VI. Micro application by using Picospritzer

Procedure

1. Set up the Picospritzer III (Parker) as the manual.

- Connet the wall pressure line to the valve of Picospritzer

- Set the control line and mid-boxes

- Connet the pressure line of Picospritzer to pipette holders

- Connet the external trigger line of ITC-18 to Picospritzer

- Set the pressure level less then 10PSI

2. Make the micropipette with glass electrode (Warner, G200F-3) for drug applications(SCJ Ver. By using the puller, make the tip size of micropipette 2-3 um. The length is about 5 cm).

3. Fill the micropipette with drugs that you want to apply by using MicroFill (WPI MF34F-5).

4. Hold the micropipette in the holder connected to Picospritzer and shot the pressure to check if the tip is not blocked.

5. Move the micropipette to the recording chamber and locate it 20-30 um away from cell body. Don’t touch neurons or the cover slip.

6. Make a whole cell for recording.

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

 8. Determine the wash-out time.

For this, increase the external solution circulation rate (infusion pump speed) and check the valve close point. When the valve is closed, the current should be dramatically disappeared (SCJ. Ver. In the case of NMDA current, decay time is very important to identify the property. So fast circulation was necessary to wash out the drug applied via micropipettes, see the Jung et al 2008 paper Figure 5B)

Some channels may have the sensitive open and close properties, so it is important to determine the pressure intensity and wash-out rate (circulation rate).

9. Analyze what you recorded.

Tips

1. Strong intensity of pressure (over 10 PSI) can blow out neurons away, so use lower PSI first and increase up to the proper level.

2. If you need to use DMSO to solve some drugs and if its rate is over 1%, don’t try to apply the drug via micropipette connected to Picospritzer. Cells can be gone without giving any data.

3. Duration of external trigger pulse (means the duration of drug application) is better if it is less then 2 sec (According to SCJ Ver.). Other groups usually use it with 500 ms – 1sec duration.

4. Don’t use Carbo. or Gas valve as a pressure line.