Materials and Reagents

Materials

1. For the ‘nest Beaker’
   1. Nylon tights
      
   2. Instant superglue (such as Scotch Super Glue, 3M, catalog number: AD124 )
      
   3. 15 ml tubes (such as VWR, catalog number: 89039-670 US, 525-0450 Europe)
      
   4. Disposable 6 cm diameter plastic Petri dish (such as Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 123TS1 )
   
   
2.  For dissection
   1. Large kitchen scissors or guillotine
      
   2. Straight fine scissors (such as Fine Science Tools, catalog number: 14060-11 )
      
   3. Curved spatula (such as Fine Science Tools, catalog number: 10092-12 )
      
   4. Scalpel (such as Fine Science Tools, catalog number: 91003-12 )
      
   5. Glass disposable Pasteur pipet (such as Fisher Scientific, FisherBrand, catalog number: 13-678-6A )
      
   6. Dropper bulb (such as Fisher Scientific, FisherBrand, catalog number: 03-448-25 )
      
   7. Plastic container, about 2.5 cm high and 150 ml, such as the lid of a pipet tip box or a large glass Petri dish (Cole-Parmer Instrument, catalog number: EW-34551-06 )
      
   8. Whatman paper (GE Healthcare, Whatman, catalog number: 1001-090 )
      
   9. Disposable Razor blade (such as Personna Double Edge Razor Blades [Amazon, PERSONNA, catalog number: BP9020 ])
   
   
3. For conditioned medium procedure
   1. Disposable 6 cm diameter (or larger) plastic Petri dish (such as Thermo Fisher, catalog number: 123TS1 )
      
   2. 1.7 to 2 ml microtubes (such as Sorenson BioScience, catalog number: 16070 )
   
   
4. For biosensors holders
   1. 2 ml Falcon pipette (Fisher Scientific, catalog number: 13-678-11C )
      
   2. Heat-Shrink tubes (from RadioShack)
      

Reagents

1. Glucose (Sigma-Aldrich, catalog number: G7021 )
   
2. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
   
3. Sodium phosphate monobasic anhydrous (VWR, catalog number: 470302-666 )
   Manufacturer: ALDON, catalog number: SS0756-500GR .
   
4. Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: S5761 )
   
5. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
   
6. Magnesium chloride solution (1 M) (Sigma-Aldrich, catalog number: 63069 )
   
7. Calcium chloride solution (1 M) (Sigma-Aldrich, catalog number: 21115 )
   
8. D-serine (Sigma-Aldrich, catalog number: S4250 )
   
9. Stock artificial cerebrospinal fluid (ACSF) solution (see Recipes)
   
10. Ice-cold Slicing ACSF stock solution (see Recipes)
    
11. Recovery ACSF stock solution (see Recipes)
    
12. Experimental ACSF stock solution (see Recipes)
    

Equipment

1. 250 ml Pyrex beaker (such as VWR, catalog number: 10754-952 )
   
2. Straight spring scissors (such as Fine Science Tools, catalog number: 15018-10 )
   
3. Curved fine forceps (such as Fine Science Tools, catalog number: 11152-10 )
   
4. 600 ml Pyrex beaker (such as VWR, catalog number: 10754-956 )
   
5. 95% O₂/5% CO₂ tank (such as AirGas, catalog number: Z02OX9522000043 )
   
6. Vibratome (such as Leica, model: Leica VT 1200 S , catalog number: 14048142066)
   
7. Bath heater (such as Thermo Fisher Scientific, Thermo Scientific, model: Precision 180 , catalog number: 51221073)
   
8. Digi IVY DY2023 Bipotentiostat (Digi-Ivy, model: DY2023 ) (www.digi-ivy.com)
   
9. Connecting cables (provided with bipotentiostat)
   
10. Computer connected to the bipotentiostat
    
11. Sarissa Probe D-serine biosensor (Sarissa Biomedical, catalog number: SBS-DSER-05-50 )
    
12. Sarissa Probe Null Sensor (Sarissa Biomedical, catalog number: SBS-NUL-05-50 )
    
13. Minimal components of a classic electrophysiology rig (Figure 1):
    Perfusion/immersion chamber (such as Warner Instruments, model: RC-26G , catalog number: 64-0235)
    
    
    Figure 1. Setup needed to mount the biosensors (see text). For clarity, there is no liquid perfused through the chamber on the above image and only one biosensor was mounted.
    
    
14. Custom-made perfusion line (gravity or pump-operated) using flexible plastic tubing (such as Cole-Parmer Instrument, Masterflex, catalog numbers: ZM-96400-13 and ZM-96400-14 )
    Note: If the perfusion system is gravity-based, a vacuum line connected to the exit end of the chamber will be required to suction the liquid from the chamber and maintain a steady state immersion level in the chamber.
    1. Chamber temperature controller (such as Warner Instruments, model: TC-344C , catalog number: 64-2401)
       
    2. Microscope or any time of magnifying binocular allowing a 5x magnification (and lighting)
       
    3. Manual manipulators to allow the placement of the sensors (such as NARISHIGE, model: M-152 )
       
    4. Home-made connector to plug the sensors to the cables provided with the bipotentiostat and allowing placement in the manipulators (see Figure 2)
       
    5. Biosensor holder. Using a 2 ml Falcon pipette, cut the desired length (we recommend 10 cm), insert the biosensor connecting cable, wrap in heat-shrink tube (from RadioShack) as shown in Figure 2 and gently heat with a lighter or a soldering iron
       
       
       Figure 2. Making a biosensor holder. This will help manipulation and placement of the biosensors with manual manipulators.
       

Software

1. DY2000EN software (provided with bipotentiostat)
   
2. Clampfit (version 9.2 minimum)
   Note: If experiments are run on an electrophysiology set-up. Note that the DY2000EN software provides the sufficient analytical capabilities, but they were not used in our protocol.
   

Procedure