V. Isolation of AMPA and NMDAR EPSC

Procedure

1. Set up the slices in the recording chamber for whole cell recording.

2. Locate the stimulating electrode on Schaffer collateral pathway.

- stimulating electrode : using theta glass pipette (WPI, TST150-6), pull the pipette with 10 ~ 20 um tip-size. And inset sliver wires to the pipette (one wire in each tunnel) and hold it in the electrode holder

- Move the pipette to the recording chamber.

- Suck ext. solution with the syringe connected to the holder right after the tip of the simulating pipette touches the ext. solution.

3. Make a whole cell.

4. Check the recording parameters (Vm. Capacitance, resistance…).

5. 5~10 min after making whole cell, apply the stimulation to induce EPSCs.

6. Determine the intensity of stimulation.

- General method : Find the minimum intensity to fire APs and then use 1/3 ~ ½ of minimum intensity. This is usual for acute slices.

- SCJ version : Find the proper intensity to fire 100 pA EPSC at -60 mV holding potential. The minimum intensity to fire APs is very variable in organotypic slices.

7. Record EPSCs at – 60 mV holding potential and make it sure that EPSC waves should be isolated from artifacts of stimulation. 

The mix of artifacts and EPSCs indicates that the stimulation point is too close to the soma. This is not good for dendritic function experiments. A single EPSC peak should be observed (Jung et al., 2008)

8. After checking good EPSCs at – 60 mV holding potential, change the holding potential to + 40 mV(SCJ Ver. Don’t change membrane potential suddenly. Increase gradually the holding potential up to + 40 mV, likely -60, -50, -40 …….. 0…+20, +30, + 40).

9. 2-3 min after changing holding potential, check the current injection. 

For holding + 40 mV, current injection should be 300~500 pA in uninfected and infected neurons of DIV 6-7 organotypic slices. IF not, don’t use them. Sometimes, more time (over 5 min) will be necessary to set the stable current injection for + 40 mV holding. Don’t use neurons in this case. Normal neurons should show the short stabilizing procedure.

10. After checking the stable current injection, record total EPSCs(control, EPSCs at + 40 mV include all currents of NMDA AMPA and other recepors such as kinate. According to the your own recording protocol, save total EPSC waves for 5~10 min (SCJ ver. 0.1 Hz stimulation, 5 min, total 30 sweeps)).

11. After recording total EPSCs, add DNQX (10 uM) to the external solution to block AMPAR EPSCs. 

NBQX or CNQX can be placed for DNQX (SCJ ver. DNQX has not much effect on Ca-mediated channels)

12. After adding DNQX (~5 min), check EPSCs at -60 mV holding. 

No EPSCs should be observed at – 60 mV if DNQX is working well.

13. After blocking AMPAR EPSC with DNQX, record NMDAR EPSC at + 40 mV and save EPSC waves(SCJ ver. 30 sweeps for 5 min, twice).

14. After recording NMDAR EPSCs, add ifenprodil (3 uM) to the external solution to block NR2B containing NMDARs(DNQX should still block AMPARs).

15. After 5 min, record NMDAR EPSCs with adding ifenprodil. From now, record NMDAR EPSCs for 30 min(SCJ ver. save waves every 5 min for 30 min and compare the peak of averaged wave to check if ifenprodil is working). 

Use the data of last 5 min if ifenprodil is working.

16. Compare control, DNQX and DNQX + ifenprodil and analyze their peak and charge.

17. DNQX and ifenprodil are not washable, so don’t use the same neurons after one experiment.

18. Jung et al., 2008 is a great paper, which you can refer.

* You can use another antagonist to block some receptors. But check the properties of antagonist and make your own protocol to record what you want (Dose-dependence, Use-dependence, Permeability, Time-dependence, etc).