Mini analysis

Overview

Goal:

Identify, annotate, and analyze spontaneous mini synaptic events (mEPSP, mEPSC, mEJP, mEJC) in the recording

Output:

  • Visual markers for individual minis

  • Exportable table of mini data (csv)

  • Data stored for future use

  • Summary

Plugins used:

Set up

Open a recording from file menu -> Open recording or Alt+o. Set the view mode to continuous by View menu -> continuous Select the channel to analyze in the channel dropdown menu.

Open plugins to use by Plug-ins menu -> select the desired plugin names. Plugins typically have user-input forms that appear in the control-panel as individual tabs.

Use the Navigation Tools located around the plot to adjust the x- and y-axes.

Alternatively, use the Navigation plugin to set the desired x- and y-axes limits.

Filtering (optional)

If noise is high in the raw recording data, a filter may be applied.

The Process Recording plugin offers ‘Lowpass Boxcar’ and ‘Lowpass Bessel’ filtering. Under the Filtering section, select the desired filtering algorithm and enter the required parameters. Save the filtered recording data by file menu -> Save recording data as.

Caution

.abf files cannot be overwritten by SimplyFire. Filtered data should be stored as new files.

Alternatively, a filter may be applied outside of the SimplyFire environment. Ensure that the filtered data is saved in .abf format. Open the filtered recording using SimplyFire.

Analysis

Analysis is performed by Mini Analysis Plugin.

The Mini Analysis Plugin uses several parameters to analyze minis. The basic parameters are described here. For more information on all the parameters that can be adjusted, see the Mini Analysis Plugin page.

Parameters

Direction

Indicates the expected sign of the minis. Typically, the direction is positive for potential and negative for current recordings.

Search radius in % of the visible x-axis (Manual)

Used for manual mini analysis. Indicates the x-axis radius that is considered for analysis when the mouse is clicked. The radius is represented as a percentage of the total x-axis. In general, the lower number in this parameter, the higher the precision of the mouse-click.

Search window in ms (Auto)

Used for automated mini analysis. Indicates the window of x-axis that is considered at a time. The window traverses from left to right, sampling the specified window at a time. In general, the lower number in this parameter, the fewer minis are skipped but also the slower the analysis.

Caution

The x-axis window considered for each mini analysis must be larger than at least of 20 data points worth of x-axis range (i.e. for a 10kHz recording, the window must be larger than 2ms) Ideally, each mini analysis covers at least the approximate width of a single mini event.

Minimum amplitude (absolute value)

Located under Filtering parameters. Specify the minimum amplitude required for a mini. Setting this to 0 (without other filtering parameters) will result in most noise being annotated as a mini.

Automated analysis

Two automated analysis modes are available: Find all searches through the entire trace. Find in window searches the visible x-axis.

All discovered minis will be annotated on the plot, and the details should appear in the data-panel located below the plot.

Caution

Find in window only uses the x-axis to exclude parts of the trace from analysis. Parts of the y-axis that are out of view may still be included in the analysis.

Manual analysis

Navigate through the trace and click on the plot near the desired mini events.

Caution

Ensure that the pan/zoom and rect to zoom options are turned off in the matplotlib toolbar. The mouse should have the default cursor shape.

If the software detects a mini near the clicked location, it will annotate the event on the plot. Details of the minis will be added to the table below the plot as new minis are discovered.

Delete minis

Discovered minis can be discarded using several methods.

Delete buttons

Delete all button clears all minis found within the channel.

Delete in window button clears data for minis found within the visible x-axis.

Caution

Delete in window only uses the x-axis to select minis to discard. Minis that are out of the visible y-axis may still be discarded if it lands within the visible x-axis boundaries.

Select minis on the plot

Clicking on the peak marker for a mini highlights it. Clicking on the peak marker while holding the Shift key highlights multiple peaks. Alternatively, left-click + drag to highlight minis found within the rectangle. Use the Escape/q key to remove the highlights. Use Ctrl+a to select all minis.

Hitting the Delete/Backspace/e key deletes the data for the mini.

Select minis from the table

Selecting entries in the data-panel highlights the corresponding mini markers on the plot. Press Shift and left-click to select a range of entries or press Ctrl and select multiple entries. Use Ctrl+a to select all entries. Use the Escape/q key to remove all the highlights.

Hitting the Delete/Backspace/e key deletes all highlighted minis.

Output

Details for all minis discovered appear in the data-panel located below the plot area. The panel should have a tab labeled ‘Mini’.

Mini analysis calculates/stores the following properties of each mini in the data-panel:

  • peak time

  • amplitude (signed)

  • decay constant (tau)

  • rise time (0-100)

  • halfwidth

  • baseline value

  • channel number

  • standard deviation of the baseline noise

  • direction of the mini (-1 or 1)

  • whether or not the mini is a compound mini

Each numerical column can be sorted by clicking on the column header.

Columns can be hidden or shown by toggling the checkboxes located at the bottom of the plugin’s control-panel.

Export the data

Selected entries in the datapanel can be copied onto the clipboard. Data can be copied by Ctrl+c key stroke or right-click on the data-panel -> Copy selected.

The copied data can be pasted into Excel or other programs of choice.

The data-panel can also be exported to a comma separated value (CSV) format by file menu -> Mini Analysis -> Export data table. All data visible in the data-panel are stored in the exported file.

Save the mini data

The annotations and details for the discovered minis can be stored in a format that can be opened by SimplyFire later.

Go to file menu -> Mini Analysis -> Save minis as... and follow the file save prompt.

The default extension for the mini data is .mini. The files can also be saved as .csv files and opened in other programs. Mini data saved this way contains more details than data exported from the data-panel.

Open previously analyzed minis

Previously analyzed minis can be opened by file menu -> Mini Analysis -> Open mini file. Doing so will discard any changes in the unsaved mini data.

Summarize the data

A summary of the discovered minis can be added to the results-display. The results-display is found in the results tab under the plot.

Right-click on the data-panel -> Report all or Report selected or press the Report stats on the control-panel to calculate the averages and standard deviations of numerical properties of minis. Additionally, the frequency of the minis between the first and last mini is calculated.

The result can be found in the results-display.