PPR Analysis Using ClampFit

Save Default View

To customize the default view to your specific needs (number of cursor pairs, cursor locations, etc.), do the following-

1. Open ClampFit software. Drag and drop the first file to be analyzed.
2. Double-click anywhere on the opened file (this is your analysis window). Go to "General" and set the number of cursor pairs you need. Go to "X Axis" and change the units to milliseconds (or any of the other options).
3. To change the cursor locations, you can either drag the cursor to your desired location or double click on the cursor to manually type the location you want.
4. Once all cursor locations are set, go to "View > Window Defaults... > Save selected Analysis Window settings as default > OK".
5. Now if you close the program, your settings will be the same as last saved when you re-open it.

Quality Control

1. Check all sweeps and make sure to hide (shortcut: "delete") any of the traces that have holding currents larger than 200 pA (i.e., more negative than -200 pA). These cells are likely unhealthy.
2. Hide any of the traces that are poly-synaptic or have voltage escapes in the peak detection window that you set.

3. Average the remaining visible traces by going to "Analyze > Average Traces...".

Get the PPR

1. You should have 4 cursor pairs, where cursors 1-2 captures the baseline region before the first pulse, cursors 3-4 captures the first response, cursors 5-6 captures the baseline region between the two responses, and cursors 7-8 captures the second response.
2. Go to "Analyze > Statistics".
3. For Trace Selection, make sure "All visible signals" and "All visible traces" are selected.
4. For Peak Polarity, check "Apply to all signals" and select "Negative-going"
5. Select your baseline region. Depending on which peak amplitude you are trying to obtain, select either cursors 1-2 or cursors 5-6 for the respective baseline mean level.
6. Check Search Region and set it to 1. Depending on which peak amplitude you are trying to obtain, select either cursors 3-4 or cursors 7-8 for your range.
7. In your measurements, select at least "Peak amplitude (pA)" and "Baseline (pA)".
8. For destination option, I recommend selecting "Append results to sheet".
9. Click "OK", then you can copy and paste your data into your own excel sheet.
10. PPR = peak2/peak1. For data that are noisy, the output from the averaged trace should be used as the datapoint for that cell.