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NeuroExpress program for analyzing patch-clamp data
Method · August 2022
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NeuroExpress program for analyzing patch-clamp data
** Introduction to the Windows 10 version **
NeuroExpress is a Windows-based program designed to perform analysis of electrophysiological
recordings made in whole-cell patch clamp experiments or using sharp electrodes. It has various
modules for analyzing different types of data, such as current step responses (I-V data), miniature
postsynaptic currents or spike arrival times. The program is a standalone executable (NeuroExpress.exe)
and it can run in Windows 7 or higher operating systems. The suggested memory size is 4 GB, but it can
likely run with less RAM. If you experience the error message ‘Out of memory’, please contact the author.
The program should be placed into a local folder, which is not designated as Read-only. One example is
c:\Program files\NeuroExpress. The program can read Axon Binary files (ABF) directly. It can also read
ATF, TXT and ASCII files but those need to be in a format that is compatible with the program. Time,
voltage and current channels need to be in separate columns.
When opening files, the user has to select the requested data format in the combobox. If the file is
readable, the program performs the analysis immediately. The content of the file will be displayed in the
upper panel(s) and analyzed data will appear in the bottom plot panels. The user can change settings for
the analysis (right side panel) or save the calculated parameters into an Excel worksheet (press the ‘Send’
button). Also, the content (event list) of the plot panels can be exported into Excel. All functions can be
accessed through popup menus, that are associated with the buttons in the right upper corner of the
display panels (‘Func’).
NeuroExpress program for analyzing patch-clamp data
** Usage feedback **
The program can send a short data packet to the developer that contains information on the number of
successfully/unsuccessfully opened files and the type of analysis performed by the user. This information
is sent via internet when the user exits NeuroExpress. The program collects absolutely no personal
information, names of opened files or parameters calculated by it. The developer would appreciate if the
users contact the author in case of crashes or when experiencing difficulties, so the program can be
further improved, and bugs uncovered. E-mail: [email protected]
** Work folder **
The program will create a work folder at c:\Users\username\AppData\Local\NeuroExpress. The program
configuration file NeuroExpress.ini is stored here. Also, the program will save the settings and manual
correction data for each analyzed file when the menu ‘Autosave analysis settings’ is checked. Such data
can be reloaded when the menu ‘Load last settings’ is checked.
NeuroExpress program for analyzing patch-clamp data
** User experience, smart features **
The author tries to make the program user friendly, fast and robust. Calculation time for most tasks
remains under 1 s. As an example, current step data from an ABF file containing 50 sweeps each 1 s long
and acquired at 20 kHz are loaded and fully analyzed well within 1 s. The program will perform all the
analysis tasks including spike detection, search for extrema, fitting of voltage traces automatically and all
the results can be saved into an Excel worksheet or TXT file. Analysis of mini events is also fast. A current
trace of 200 s containing a few hundred events and acquire at 20 kHz is analyzed within 1 s.
Parameters for the analysis can be set manually or using the mouse wheel (incrementing or
decrementing, try it). Whenever a parameter is changed the mini analysis is re-ran and the results are
shown immediately.
If the mini recordings contain artefacts or test pulses to be excluded, the user can bring up selection
cursors and set the range to exclude using the mouse (click and drag the cursor). Such sections can be
ignored and the data outside of such regions are used for the statistics. Ignored sections will turn gray.
Analysis of physiological properties
** General instructions **
To perform analysis of voltage traces evoked by current step stimulation the recording must be in a
format that is suitable for the program. ABF and ATF files containing 2 channel recordings can be
analyzed. The acquisition mode of the recording must be episodic stimulation for this analysis.
Measurement units of the voltage channel must be mV and pA or nA for the current channel. The first
channel contains the voltage response of the neuron while the second channel contains the current that
is injected. These are rectangular current steps starting at a negative level and incremented in small
steps to more depolarizing levels. As an example, a recording containing step responses starting from -
200 pA, incremented by +10 pA and ending at +200 pA is OK. The length of each episode can be 0.1 – 4.5
s at 20 kHz sampling rate for the recording. The maximal number of current steps in a recording can be
192. The program will load the ABF of ATF file into the memory and then immediately extracts dozens of
physiological parameters from the voltage traces. Voltage deflection data, resistance, time constant,
estimated membrane capacitance and other parameters will be displayed as functions of the injected
current. These will appear in the bottom plot panels. The content of each panel can be selected by the
popup/dropdown menu (use the right mouse button to explore freely). Linear or exponential functions
will be used automatically to fit various relationships, e.g. the resistance vs. current plot or the spike
number vs. current relationship.
Analysis of physiological properties
Panel showing
action potentials
during the step.
Initial part of the The entire recorded
voltage traces are voltage trace is shown.
fitted automatically.
The two resistance
curves are fitted
with linear functions.
Input-output Select file
relationship. format here.
The voltage sag and the
afterdepolarization is plotted You can open 2-channel ABF or
against the current. ATF files in episodic stim. mode.
The first channel is the voltage,
the second is the current.
Analysis of physiological properties
** Settings and controls **
These are parameters for the analysis of current step responses. Plots will be updated when the user
enters a new value for a selected parameter. All parameters are accessible via the Settings dialog box.
Current reject range: No voltage traces are used for resistance calculation within this current range
centered at I=0 pA. Estimation of resistance values can be increasingly less
accurate when applying current steps with smaller amplitude.
Resistance midpoint: This is the midpoint (breakpoint) current level that separates the 2 linear parts
of the resistance curves.
Vsag/ADP midpoint: This is the midpoint (breakpoint) current level that separates the 2 linear parts
of the voltage sag and afterdepolarization curves.
I-O spike # fit limit: The I-O curve is considered as linear up to this spike count level.
Spike counting limit: Spikes are counted and summed up to this current current level to calculate
cumulative spike counts.
Analysis of physiological properties
** Cell info **
Nervous system/area: Use the combobox to select the preparation or brain area where the recordings
were made.
Cell type: This is the phenotype of the cell recorded, e.g. fast spiking, pyramidal,
PV interneuron, medium spiny neuron, etc.
Treatment group: This is the treatment group, e.g. control, LTP, chronic drug group, etc.
** Main panel **
Results saved into: The user can select the destination where calculated parameters are saved.
Send: Pressing this button the user saves the calculated parameters for the current
data file.
Del: When pressed, the program deletes the last row in the Excel worksheet.
Lst: When pressed, the program lists the event data into the Excel table or text file.
Auto: Button to select fully automatic calculation vs. manual adjustment mode.
Reset: When pressed, the correction data for the current file will be deleted.
Open: The most used button, this will bring up the dialog box to open single data file.
Analysis of physiological properties
The selected
voltage trace
is shown. Maximal
Exponential fit overshoot after
the current step
Voltage deflection at
the end of the step
Maximal voltage
deflection
Func button brings up the
popup menu. You can select
the parameter to be displayed.
Two resistance curves are
calculated from voltage Button to select automatic
deflection points. analisys vs. Manual
correction mode.
Analysis of physiological properties
** Correcting data **
Voltage traces are often contaminated by EPSPs or IPSPs received by the neuron. Other type of noise or
transients can appear in the recordings that are usually interfere with the analysis. One example is when
an EPSS arrives just before the current step, so the resting membrane potential will be inaccurately
obtained. The user can override this problem by manually moving the datapoint that is associated with
the resting membrane potential. This is a gray colored symbol shown in each voltage trace and appearing
before the onset of the current step. The user can check the box ‘Manual correction’ in the lower right
panel and then move the datapoints freely. First, the contaminated voltage trace is selected by moving
the mouse within the plot box that shows the resistance or voltage sag. When the contaminated trace is
displayed in the upper panel, the user can click the left mouse button to make the selection and the gray
datapoint can be grabbed and moved by the user. The same can be applied to the datapoints associated
with the maximal voltage deflection, voltage at the end of the step and the afterdepolarization. Moving
the datapoints will cause the associated parameters to be recalculated and the plot boxes refreshed
immediately. A completely wrong datapoint can be erased by moving it outside of the ‘Voltage traces’
panel.
Analysis of physiological properties
Spike phase
trajectories
(dVm/dt vs. Vm)
Red points indicate
most negative Vm
between spikes.
Time constant is plotted
against the stimulus current.
Linear extrapolation us used
for membrane time constant.
The latency curve
is also fitted
automatically.
The resistance curve
is split into two parts
defined by the
midpoint parameter.
Photo of the cell
when available.
Voltage sag is red, ADP Spike latency as a
is orange. Linear fits function of the current
are used to calculate level (data for rebound
slope parameters. spikes are also shown)
Analysis of physiological properties
** Exporting calculated parameters **
The program saves the physiological parameters calculated from the current step responses into an Excel
worksheet. The ‘Link to Excel’ checkbox needs to be checked first. Input resistance, resting membrane
potential, sag ratio, rheobase and many other parameters are saved just by pressing the ‘Send’ button in
the main panel. The user can check the ‘Cell comments in Excel’ box in order to have descriptions of the
calculated parameters as comments appearing in the Excel worksheet.
Analysis of miniature EPSCs
** General instructions **
To perform analysis of miniature postsynaptic currents first open the file to be analyzed. ABF and ATF files
containing 1 or 2 channel recordings can be analyzed. The acquisition mode of the recording has to be
gap-free for this analysis. Measurement units of the channels should be pA or nA for the current channel
and mV for the voltage channel. If the 'Estimate noise level' checkbox is checked, the program will
attempt to calculate the mean noise floor for the recording and sets the value of the corresponding edit
box. After this, events are detected automatically by running one of the 3 algorithms specified in the gray
colored group box below. To achieve the most accurate analysis, the Estimated noise, Max. EPSC rise
time and Max. EPSC decay time parameters should be set by the user. Noise should be set to a level close
to the amplitude of baseline peak-to-peak fluctuations in the signal. Max. EPSC rise time parameter
should be set to a level that is up to 2-3 times greater than the visually determined rise time of the mini
events. Max. decay time should be set in a way that most of detected mEPSC events can be entirely
displayed in the upper right panel (mEPSC traces). Detected events will be indicated by red symbols in
the trace panels. If baseline points are shown, those are indicated by cyan colored symbols. Use the
zoom feature to check the quality of event detection and change the parameters listed above to get the
best analysis. Here, you use the mouse in the Full trace panel and define a rectangle in which the data
will be displayed and analyzed separately. The uppermost panel (Selection from full trace) will show the
section of the trace you selected. Statistical parameters will be calculated for this selected section, so the
rest of the trace is not included. Of course, you can use the entire trace for analysis when you unzoom
(just click outside of the grayed rectangle or press the Full button).
Analysis of miniature EPSCs
This will show the selected
(zoomed) part of the recording.
The bottom 4 panels will show
data from this selection.
Up to 20 detected
mEPSCs are shown
in this panel.
The entire recording is
shown here. You can
zoom into this panel!
Settings for the analysis. Plots
are immediately updated
after changing a parameter.
Each plot panel has a
popup menu where
you can select various
functions.
Statistical information You can open 2-channel ABF or
is shown in the ATF files in gap-free mode. The
bottom info panel. first channel is the current, the
second is the holding potential.
Analysis of miniature EPSCs
Minis are fitted with
mono- or biexponential Average event
functions. waveform.
This is the selection
window that is used for the
statistical calculations.
Statistical data can be
Interevent interval quickly exported into an
histogram (cumulative Excel worksheet when
is also shown). you press Send.
Analysis of miniature EPSCs
** Settings and controls **
These are parameters for the analysis of mini EPSCs or IPSCs. Plots will be updated when the user enters
a new value for a selected parameter and then clicks in an adjacent edit box (light-blue colored edit box is
left).
Estimated noise level: Estimated peak-to-peak noise in the recording.
Max. mEPSC rise time: Maximal rise time of the mini EPSC in ms.
Max. mEPSC decay: The approximate decay time of the slowest mini EPSC in the recording.
Smoothing parameter: This parameter is used for moving average smoothing or Savitzky-Golay
smoothing of the current trace. Higher values will perform stronger smoothing.
IEI hist. max/bin: The width and binsize of the interevent interval histogram.
Amp. hist. max/bin: The width and binsize of the amplitude histogram.
Analysis of miniature EPSCs
Auto estimate: Turn it ON for automatic noise level estimation based on standard deviation of
the signal in the beginning of the recording.
Biexp. fitting: Turn it ON to use biexponential fitting of events instead of monoexponentials.
Outward minis: When checked, IPSCs are detected instead of EPSCs.
Sav-Gol filter: When checked, the program uses Savitzky-Golay filtering instead of simple
moving average filtering of data.
Parzen: When checked, the program calculates histograms based on kernel estimation
algorithm (Parzen-estimation).
Detection algorithm: There are 3 slightly different algorithms to detect mini EPSCs. Use the first
when the data are low-pass filtered and smooth. The second is better for
data with high-frequency noise, but it performs slower. Optimal setting for
estimated noise level can be different for the different algorithms.
Analysis of miniature EPSCs
Cyan colored points
indicate the baseline
(reference) before
the EPSC event.
Estimated noise
amplitude, use this
to set limit for the
event detection.
Time constants are shown
for the fitted events shown
in the uppermost panel.
Event area histogram
calculated from the
selected part of the trace.
Analysis of miniature EPSCs
** Exporting calculated parameters **
The program saves the parameters of the miniature EPSCs/IPSCs into an Excel worksheet. The ‘Excel
worksheet’ needs to be selected in the combobox first. mEPSC interevent intervals, amplitudes, decay
time constants and many other parameters are saved just by pressing the ‘Send’ button in the main
panel. The user can check the ‘Parameter explanation in Excel’ menu under Optionsin order to have
descriptions of the calculated parameters as comments appearing in the Excel worksheet.
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