VI. Micro application by using Picospritzer

Procedure

1. Set up the Picospritzer III (Parker) according to the manual.

   • Connect the wall pressure line to the valve of the Picospritzer.

   • Set the control line and mid-boxes.

   • Connect the pressure line of the Picospritzer to the pipette holders.

   • Connect the external trigger line of ITC-18 to the Picospritzer.

   • Set the pressure level to less than 10 PSI.

2. Prepare the micropipette using glass electrodes (Warner, G200F-3) for drug application.
   (SCJ ver.: Using the puller, make a micropipette with a 2–3 µm tip; length ~5 cm.)

3. Fill the micropipette with the drug solution using MicroFil (WPI MF34F-5).

4. Hold the micropipette in the holder connected to the Picospritzer and apply pressure to confirm that the tip is not blocked.

5. Move the micropipette to the recording chamber and position it 20–30 µm away from the cell body.
   Do not touch neurons or the coverslip.

6. Establish whole-cell recording.

7. Record currents. (SCJ ver.: NMDA currents were recorded by applying NMDA (20 µM) via micropipettes.  In this case, the external solution should contain glycine (100 µM) and the glycine receptor blocker strychnine. The pressure intensity was ~5 PSI.)

8. Determine the wash-out time.
   To do this, increase the external solution circulation rate (infusion pump speed) and check the valve-close point.  When the valve is closed, the current should disappear rapidly. (SCJ ver.: For NMDA currents, decay time is critical for identifying channel properties. Therefore, fast circulation was necessary to wash out the drug applied via micropipettes; see Jung et al., 2008, Figure 5B.)
   Some channels show sensitive open-close kinetics, so it is important to determine both the appropriate pressure intensity and wash-out rate (circulation rate).

9. Analyze the recorded data.

Tips

1. Strong pressure (>10 PSI) can blow neurons away. Start with low pressure and increase gradually to an appropriate level.

2. If DMSO is required to dissolve drugs and its concentration exceeds 1%, do not apply the drug via a Picospritzer-connected micropipette. Cells may be lost without yielding usable data.

3. The duration of the external trigger pulse (drug application time) should be less than 2 s (SCJ ver.: Other groups typically use 500 ms–1 s pulses.)

4. Do not use carbon or gas valves as pressure lines.