G-needle dental vacuum)
       
   14. 50-ml syringe for inflow (gravity) with 1 home-made magnetic holder
       
   15. Peristaltic pump for outflow with a cut 23 G needle held by home-made magnetic holder (Figure 4)
       
       
       Figure 4. Magnetic holder
       
       
   16. Cotton balls
       
   17. Tissue paper, separated before use
       
   18. Lab tape
       
   19. Cotton sewing thread No 50
       
   20. Super glue with brush
       
   21. Polyethylene catheter (o.d. 0.96 x i.d. 0.58 mm)
       
   22. Lighter
       

Software

1. ClampEx pCLAMP 10 Electrophysiology Data Acquisition and Analysis Software

Procedure