Materials and Reagents

1. Embryonic hippocampal neurons (E18)
   
2. SynaptopHluorin
   Note: pHluorin-based fusion-proteins in which ecliptic pHluorin was fused to an intraluminal loop of synaptotagmin (Fernandez-Alfonso and Ryan, 2006; Wienisch and Klingauf, 2006).
   
3. 0.03% trypsin (-20 °C) (Sigma-Aldrich)
   
4. 0.1% (w/v) poly-L-lysine (-20 °C) (Peptides International)
   
5. Neurobasal medium (2-8 °C) (Life Technologies, Gibco^®)
   
6. B-27 (-20 °C) (Life Technologies, Gibco^®)
   
7. Glutamine (final concentration: 0.5 mM, -20 °C) (Life Technologies, Gibco^®)
   
8. Glutamate (final concentration: 0.025 mM) (Sigma-Aldrich)
   
9. Fetal bovine serum (FBS) (-20 °C) (Life Technologies, Gibco^®)
   
10. Fluo-4 AM calcium indicator (-20 °C) (Life Technologies, Molecular Probes^®, catalog number: F14201 )
    
11. DL-2-Amino-5-phosphonopentanoic acid (APV) (final concentration: 50 µM, solution in 4 °C) (Sigma-Aldrich, catalog number: A5282 )
    
12. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (final concentration: 10 µM , -20 °C) (Sigma-Aldrich, catalog number: C127 )
    
13. Bafilomycin A1 (final concentration: 5 µM, 20 °C) (Calbiochem^®, catalog number: 196000 )
    
14. Vacuum grease (Dow-Corning, for mounting the coverslip)
    
15. HEPES-buffered saline (HBS) extracellular solution (4 °C) (see Recipes)
    
16. 2x HeBS (see Recipes)
    

Equipment

1. Pasteur pipette
   
2. No. 1, 12-mm diameter glass coverslips (Warner Instruments)
   Note: No.1 glass coverslips are washed with HCl and ethanol, then autoclaved. Autoclaved glass coverslips are treated in 0.1% (w/v) poly-L-lysine for overnight. poly-L-lysine is aspirated on the next day, and glass coverslips are washed with sterile water three times.
   
3. Inverted microscope (63x oil immersion lens, Mercury arc lamp, filters for EGFP) (ZEISS)
   
4. Computer with time-lapse imaging system
   
5. Electric field stimulation chamber (two parallel platinum wire bath electrodes) (Warner Instruments)
   
6. Perfusion pump (Ismatec^® REGLO Digital 2-Ch Var-Speed Pump; 0.005 to 59 ml/min) (Cole-Parmer, catalog number: WU-78016-40 )
   

Software

1. Axiovision LE software
   
2. Microsoft Excel
   

Procedure