SPECIAL COMMUNICATION
LabPatch, an acquisition and analysis program for patch-clamp electrophysiology

Tim Robinson, Lars Thomsen, and Jan D. Huizinga

Intestinal Disease Research Program and Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

An acquisition and analysis program, "LabPatch," has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/.

voltage clamp; data acquisition; database; Windows 95/98


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE PATCH-CLAMP TECHNIQUE allows for measurement of ionic currents with a very high signal-to-noise ratio and thereby allows for characterization of the currents. Currently, most data acquisition and analysis programs are expensive and time consuming to use. Patch programs written for older operating systems do not have direct connections with powerful programs such as Quattro Pro, Microsoft Access, or Excel. Working in the Windows 95/98 operating system allows more than one program to be open at a time, cut and paste from one program to another, and use of the latest versions of commercial word processors, spreadsheets, and databases. Connections from the patch program to databases and spreadsheets can be made automatic in Windows 95/98. We developed an analysis and acquisition program, "LabPatch," that was written using the graphical programming language Labview 4.1 (National Instruments, Austin, TX). Several electrophysiological programs have already been written using Labview (2, 3, 5). Our objective was to design a program for use in single electrode whole cell patch-clamp experiments that was straightforward, user friendly, free of charge, and integrated into the Windows 95/98 operating system. Specific objectives for the program were the acquiring and recording of data, running voltage protocols, analyzing data, and communicating with spreadsheet and database programs.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Equipment and Setup

The system requirements for LabPatch include a PC with at least a Pentium 100-MHz processor, Windows 95 or 98, and at least 16 MB RAM. A hard drive capacity of at least 500 MB is recommended, although the program itself requires only 22 MB. Users of LabPatch will need a data acquisition (DAQ) board and digital-to-analog (D/A) converter from National Instruments, as well as any commercial patch-clamp amplifier. We used the PCI-MIO-16XE-10 DAQ board, which is capable of sampling at 1 × 105 samples/s. The 16-bit PCI card provides high resolution for recording, although file sizes are larger. We used the BNC-2090 D/A converter, although others are available from National Instruments. Users of LabPatch must have NiDAQ installed on their computer, which comes with every DAQ board from National Instruments.

Verification of Program Function

LabPatch was used to record potassium currents in interstitial cells of Cajal (ICC). A primary cell culture from the murine small intestine was developed, using the method described previously (6), to obtain isolated ICC. The external bath solution used while in whole cell patch configuration contained the following (in mM): 135 NaCl, 5.4 KCl, 0.33 NaH2PO4 · 7H2O, 5 HEPES, 0.8 MgCl2, 5.5 glucose, and 2 CaCl2. The internal patch solution within the electrode contained the following (in mM): 100 potassium aspartate, 30 KCl, 5 HEPES, 1 MgCl2, 5 EGTA, 5 ATP, and 0.1 GTP. Patch-clamp recordings were made from morphologically identified ICC, using 4- to 5-MOmega electrodes and the Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Recordings obtained with LabPatch were compared with recordings made using pCLAMP5 (Axon Instruments). Additionally, the input and output from LabPatch was tested throughout the program's development using an oscilloscope and a model cell, to verify every step of the way that voltages were correctly communicated between LabPatch and the amplifier.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Program Overview

LabPatch consists of four programs: LabPatch Data Acq, LabPatch Protocol Generator, LabPatch Reader, and LabPatch Save Parameters. A flow chart (Fig. 1) describes the purpose and capabilities of each component of LabPatch and shows the general order of program flow. LabPatch Save Parameters need only be run once by the user, unless hardware is changed, and thus the user normally works with only three of the four programs. LabPatch Data Acq is the main patch-clamp program, which is used to acquire and record electrical data while controlling the patch-clamp amplifier. LabPatch Protocol Generator is used to design voltage protocols that can be executed in LabPatch Data Acq. Finally, LabPatch Reader is used to view and analyze recorded data files. LabPatch also comes with a Microsoft Access database file for automatic storage of experimental parameters and notes in Microsoft Access. Other databases can be used, since LabPatch creates an ASCII text file that can easily be read by most databases.


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Fig. 1.   Flow chart describing the purpose and capabilities of each component of LabPatch. Arrows indicate the general order in which each component of LabPatch is used during experimentation.

Configuration

The purpose of LabPatch Save Parameters is to save a configuration file for LabPatch, which contains parameter values specific for the amplifier in use. This program need only be run once, i.e., anytime before LabPatch Data Acq is run for the first time. However, if the user changes amplifiers, or the parameter files are accidentally deleted, the program should be run again to save new parameter files. These settings enable the user to customize how LabPatch interprets voltages output from the amplifier. The user is able to customize settings for the specific DAQ board installed, including the timer capability and voltage range. Additionally, the user can specify path locations of data and parameter files.

LabPatch Data Acquisition

Overview. Patch-clamp experiments are performed using LabPatch Data Acq. This program has three functions built into one program: "test seal" mode, "acquire data" mode (Fig. 2A) for manual viewing and recording, and "protocol" mode for running voltage protocols. Test seal mode is used to obtain a tight seal between the patch electrode and the cell, before electrical recording can begin. Once the desired seal resistance has been obtained, the user can switch to either acquire data or protocol mode. In acquire data mode, the user can view or record data while manually adjusting the holding potential or current. In protocol mode, LabPatch runs voltage protocols and records the response from the cell. Voltage protocols can be designed and saved by LabPatch Protocol Generator.


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Fig. 2.   When the program switch is set to acquire data, LabPatch Data Acq outputs the desired holding command and inputs data returning from the amplifier, with the option of recording the incoming data (A). LabPatch Protocol Generator (B) is used to design and save a voltage protocol ready for use in LabPatch Data Acq. The purpose of the patchdata database is to keep track of binary data files, protocol files, ASCII files (spreadsheet data files), and analysis files, by relating them to the binary data file, and to display key information from these files (C).

Test seal. When the "program" switch in LabPatch Data Acq is set to test seal, LabPatch generates a square wave pulse to test the resistance of the patch electrode and to guide the user into obtaining a high resistance seal. An oscilloscope graph appears in the upper half of the screen. The "zap" function, which is useful for breaking the membrane to gain access to the cell, has been included. The zap function produces a large depolarizing pulse of 1.3 V for 30 ms.

Acquire data. When the program switch is set to acquire data, LabPatch outputs the desired holding command and inputs data returning from the amplifier, with the option of recording the incoming data (Fig. 2A). A waveform chart appears in the upper half of the screen. The x-axis is always real time, in seconds, while the y-axis can be in either picoamperes or millivolts, depending on whether the amplifier is in voltage-clamp or current-clamp mode, respectively. The user can change the rate at which data points are collected (between 1,000 and 10,000 data points/s).

When the mode switch is set to "record," LabPatch begins recording the data to file. LabPatch automatically names the file according to the date and time at that instant. Certain amplifier settings are saved along with the recorded file, including the mode (for example voltage clamp, or current clamp), gain, filter frequency, and cell capacitance. As well, the user is given the opportunity to type additional notes about the experiment, which is saved at the beginning of the file in the header. For example, the user could type a picture number if a picture was taken of the cell being recorded from, or type the name of the drug(s) that will be applied to the cell. Notes can also be taken during and immediately following recording and are saved at the end of the file in a footer. The user may wish to comment on the quality of this recording and then search for keyword terms used in the database. The user could also point out any significant events noticed during the recording and the approximate time they occurred.

If the user injects a drug into the bath, the drug addition can be tagged into the file being recorded by entering the drug concentration and then clicking on the Insert button. When the Insert button is clicked, the drug concentration and approximate time (accurate to 0.1 s) are recorded. All changes in the holding command are also stored, so that the user can keep track of all the stimuli applied to the cell during recording. These automatic features help reduce the amount of time the user spends writing notes down by hand. All amplifier settings, user notes, drug additions, and holding potential changes recorded within the file can automatically be transferred to the Microsoft Access database or can be indirectly imported into a different database. Thus the user can perform searches within the database to find particular settings or notes made during experiments. These features greatly help to increase the user's organization of data and help the user retrieve and relate experiments quickly and easily.

Protocol. At any point while in acquire data mode, the user can set the "experiment" switch to protocol, to run a voltage protocol, in which the output from LabPatch follows a specific program designed by the user. The user is first prompted to either open an existing protocol or design a new one using LabPatch Protocol Generator (Fig. 2B; a more detailed explanation of protocol design follows below). After the protocol has been either selected or designed, LabPatch begins to output the protocol, and the data input from the amplifier is displayed in a waveform chart. Protocol data are always saved to a file automatically named by LabPatch. Once the protocol is finished and all data have been collected, the recording ends automatically, and the user is prompted to type any final comments into a dialog box. Just as described above for the manual file, all amplifier settings and user comments recorded in the protocol file can be automatically transferred to the Microsoft Access database or indirectly imported into a different database.

Outward rectifying potassium currents were recorded from ICC using LabPatch Data Acq in protocol mode in whole cell patch-clamp configuration (see METHODS). A sample recording is depicted in Fig. 3, the voltage protocol used to make the recording is depicted in Fig. 2C, and the current-voltage (I-V) curve generated from the recording is depicted in Fig. 4. The outward current had a fast activation before proceeding to steady state, followed by large tail currents (Fig. 3). The I-V curve (Fig. 4) shows a sharp voltage activation takeoff at -40 mV. Normalizing the tail currents revealed the relative conductances of the ICC outward currents. The above is just one example of how LabPatch can be used to collect current records and how data can be analyzed using LabPatch.


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Fig. 3.   LabPatch Reader is used to read and analyze binary data files recorded by LabPatch Data Acq. The waveform chart displays potassium currents recorded from an interstitial cell of Cajal, using a voltage protocol with a holding potential at -40 mV and 10-mV steps between -80 and +100 mV (depicted in Fig. 2C).



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Fig. 4.   Analysis table and current-voltage (I-V) curve. When the Analyze marker 1+2 button is pressed, the "analyze prompt" appears, and the user has the option of selecting 1 of 6 different types of analysis to be performed on the data that lie between markers 1 and 2. Results of the particular analysis are displayed in the analysis table, which appears over the top of the waveform graph. The I-V curve can plot any 2 columns in the analysis table and is not limited to just plotting current vs. potential. I-V curve depicted was generated from the potassium currents in Fig. 3, showing a sigmoidal pattern characteristic of this type of voltage-activated channel. Polynomial equation used to fit the points is displayed in the text box below the curve.

LabPatch Protocol Generator

The purpose of LabPatch Protocol Generator (Fig. 2B) is to design and save a voltage protocol ready for use in LabPatch Data Acq. This is a stand-alone program; however, an online version can be run right from LabPatch before starting a voltage protocol experiment. When the program first runs, the user should either design a new protocol, using the steps described below, or open an existing protocol by clicking on the Open protocol button. A sample protocol can be viewed at the bottom of the screen, by using the right scrollbar to scroll down. Protocols can be run with P/4 leakage current subtraction (1) for removal of the linear portion of the capacitive current. Conditioning pulses can also be applied before the main stimulus waveform. During execution of the protocol in a patch-clamp experiment, the main waveform is the only data saved to disk, even though leak subtraction and conditioning pulses may have been executed in the protocol. If leak subtraction is enabled, only the corrected waveform values are displayed in the waveform graph and saved to disk. It is not possible to save uncorrected data when leak subtraction is enabled in the protocol.

There are three possible waveforms available in a voltage protocol, each of which can be either a step or a ramp and can be customized with great flexibility by the user. The # Active Columns control can be used to select the number of waveforms in the protocol. The starting Y value, Y1 (voltage or current), can be specified by the user and will be either held or ramped for the time specified to the Y1 of the next waveform. The change in time (delta time) is the amount of time added or subtracted at each episode. The change in Y value (delta Y) is the amount added or subtracted from the Y1 at each episode. The # Episodes control can be used to set the number of episodes in the protocol. The HO control can be used to set the holding command of the episode. Leak subtraction can be enabled or disabled, and the number of pulses to determine the leak current and their polarity can be selected. Conditioning pulses can be enabled or disabled, and the number of pulses selected.

Each protocol can automatically be transferred to the Microsoft Access database. A picture of the protocol can be manually transferred to the database, to provide visual cues to the user as to what each protocol encompasses and to help in selecting protocols for future experiments. Thus patch-clamp recordings made in protocol mode are linked within the database to the protocol used to make the recording.

LabPatch Reader

Data files recorded by LabPatch Data Acq can be read and analyzed using LabPatch Reader (Fig. 3). The user can view data within the data file as blocks that can be adjusted in size (real time) and displayed in a waveform graph. The waveform graph can display data as a single trace or compiled into episodes in the case of protocol files. The user can move forward and backward through the file, zooming in and out, while viewing and analyzing the data. LabPatch Reader allows the user to detect peaks and valleys and to determine baseline levels, time intervals, as well as height, width, and frequency of peaks or valleys. Traces can be filtered, and analyzed for amplitudes and frequencies. All numbers generated during the analysis are stored in an analysis table for plotting (such as I-V curves) and so that no numbers need be written down by hand on paper. The analysis table is fully editable by the user and can be exported to spreadsheets such as Microsoft Excel or Quattro Pro for generation of graphs and further analysis if desired. Recorded traces can also be exported to spreadsheets or presentation software.

Clicking on the Analyze marker 1+2 button will produce the analysis prompt to analyze data located after marker 1 and before marker 2. The type of numbers that can be determined and stored in the analysis table (Fig. 4) by analyzing the data located between markers 1 and 2 include the time point at markers 1 and 2 (Time 1, Time 2), the difference in time (dTime), the voltage or current Y value (Y1, Y2), the difference in Y value (dY), the mean Y value (mean Y), the slope, the number of peaks or valleys, the frequency of peaks or valleys, the holding potential (HO), the concentration of a drug ([drug]), and the plot number (for protocol files that have multiple superimposed plots). While the analysis table is visible, clicking on Save Table to ASCII will save the data in the analysis table to an ASCII file that can be read by spreadsheet programs. Data can also be imported into the analysis table by clicking on Open ASCII File. A plot can be generated from any two columns in the analysis table, for example to generate IV curves. Any of the fields described above (for example dY vs. [drug] to give a dose-response curve) can be plotted, and thus the plot is not just limited to I-V curves. The points in the plot can be fitted with a linear, exponential, or polynomial equation, and the equation of the fitted line is displayed below the plot. Unwanted points in the plot can be removed by deleting them in the analysis table.

Patchdata Database in Microsoft Access

LabPatch gives the user the option of automatically transferring information to a database, such as Microsoft Access, to keep track of all data and protocol files. This transfer of information can be turned on or off by a switch, to enable or disable this feature. If this feature is enabled, LabPatch can link to the "patchdata" database (included in the LabPatch package, Fig. 2C), which runs in Microsoft Access. The user must have Access to run the Patchdata.mdb file. If the user has a different database or does not have any database program at all, then there are alternative options. All the information that gets stored in the patchdata database is first saved as text files. These text files can be read by other database programs and stored in a format similar to that in the Microsoft Access database. If the user does not have a database program at all, the text files themselves can be used as databases, since they are fully searchable in even the most simple word processing program (such as Windows NotePad).

The patchdata database stores all the pertinent information about the experiment for each data file recorded, such as the mode, holding commands, drug additions, user header, and final comments. In the case of protocol data files, an image of the protocol and all protocol details are stored. The database if fully searchable, using the tools provided by Microsoft Access, thus allowing the user to quickly and easily search for certain experiments and group-related experiments together.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LabPatch is a software package for use in single electrode whole cell or single-channel patch-clamp experiments. LabPatch was tested throughout development using an oscilloscope and model cell to verify input/output voltages, as well as by recording and analyzing potassium currents in ICC. Characteristics of the recorded current, such as takeoff (activation) voltage, large tail currents, half-activation voltage, slope factor, etc., were the same as those recorded by Lee et al. (4) using the commercial program pCLAMP5 (Axon Instruments) under similar recording conditions. These data indicate that LabPatch accurately controls and inputs data from the patch-clamp amplifier.

We had three objectives in mind while designing LabPatch: first, make LabPatch straightforward and user friendly; second, make LabPatch run in the Windows 95/98 operating system; third, reduce the start-up costs of patch-clamp research by providing LabPatch free of charge.

Our first objective was satisfied almost immediately by our choice of programming language. The Labview graphical programming language provides simple controls and indicators that are arranged on the screen by the programmer to emulate the front panel of an instrument. The user controls switches, sliding bars, and dials just as if operating a real instrument such as an oscilloscope. The layout of LabPatch was designed to ensure that all important controls and indicators are on-screen at all times, so that the user need not flip around to other screens to operate the program. Controls can be accessed and manipulated via the mouse or keyboard. Although LabPatch is composed of more than one program, the divisions between programs are logical and based on function. For example, pCLAMP6 from Axon Instruments divides data acquisition between ClampEx6 and FetchEx6 programs to perform manual and protocol experiments, respectively. Both these components are contained within one program in LabPatch, called LabPatch Data Acq. The fact that all controls are available on one screen makes LabPatch more user friendly than most other patch-clamp programs.

Our second objective was to make LabPatch run in the Windows 95/98 operating system. Working in Windows 95/98, the patch computer becomes much more useful because it allows integration of patch clamp data with word processing, internet communication, design of presentations, databases, etc., which is not possible in the older operating systems. LabPatch is connectable to spreadsheet and database programs that operate in Windows 95/98, such as Quattro Pro or Excel and Access within the Microsoft Office package. Although LabPatch can perform analysis on data, it can also save the data or the numbers generated in the analysis to a spreadsheet-readable ASCII file for graphing or analysis in the spreadsheet environment. The connection to spreadsheet programs was necessary, since it was recognized that graphs and analysis need be highly customizable in terms of axis, colors, fonts, text locations, etc., a quality that is unparalleled in the more popular spreadsheet programs. The intimate interaction with these powerful Win95/98 programs adds to the user-friendly design of LabPatch, allowing for rapid cut and paste between applications, the ability to customize graphs and analysis, and the ability to keep track of data files using the database.

LabPatch can automatically pass information to Microsoft Access, and indirectly to other databases, to keep track of data files and allow searches to be performed on the information contained in these files. This relational database keeps track of all analysis files related to each binary data file and displays key information from all of these files. Holding command changes, as well as concentrations of drugs added to the bath, are time stamped and recorded within the file. This feature conveniently binds parameters and comments together with the data, thus preventing loss of information or confusion about what parameters were used to record the data. The user can also type information into the file header before recording is started, such as a description of the cell being patched, a picture number if one was taken, or any other useful information. During and after recording, the user can type in any comments about the recording in a file footer, such as a description of the general quality of the recording or interesting events within the recording to be referred to later.

LabPatch can be downloaded from the Internet free of charge, at http://www.fhs.mcmaster.ca/huizinga/. The source code cannot be provided along with the program, due to a copyright agreement implicit in using Labview as a development tool.


    ACKNOWLEDGEMENTS

Present address of L.Thomsen: AstraZeneca Mölndal, Gastrointestinal Pharmacology, S-431 83 Mölndal, Sweden.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. D. Huizinga, McMaster Univ., HSC-3N5C, 1200 Main St. West, Hamilton, ON, Canada L8N 3Z5 (E-mail: huizinga{at}mcmaster.ca).

Received 13 November 1998; accepted in final form 14 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bezanilla, F, and Armstrong CM. Inactivation of the sodium channel. I. Sodium current experiments. J Gen Physiol 70: 549-566, 1977[Abstract/Free Full Text].

2.   Budai, D, Kehl LJ, Poliac GI, and Wilcox GL. An iconographic program for computer-controlled whole-cell voltage clamp experiments. J Neurosci Methods 48: 65-74, 1993[ISI][Medline].

3.   Kunze, WA, Furness JB, Bertrand PP, and Bornstein JC. Intracellular recording from myenteric neurons of the guinea-pig ileum that respond to stretch. J Physiol (Lond) 506: 827-842, 1998[Abstract/Free Full Text].

4.   Lee, JCF, Thuneberg L, Berezin I, and Huizinga JD. The generation of slow waves in membrane potential is an intrinsic property of interstitial cells of Cajal. Am J Physiol Gastrointest Liver Physiol 277: G409-G423, 1999[Abstract/Free Full Text].

5.   Nordstrom, MA, Mapletoft EA, and Miles TS. Spike-train acquisition, analysis and real-time experimental control using a graphical programming language (LabView). J Neurosci Methods 62: 93-102, 1995[ISI][Medline].

6.   Thomsen, L, Robinson TL, Lee JCF, Farraway L, Hughes MJG, Andrews DW, and Huizinga JD. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med 4: 848-851, 1998[ISI][Medline].


Am J Physiol Cell Physiol 278(5):C1055-C1061
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society




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